CN113462671B - CRISPR-Cas12j酶和系统 - Google Patents

CRISPR-Cas12j酶和系统 Download PDF

Info

Publication number
CN113462671B
CN113462671B CN202110475316.6A CN202110475316A CN113462671B CN 113462671 B CN113462671 B CN 113462671B CN 202110475316 A CN202110475316 A CN 202110475316A CN 113462671 B CN113462671 B CN 113462671B
Authority
CN
China
Prior art keywords
lys
leu
ser
glu
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110475316.6A
Other languages
English (en)
Other versions
CN113462671A (zh
Inventor
赖锦盛
周英思
李英男
张继红
王莹莹
吕梦璐
张湘博
赵海铭
宋伟彬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
China Agricultural University
Original Assignee
China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by China Agricultural University filed Critical China Agricultural University
Priority to CN202110475316.6A priority Critical patent/CN113462671B/zh
Publication of CN113462671A publication Critical patent/CN113462671A/zh
Application granted granted Critical
Publication of CN113462671B publication Critical patent/CN113462671B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明提供了CRISPR‑Cas12j酶和系统。具体而言，本发明提供一种Cas效应蛋白，包含此类蛋白的融合蛋白，以及其编码核酸分子。还提供用于核酸编辑的复合物和组合物，例如基因或基因组编辑的复合物和组合物，其包含Cas效应蛋白或融合蛋白，或其编码核酸分子。还提供用于核酸编辑的方法，例如基因或基因组编辑的方法，其使用Cas效应蛋白或融合蛋白。

Description

CRISPR-Cas12j酶和系统
本申请是申请号为201980014005.0、申请日为2019年11月15日、发明名称为“CRISPR-Cas12j酶和系统”的发明申请的分案申请。
技术领域
本发明涉及核酸编辑领域，特别是规律成簇的间隔短回文重复(CRISPR)技术领域。具体而言，本发明涉及Cas效应蛋白，包含此类蛋白的融合蛋白，以及编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的复合物和组合物，其包含本发明的蛋白或融合蛋白，或编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的方法，其使用包含本发明的蛋白或融合蛋白。
背景技术
CRISPR/Cas技术是一种被广泛使用的基因编辑技术，它通过RNA引导对基因组上的靶序列进行特异性结合并切割DNA产生双链断裂，利用生物非同源末端连接或同源重组进行定点基因编辑。
CRISPR/Cas9系统是最常用的II型CRISPR系统，它识别3’-NGG的PAM基序，对靶标序列进行平末端切割。CRISPR/Cas Type V系统是一类近两年新发现的CRISPR系统，它具有5’-TTN的基序，对靶标序列进行粘性末端切割，例如Cpf1,C2c1,CasX,CasY。然而目前存在的不同的CRISPR/Cas各有不同的优点和缺陷。例如Cas9,C2c1和CasX均需要两条RNA进行导向RNA，而Cpf1只需要一条导向RNA而且可以用来进行多重基因编辑。CasX具有980个氨基酸的大小，而常见的Cas9，C2c1，CasY和Cpf1通常大小在1300个氨基酸左右。此外，Cas9，Cpf1，CasX，CasY的PAM序列都比较复杂多样，而C2c1识别严谨的5’-TTN,因此它的靶标位点比其他系统容易被预测从而降低了潜在的脱靶效应。
总之，鉴于目前可获得的CRISPR/Cas系统都受限于一些缺陷，开发一种更稳健的、具有多方面良好性能的新型CRISPR/Cas系统对生物技术的发展具有重要意义。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现了一种新型RNA指导的核酸内切酶。基于这一发现，本发明人开发了新的CRISPR/Cas系统以及基于该系统的基因编辑方法。
Cas效应蛋白
因此，在第一方面，本发明提供了多种蛋白，其具有SEQ ID NOs：1-20、107、108任一项所示的氨基酸序列或其直系同源物、同源物、变体或功能性片段；其中，所述直系同源物、同源物、变体或功能性片段基本保留了其所源自的序列的生物学功能。
在本发明中，上述序列的生物学功能包括但不限于，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性。
在某些实施方案中，所述直系同源物、同源物、变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性。
在某些实施方案中，所述直系同源物、同源物、变体与SEQ ID NOs：1-20、107、108任一项所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的生物学功能(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。
在某些实施方案中，所述蛋白是CRISPR/Cas系统中的效应蛋白。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NOs：1-20、107、108任一项所示的序列；
(ii)与SEQ ID NOs：1-20、107、108任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NOs：1-20、107、108任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：1所示的序列；
(ii)与SEQ ID NO：1所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：2所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：2所示的序列；
(ii)与SEQ ID NO：2所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：2所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：3所示的序列；
(ii)与SEQ ID NO：3所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：3所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：4所示的序列；
(ii)与SEQ ID NO：4所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：4所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：5所示的序列；
(ii)与SEQ ID NO：5所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：5所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：5所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：6所示的序列；
(ii)与SEQ ID NO：6所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：6所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：6所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：7所示的序列；
(ii)与SEQ ID NO：7所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：7所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：8所示的序列；
(ii)与SEQ ID NO：8所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：8所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：9所示的序列；
(ii)与SEQ ID NO：9所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：9所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：9所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：10所示的序列；
(ii)与SEQ ID NO：10所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：10所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：10所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：11所示的序列；
(ii)与SEQ ID NO：11所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：11所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：11所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：12所示的序列；
(ii)与SEQ ID NO：12所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：12所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：12所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：13所示的序列；
(ii)与SEQ ID NO：13所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：13所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：13所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：14所示的序列；
(ii)与SEQ ID NO：14所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：14所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：14所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：15所示的序列；
(ii)与SEQ ID NO：15所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：15所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：15所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：16所示的序列；
(ii)与SEQ ID NO：16所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：16所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：16所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：17所示的序列；
(ii)与SEQ ID NO：17所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：17所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：17所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：18所示的序列；
(ii)与SEQ ID NO：18所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：18所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：18所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：19所示的序列；
(ii)与SEQ ID NO：19所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：19所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：19所示的氨基酸序列。
在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：20所示的序列；
(ii)与SEQ ID NO：20所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：20所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
在某些实施方案中，本发明的蛋白具有SEQ ID NO：20所示的氨基酸序列。
衍生的蛋白
本发明的蛋白可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，蛋白的衍生化(例如，标记)不会不利影响该蛋白的期望活性(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。因此，本发明的蛋白还意欲包括此类衍生化的形式。例如，可以将本发明的蛋白功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个蛋白或多肽，检测试剂，药用试剂等。
特别地，可以将本发明的蛋白连接其他功能性单元。例如，可以将其与核定位信号(NLS)序列连接，以提高本发明的蛋白进入细胞核的能力。例如，可以将其与靶向部分连接，以使得本发明的蛋白具有靶向性。例如，可以将其与可检测的标记连接，以便于对本发明的蛋白进行检测。例如，可以将其与表位标签连接，以便于本发明的蛋白的表达、检测、示踪和/或纯化。
缀合物
因此，在第二方面，本发明提供了一种缀合物，其包含如上所述的蛋白和修饰部分。
在某些实施方案中，所述修饰部分选自另外的蛋白或多肽、可检测的标记或其任意组合。
在某些实施方案中，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，具有选自下列的活性的结构域：核苷酸脱氨酶，甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合。
在某些实施方案中，本发明的缀合物包含一个或多个NLS序列，例如SV40病毒大T抗原的NLS。在某些示例性实施方案中，所述NLS序列如SEQ ID NO:81所示。在某些实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的末端(例如，N端或C端)。在某些示例性实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的C端。
在某些实施方案中，本发明的缀合物包含表位标签(epitope tag)。这类表位标签是本领域技术人员熟知的，其实例包括但不限于His、V5、FLAG、HA、Myc、VSV-G、Trx等，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的表位标签。
在某些实施方案中，本发明的缀合物包含报告基因序列。这类报告基因是本领域技术人员熟知的，其实例包括但不限于GST、HRP、CAT、GFP、HcRed、DsRed、CFP、YFP、BFP等。
在某些实施方案中，本发明的缀合物包含能够与DNA分子或细胞内分子结合的结构域，例如麦芽糖结合蛋白(MBP)、Lex A的DNA结合结构域(DBD)、GAL4的DBD等。
在某些实施方案中，本发明的缀合物包含可检测的标记，例如荧光染料，例如FITC或DAPI。
在某些实施方案中，本发明的蛋白任选地通过接头与所述修饰部分偶联、缀合或融合。
在某些实施方案中，所述修饰部分直接连接至本发明的蛋白的N端或C端。
在某些实施方案中，所述修饰部分通过接头连接至本发明的蛋白的N端或C端。这类接头是本领域熟知的，其实例包括但不限于包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Glu或Ser)或氨基酸衍生物(如，Ahx、β-Ala、GABA或Ava)的接头，或PEG等。
融合蛋白
在第三方面，本发明提供了一种融合蛋白，其包含本发明的蛋白以及另外的蛋白或多肽。
在某些实施方案中，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，具有选自下列的活性的结构域：核苷酸脱氨酶，甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合。
在某些实施方案中，本发明的融合蛋白包含一个或多个NLS序列，例如SV40病毒大T抗原的NLS。在某些实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的末端(例如，N端或C端)。在某些示例性实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的C端。
在某些实施方案中，本发明的融合蛋白包含表位标签。
在某些实施方案中，本发明的融合蛋白包含报告基因序列。
在某些实施方案中，本发明的融合蛋白包含能够与DNA分子或细胞内分子结合的结构域。
在某些实施方案中，本发明的蛋白任选地通过接头与所述另外的蛋白或多肽融合。
在某些实施方案中，所述另外的蛋白或多肽直接连接至本发明的蛋白的N端或C端。
在某些实施方案中，所述另外的蛋白或多肽通过接头连接至本发明的蛋白的N端或C端。
在某些示例性实施方案中，本发明的融合蛋白具有选自下列的氨基酸序列：SEQID NOs:82-101。
本发明的蛋白、本发明的缀合物或本发明的融合蛋白不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。
同向重复序列
在第四方面，本发明提供了一种分离的核酸分子，其包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NOs：41-60任一项所示的序列；
(ii)与SEQ ID NOs：41-60任一项所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NOs：41-60任一项所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列；
并且，(ii)-(v)中任一项所述的序列基本保留了其所源自的序列的生物学功能，所述序列的生物学功能是指，作为CRISPR-Cas系统中的同向重复序列的活性。
在某些实施方案中，所述分离的核酸分子是CRISPR-Cas系统中的同向重复序列。
在某些实施方案中，所述核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NOs：41任一项所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)(a)中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：41所示的序列；
(ii)与SEQ ID NO：41所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：41所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：41所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：41所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：42所示的序列；
(ii)与SEQ ID NO：42所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：42所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：42所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：42所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：43所示的序列；
(ii)与SEQ ID NO：43所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：43所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：43所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：43所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：44所示的序列；
(ii)与SEQ ID NO：44所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：44所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：44所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：44所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：45所示的序列；
(ii)与SEQ ID NO：45所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：45所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：45所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：45所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：46所示的序列；
(ii)与SEQ ID NO：46所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：46所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：46所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：46所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：47所示的序列；
(ii)与SEQ ID NO：47所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：47所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：47所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：47所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：48所示的序列；
(ii)与SEQ ID NO：48所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：48所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：48所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：48所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：49所示的序列；
(ii)与SEQ ID NO：49所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：49所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：49所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：49所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：50所示的序列；
(ii)与SEQ ID NO：50所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：50所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：50所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：50所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：51所示的序列；
(ii)与SEQ ID NO：51所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：51所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：51所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：51所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：52所示的序列；
(ii)与SEQ ID NO：52所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：52所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：52所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：52所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：53所示的序列；
(ii)与SEQ ID NO：53所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：53所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：53所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：53所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：54所示的序列；
(ii)与SEQ ID NO：54所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：54所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：54所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：54所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：55所示的序列；
(ii)与SEQ ID NO：55所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：55所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：55所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：55所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：56所示的序列；
(ii)与SEQ ID NO：56所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：56所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：56所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：56所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：57所示的序列；
(ii)与SEQ ID NO：57所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：57所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：57所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：57所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：58所示的序列；
(ii)与SEQ ID NO：58所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：58所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：58所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：58所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：59所示的序列；
(ii)与SEQ ID NO：59所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：59所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：59所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：59所示的核苷酸序列的互补序列。
在某些实施方案中，所述分离的核酸分子是RNA。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(i)SEQ ID NO：60所示的序列；
(ii)与SEQ ID NO：60所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；
(iii)与SEQ ID NO：60所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；
(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或
(v)(i)-(iii)任一项中所述的序列的互补序列。
在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：
(a)SEQ ID NO：60所示的核苷酸序列；
(b)在严格条件下与(a)中所述的序列杂交的序列；或
(c)SEQ ID NO：60所示的核苷酸序列的互补序列。
CRISPR/Cas复合物
在第五方面，本发明提供了一种复合物，其包含：
(i)蛋白组分，其选自：本发明的蛋白、缀合物或融合蛋白，及其任意组合；和
(ii)核酸组分，其从5’至3’方向包含如上文所述的分离的核酸分子和能够与靶序列杂交的导向序列，
其中，所述蛋白组分与核酸组分相互结合形成复合物。
在某些实施方案中，所述导向序列连接于所述核酸分子的3’端。
在某些实施方案中，所述导向序列包含所述靶序列的互补序列。
在某些实施方案中，所述核酸组分是CRISPR-Cas系统中的导向RNA。
在某些实施方案中，所述核酸分子是RNA。
在某些实施方案中，所述复合物不包含反式作用crRNA(tracrRNA)。
在某些实施方案中，所述导向序列在长度上为至少5个、至少10个、至少14个。在某些实施方案中，所述导向序列在长度上为10-30个、或15-25个、或15-22个、或19-25个、19-22个核苷酸或14-28个核苷酸。
在某些实施方案中，所述分离的核酸分子在长度上为55-70个核苷酸，例如55-65个核苷酸，例如60-65个核苷酸，例如62-65个核苷酸，例如63-64个核苷酸。在某些实施方案中，所述分离的核酸分子在长度上为15-30个核苷酸，例如15-25个核苷酸，例如20-25个核苷酸，例如22-24个核苷酸，例如23个核苷酸。
编码核酸、载体及宿主细胞
在第六方面，本发明提供了一种分离的核酸分子，其包含：
(i)编码本发明的蛋白或融合蛋白的核苷酸序列；
(ii)编码如第四方面所述的分离的核酸分子；或
(iii)包含(i)和(ii)的核苷酸序列。
在某些实施方案中，(i)-(iii)任一项中所述的核苷酸序列经密码子优化用于在原核细胞中进行表达。在某些实施方案中，(i)-(iii)任一项中所述的核苷酸序列经密码子优化用于在真核细胞中进行表达。
在第七方面，本发明还提供了一种载体，其包含如第六方面所述的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在某些实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，柯斯质粒等等。在某些选实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的蛋白、融合蛋白、如第四方面所述的分离的核酸分子或如第五方面所述的复合物。
在第八方面，本发明还提供了包含如上所述的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
组合物及载体组合物
在第九方面，本发明还提供了一种组合物，其包含：
(i)第一组分，其选自：本发明的蛋白、缀合物、融合蛋白、编码所述蛋白或融合蛋白的核苷酸序列，以及其任意组合；和
(ii)第二组分，其为包含导向RNA的核苷酸序列，或者编码所述包含导向RNA的核苷酸序列的核苷酸序列；
其中，所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；
所述导向RNA能够与(i)中所述的蛋白、缀合物或融合蛋白形成复合物。
在某些实施方案中，所述同向重复序列是如第四方面所定义的分离的核酸分子。
在某些实施方案中，所述导向序列连接至所述同向重复序列的3’端。在某些实施方案中，所述导向序列包含所述靶序列的互补序列。
在某些实施方案中，所述组合物不包含tracrRNA。
在某些实施方案中，所述组合物是非天然存在的或经修饰的。在某些实施方案中，所述组合物中的至少一个组分是非天然存在的或经修饰的。在某些实施方案中，所述第一组分是非天然存在的或经修饰的；和/或，所述第二组分是非天然存在的或经修饰的。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-ATG所示的序列。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-TTN所示的序列，其中，N选自A、G、T、C。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-KTR所示的序列。
在某些实施方案中，当所述靶序列为RNA时，所述靶序列不具有PAM结构域限制。
在某些实施方案中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列。在某些实施方案中，所述靶序列是非天然存在的DNA或RNA序列。
在某些实施方案中，所述靶序列存在于细胞内。在某些实施方案中，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是原核细胞。
在某些实施方案中，所述蛋白连接有一个或多个NLS序列。在某些实施方案中，所述缀合物或融合蛋白包含一个或多个NLS序列。在某些实施方案中，所述NLS序列连接至所述蛋白的N端或C端。在某些实施方案中，所述NLS序列融合至所述蛋白的N端或C端。
在第十方面，本发明还提供了一种组合物，其包含一种或多种载体，所述一种或多种载体包含：
(i)第一核酸，其为编码本发明的蛋白或融合蛋白的核苷酸序列；任选地所述第一核酸可操作地连接至第一调节元件；以及
(ii)第二核酸，其编码包含导向RNA的核苷酸序列；任选地所述第二核酸可操作地连接至第二调节元件；
其中：
所述第一核酸与第二核酸存在于相同或不同的载体上；
所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；
所述导向RNA能够与(i)中所述的效应蛋白或融合蛋白形成复合物。
在某些实施方案中，所述同向重复序列是如第四方面所定义的分离的核酸分子。
在某些实施方案中，所述导向序列连接至所述同向重复序列的3’端。在某些实施方案中，所述导向序列包含所述靶序列的互补序列。
在某些实施方案中，所述组合物不包含tracrRNA。
在某些实施方案中，所述组合物是非天然存在的或经修饰的。在某些实施方案中，所述组合物中的至少一个组分是非天然存在的或经修饰的。
在某些实施方案中，所述第一调节元件是启动子，例如诱导型启动子。
在某些实施方案中，所述第二调节元件是启动子，例如诱导型启动子。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-ATG所示的序列。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-TTN所示的序列，其中，N选自A、G、T、C。
在某些实施方案中，当所述靶标序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-KTR所示的序列。
在某些实施方案中，当所述靶序列为RNA时，所述靶序列不具有PAM结构域限制。
在某些实施方案中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列。在某些实施方案中，所述靶序列是非天然存在的DNA或RNA序列。
在某些实施方案中，所述靶序列存在于细胞内。在某些实施方案中，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是原核细胞。
在某些实施方案中，所述蛋白连接有一个或多个NLS序列。在某些实施方案中，所述缀合物或融合蛋白包含一个或多个NLS序列。在某些实施方案中，所述NLS序列连接至所述蛋白的N端或C端。在某些实施方案中，所述NLS序列融合至所述蛋白的N端或C端。
在某些实施方案中，一种类型的载体是质粒，其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。在重组DNA技术中使用的普通表达栽体通常是质粒形式。
重组表达载体可包含处于适合于在宿主细胞中的核酸表达的形式的本发明的核酸分子，这意味着这些重组表达载体包含基于待用于表达的宿主细胞而选择的一种或多种调节元件，所述调节元件可操作地连接至待表达的核酸序列。
递送及递送组合物
本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物，可以通过本领域已知的任何方法进行递送。此类方法包括但不限于，电穿孔、脂转染、核转染、显微注射、声孔效应、基因枪、磷酸钙介导的转染、阳离子转染、脂质体转染、树枝状转染、热激转染、核转染、磁转染、脂转染、穿刺转染、光学转染、试剂增强性核酸摄取、以及经由脂质体、免疫脂质体、病毒颗粒、人工病毒体等的递送。
因此，在另一个方面，本发明提供了一种递送组合物，其包含递送载体，以及选自下列的一种或多种：本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物。
在某些实施方案中，所述递送载体是粒子。
在某些实施方案中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、微泡、基因枪或病毒载体(例如，复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。
试剂盒
在另一个方面，本发明提供了一种试剂盒，其包含如上所述的组分中的一种或多种。在某些实施方案中，所述试剂盒包含一种或多种选自下列的组分：本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物。
在某些实施方案中，本发明的试剂盒包含如第九方面所述的组合物。在某些实施方案中，所述试剂盒还包含使用所述组合物的说明书。
在某些实施方案中，本发明的试剂盒包含如第十方面所述的组合物。在某些实施方案中，所述试剂盒还包含使用所述组合物的说明书。
在某些实施方案中，本发明的试剂盒中包含的组分可以被提供于任何适合的容器中。
在某些实施方案中，所述试剂盒还包含一种或多种缓冲液。缓冲液可以是任何缓冲液，包括但不限于碳酸钠缓冲液、碳酸氢钠缓冲液、硼酸盐缓冲液、Tris缓冲液、MOPS缓冲液、HEPES缓冲液及其组合。在某些实施方案中，该缓冲液是碱性的。在某些实施方案中，该缓冲液具有从约7至约10的pH。
在某些实施方案中，该试剂盒还包括一个或多个寡核苷酸，该一个或多个寡核苷酸对应于一个用于插入进载体中的导向序列，以便可操作地连接该导向序列和调节元件。在某些实施方案中，该试剂盒包括同源重组模板多核苷酸。
方法及用途
在另一个方面，本发明提供了一种修饰靶基因的方法，其包括：将如第五方面所述的复合物、如第九方面所述的组合物或如第十方面所述的组合物与所述靶基因接触，或者递送至包含所述靶基因的细胞中；所述靶序列存在于所述靶基因中。
在某些实施方案中，所述靶基因存在于细胞内。在某些实施方案中，所述细胞是原核细胞。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。在某些实施方案中，所述细胞选自非人灵长类动物、牛、猪或啮齿类动物细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽或鱼等。在某些实施方案中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。
在某些实施方案中，所述靶基因存在于体外的核酸分子(例如，质粒)中。在某些实施方案中，所述靶基因存在于质粒中。
在某些实施方案中，所述修饰是指所述靶序列的断裂，如DNA的双链断裂或RNA的单链断裂。
在某些实施方案中，所述断裂导致靶基因的转录降低。
在某些实施方案中，所述方法还包括：将编辑模板与所述靶基因接触，或者递送至包含所述靶基因的细胞中。在此类实施方案中，所述方法通过与外源模板多核苷酸同源重组修复所述断裂的靶基因，其中所述修复导致一种突变，包括所述靶基因的一个或多个核苷酸的插入、缺失、或取代。在某些实施方案中，所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。
因此，在某些实施方案中，所述修饰还包括将编辑模板(例如外源核酸)插入所述断裂中。
在某些实施方案中，所述的蛋白、缀合物、融合蛋白、分离的核酸分子、复合物、载体或组合物包含于递送载体中。
在某些实施方案中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、病毒载体(如复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。
在某些实施方案中，所述方法其用于改变靶基因或编码靶基因产物的核酸分子中的一个或多个靶序列来修饰细胞、细胞系或生物体。
在另一个方面，本发明提供了一种改变基因产物的表达的方法，其包括：将如第五方面所述的复合物、如第九方面所述的组合物或如第十方面所述的组合物与编码所述基因产物的核酸分子接触，或者递送至包含所述核酸分子的细胞中，所述靶序列存在于所述核酸分子中。
在某些实施方案中，所述核酸分子存在于细胞内。在某些实施方案中，所述细胞是原核细胞。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。在某些实施方案中，所述细胞选自非人灵长类动物、牛、猪或啮齿类动物细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽或鱼等。在某些实施方案中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。
在某些实施方案中，所述核酸分子存在于体外的核酸分子(例如，质粒)中。在某些实施方案中，所述核酸分子存在于质粒中。
在某些实施方案中，所述基因产物的表达被改变(例如，增强或降低)。在某些实施方案中，所述基因产物的表达被增强。在某些实施方案中，所述基因产物的表达被降低。
在某些实施方案中，所述基因产物是蛋白。
在某些实施方案中，所述的蛋白、缀合物、融合蛋白、分离的核酸分子、复合物、载体或组合物包含于递送载体中。
在某些实施方案中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、病毒载体(如复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。
在某些实施方案中，所述方法其用于改变靶基因或编码靶基因产物的核酸分子中的一个或多个靶序列来修饰细胞、细胞系或生物体。
在另一个方面，本发明涉及如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物，用于核酸编辑的用途。
在某些实施方案中，所述核酸编辑包括基因或基因组编辑，例如修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或插入多核苷酸。
在另一个方面，本发明涉及如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物，在制备制剂中的用途，所述制剂用于：
(i)离体基因或基因组编辑；
(ii)离体单链DNA的检测；
(iii)编辑靶基因座中的靶序列来修饰生物或非人类生物；
(iv)治疗由靶基因座中的靶序列的缺陷引起的病症。
细胞及细胞子代
在某些情况下，由本发明的方法引入到细胞的修饰可以使得细胞和其子代被改变以改进其生物产物(如抗体、淀粉、乙醇或其他期望的细胞输出物)的产生。在某些情况下，由本发明的方法引入到细胞的修饰可以使得细胞和其子代包括使所生产生物产物发生变化的改变。
因此，在另一方面，本发明还涉及如上所述的方法获得的细胞或其子代，其中所述细胞含有在其野生型中不存在的修饰。
本发明还涉及如上所述的细胞或其子代的细胞产物。
本发明还涉及一种体外的、离体的或体内的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物。
在某些实施方案中，所述细胞是原核细胞。
在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。某些实施方案中，所述细胞是非人哺乳动物细胞，例如非人灵长类动物、牛、羊、猪、犬、猴、兔、啮齿类(如大鼠或小鼠)的细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽鸟类(如鸡)、鱼类或甲壳动物(如蛤蜊、虾)的细胞。在某些实施方案中，所述细胞是植物细胞，例如单子叶植物或双子叶植物具有的细胞或栽培植物或粮食作物如木薯、玉米、高粱、大豆、小麦、燕麦或水稻具有的细胞，例如藻类、树或生产植物、果实或蔬菜(例如，树类如柑橘树、坚果树；茄属植物、棉花、烟草、番茄、葡萄、咖啡、可可等)。
在某些实施方案中，所述细胞是干细胞或干细胞系。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本发明中，表述“Cas12j”是指，本发明人首次发现并鉴定的一种Cas效应蛋白，其具有选自下列的氨基酸序列：
(i)SEQ ID NOs：1-20、107、108任一项所示的序列；
(ii)与SEQ ID NOs：1-20、107、108任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NOs：1-20、107、108任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。
本发明的Cas12j是一种在导向RNA引导下与靶序列特定位点结合并切割的核酸内切酶，同时具有DNA和RNA内切酶活性。
如本文中所使用的，术语“规律成簇的间隔短回文重复(CRISPR)-CRISPR-相关(Cas)(CRISPR-Cas)系统”或“CRISPR系统”可互换地使用并且具有本领域技术人员通常理解的含义，其通常包含与CRISPR相关(“Cas”)基因的表达有关的转录产物或其他元件，或者能够指导所述Cas基因活性的转录产物或其他元件。此类转录产物或其他元件可以包含编码Cas效应蛋白的序列和包含CRISPR RNA(crRNA)的导向RNA，以及在CRISPR-Cas9系统中所含有的反式作用crRNA(tracrRNA)序列，或来自CRISPR基因座的其他序列或转录产物。
如本文中所使用的，术语“Cas效应蛋白”、“Cas效应酶”可互换地使用并且是指，CRISPR-Cas系统中呈现的任一种大于长度800个氨基酸的蛋白质。在某些情况下，这类蛋白是指从Cas基因座中鉴定的蛋白。
如本文中所使用的，术语“导向RNA(guide RNA)”、“成熟crRNA”可互换地使用并且具有本领域技术人员通常理解的含义。一般而言，导向RNA可以包含同向(direct)重复序列和导向序列(guide sequence)，或者基本上由或由同向重复序列和导向序列(在内源性CRISPR系统背景下也称为间隔序列(spacer))组成。在某些情况下，导向序列是与靶序列具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列。在某些实施方案中，当最佳比对时，导向序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。
在某些情况下，所述导向序列在长度上为至少5个、至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少35个、至少40个、至少45个或至少50个核苷酸。在某些情况下，所述导向序列在长度上为不超过50个、45个、40个、35个、30个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述导向序列在长度上为10-30个、或15-25个、或15-22个、或19-25个或19-22个核苷酸。
在某些情况下，所述同向重复序列在长度上为至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少56个、至少57个、至少58个、至少59个、至少60个、至少61个、至少62个、至少63个、至少64个、至少65个或至少70个核苷酸。在某些情况下，所述同向重复序列在长度上为不超过70个、65个、64个、63个、62个、61个、60个、59个、58个、57个、56个、55个、50个、45个、40个、35个、30个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述同向重复序列在长度上为55-70个核苷酸，例如55-65个核苷酸，例如60-65个核苷酸，例如62-65个核苷酸，例如63-64个核苷酸。在某些实施方案中，所述同向重复序列在长度上为15-30个核苷酸，例如15-25个核苷酸，例如20-25个核苷酸，例如22-24个核苷酸，例如23个核苷酸。在某些实施方案中，所述同向重复序列在长度上不少于32nt，例如32nt-37nt。
如本文中所使用的，术语“CRISPR/Cas复合物”是指，导向RNA(guide RNA)或成熟crRNA与Cas蛋白结合所形成的核糖核蛋白复合体，其包含杂交到靶序列上并且与Cas蛋白结合的导向序列。该核糖核蛋白复合体能够识别并切割能与该导向RNA或成熟crRNA杂交的多核苷酸。
因此，在形成CRISPR/Cas复合物的情况下，“靶序列”是指被设计为具有靶向性的导向序列所靶向的多核苷酸，例如与该导向序列具有互补性的序列，其中靶序列与导向序列之间的杂交将促进CRISPR/Cas复合物的形成。完全互补性不是必需的，只要存在足够互补性以引起杂交并且促进一种CRISPR/Cas复合物的形成即可。靶序列可以包含任何多核苷酸，如DNA或RNA。在某些情况下，所述靶序列位于细胞的细胞核或细胞质中。在某些情况下，该靶序列可位于真核细胞的一个细胞器例如线粒体或叶绿体内。可被用于重组到包含该靶序列的靶基因座中的序列或模板被称为“编辑模板”或“编辑多核苷酸”或“编辑序列”。在某些实施方案中，所述编辑模板为外源核酸。在某些实施方案中，该重组是同源重组。
在本发明中，表述“靶序列”或“靶多核苷酸”可以是对细胞(例如，真核细胞)而言任何内源或外源的多核苷酸。例如，该靶多核苷酸可以是一种存在于真核细胞的细胞核中的多核苷酸。该靶多核苷酸可以是一个编码基因产物(例如，蛋白质)的序列或一个非编码序列(例如，调节多核苷酸或无用DNA)。在某些情况下，据信该靶序列应该与原间隔序列临近基序(PAM)相关。对PAM的精确序列和长度要求取决于使用的Cas效应酶而不同，但是PAM典型地是临近原间隔序列(也即，靶序列)的2-5个碱基对序列。本领域技术人员能够鉴定与给定的Cas效应蛋白一起使用的PAM序列。
在某些情况下，靶序列或靶多核苷酸可以包括多个疾病相关基因和多核苷酸以及信号传导生化途径相关基因和多核苷酸。此类靶序列或靶多核苷酸的非限制性实例，包括分别提交于2012年12月12日和2013年1月2日的美国临时专利申请61/736,527和61/748,427、提交于2013年12月12日的国际申请PCT/US2013/074667中所列举的那些，其全部通过引用并入本文。
在某些情况下，靶序列或靶多核苷酸的实例包括与信号传导生化途径相关的序列，例如信号传导生化途径相关基因或多核苷酸。靶多核苷酸的实例包括疾病相关基因或多核苷酸。“疾病相关”基因或多核苷酸是指与非疾病对照的组织或细胞相比，在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因或多核苷酸。在改变的表达与疾病的出现和/或进展相关的情况下，它可以是一个以异常高的水平被表达的基因；或者，它可以是一个以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的，并且可以处于正常或异常水平。
如本文中所使用的，术语“野生型”具有本领域技术人员通常理解的含义，其表示生物、菌株、基因的典型形式或者当它在自然界存在时区别于突变体或变体形式的特征，其可从自然中的来源分离并且没有被人为有意地修饰。
如本文中所使用的，术语“非天然存在的”或“工程化的”可互换地使用并且表示人工的参与。当这些术语用于描述核酸分子或多肽时，其表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
如本文中所使用的，术语“直系同源物(orthologue,ortholog)”具有本领域技术人员通常理解的含义。作为进一步指导，如本文中所述的蛋白质的“直系同源物”是指属于不同物种的蛋白质，该蛋白质执行与作为其直系同源物的蛋白相同或相似的功能。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“载体”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
本领域技术人员将理解，表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽，包括由如本文所述的蛋白、融合蛋白、分离的核酸分子等(例如，CRISPR转录物，如核酸转录物、蛋白质、或酶)。
如本文中所使用的，术语“调节元件”旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)，其详细描述可参考戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRESSIONTECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥(SanDiego)，加利福尼亚州(1990)。在某些情况下，调节元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。在某些情况下，调节元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。在某些情况下，术语“调节元件”涵盖的是增强子元件，如WPRE；CMV增强子；在HTLV-I的LTR中的R-U5’片段((Mol.Cell.Biol.，第8(1)卷，第466-472页，1988)；SV40增强子；以及在兔β-珠蛋白的外显子2与3之间的内含子序列(Proc.Natl.Acad.Sci.USA.，第78(3)卷，第1527-31页，1981)。
如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。组成型(constitutive)启动子是这样的核苷酸序列：当其与编码或者限定基因产物的多核苷酸可操作地相连时，在细胞的大多数或者所有生理条件下，其导致细胞中基因产物的产生。诱导型启动子是这样的核苷酸序列，当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当对应于所述启动子的诱导物在细胞中存在时，其导致所述基因产物在细胞内产生。组织特异性启动子是这样的核苷酸序列：当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当细胞是该启动子对应的组织类型的细胞时，其才导致在细胞中产生基因产物。
如本文中所使用的，术语“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调节元件(例如，处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。
如本文中所使用的，术语“互补性”是指核酸与另一个核酸序列借助于传统的沃森-克里克或其他非传统类型形成一个或多个氢键的能力。互补百分比表示一个核酸分子中可与一个第二核酸序列形成氢键(例如，沃森-克里克碱基配对)的残基的百分比(例如，10个之中有5、6、7、8、9、10个即为50％、60％、70％、80％、90％、和100％互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％、或100％的互补程度，或者是指在严格条件下杂交的两个核酸。
如本文中所使用的，对于杂交的“严格条件”是指与靶序列具有互补性的一个核酸主要地与该靶序列杂交并且基本上不杂交到非靶序列上的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。一般而言，该序列越长，则该序列特异性地杂交到其靶序列上的温度就越高。严格条件的非限制性实例描述于蒂森(Tijssen)(1993)的《生物化学和分子生物学中的实验室技术-核酸探针杂交》(Laboratory Techniques InBiochemistryAnd Molecular Biology-Hybridization With Nucleic Acid Probes)，第I部分，第二章，“杂交原理概述和核酸探针分析策略”(“Overview of principles ofhybridization andthe strategy of nucleic acid probe assay”)，爱思唯尔(Elsevier)，纽约。
如本文中所使用的，术语“杂交”是指其中一个或多个多核苷酸反应形成一种复合物的反应，该复合物经由这些核苷酸残基之间的碱基的氢键键合而稳定化。氢键键合可以借助于沃森-克里克碱基配对、Hoogstein结合或以任何其他序列特异性方式而发生。该复合物可包含形成一个双链体的两条链、形成多链复合物的三条或多条链、单个自我杂交链、或这些的任何组合。杂交反应可以构成一个更广泛的过程(如PCR的开始、或经由一种酶的多核苷酸的切割)中的一个步骤。能够与一个给定序列杂交的序列被称为该给定序列的“互补物”。
如本文中所使用的，术语“表达”是指，藉此从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后藉此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。
如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure 2:1121-1123)。
如本文中所使用的，术语“治疗”是指，治疗或治愈病症，延缓病症的症状的发作，和/或延缓病症的发展。
如本文中所使用的，术语“受试者”包括但不限于各种动物，例如哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、兔科动物、啮齿类动物(例如，小鼠或大鼠)、非人灵长类动物(例如，猕猴或食蟹猴)或人。在某些实施方式中，所述受试者(例如人)患有病症(例如，疾病相关基因缺陷所导致的病症)。
发明的有益效果
与现有技术相比，本发明的Cas蛋白及系统具有显著的有利方面。例如，本发明的Cas效应蛋白具有严格的错配容忍度，使其可能具有更低的脱靶率。例如，本发明的Cas效应蛋白拥有更加严谨的PAM识别方式，从而显著降低脱靶效应。
附图说明
图1为cas12j蛋白对pre-crRNA加工的凝胶电泳结果。
图2A-2B为cas12j蛋白的PAM结构域分析的结果。
图3为CRISPR/Cas12j系统的DNA切割方式的鉴定结果。
图4为Cas12j.4、Cas12j.19、Cas12j.22体外切割位点分析的结果。
图5为Cas12j.19在不同温度下体外酶切活性检测的结果。
图6为CRISPR/Cas12j.19系统中不同spacer长度对酶切活性影响的结果。
图7为CRISPR/Cas12j.19系统中不同repeat长度对酶切活性影响的结果。WT表示未经截短的repeat序列。
图8为CRISPR/Cas12j.19系统对于spacer错配容忍的结果。WT表示未经突变的spacer序列。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATORY MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
以下实施例涉及的部分试剂的来源如下：
LB液体培养基：10g胰蛋白胨(Tryptone)，5g酵母提取物(Yeast Extract)，10gNaCl，定容至1L，灭菌。若需加抗生素，则待培养基冷却后加，50μg/ml的终浓度。
氯仿/异戊醇：240ml的氯仿加10ml的异戊醇，混匀。
RNP缓冲液：100mM氯化钠,50mM Tris-HCl,10mM MgCl2,100μg/ml BSA,pH 7.9。
原核表达载体pACYC-Duet-1和pUC19购自金斯瑞生物有限公司。
大肠杆菌感受态EC100购自Epicentre公司。
实施例1.Cas12j基因和Cas12j导向RNA的获得
1、CRISPR和基因的注释：使用Prodigal对将NCBI和JGI数据库的微生物基因组和宏基因组数据进行基因注释得到所有蛋白，同时用Piler-CR进行CRISPR座的注释，参数均为默认参数。
2、蛋白质的过滤：通过序列一致性对注释蛋白去冗余，去除序列完全一致的蛋白，同时将长度大于800个氨基酸的蛋白划分为大分子蛋白。由于目前发现的所有第二类CRISPR/Cas系统的效应蛋白长度多大于900个氨基酸，所以为了降低计算复杂度，我们在挖掘CRISPR效应蛋白的时候只对大于800个氨基酸的大分子蛋白进行考虑。
3、CRISPR相关大分子蛋白的获得：将每一个CRISPR座上下游延伸10Kb，将对CRISPR邻近区间内的非冗余大分子蛋白进行鉴定。
4、CRISPR相关大分子蛋白质的聚类：使用BLASTP对非冗余大分子CRISPR相关蛋白进行内部的两两比对，输出Evalue<1E-10的比对结果。使用MCL对BLASTP的输出结果进行聚类分析，CRISPR相关蛋白质家族。
5、CRISPR富集大分子蛋白质家族的鉴定：使用BLASTP对CRISPR相关蛋白质家族的蛋白比对到去除去CRISPR相关蛋白的非冗余大分子蛋白数据库，输出Evalue<1E-10的比对结果。如果一个非CRISPR相关蛋白数据库发现的同源蛋白小于100％，那么则说明这个家族的蛋白在CRISPR区域是富集的，通过这种方法我们对CRISPR富集大分子蛋白质家族进行鉴定。
6、蛋白功能和结构域的注释：利用Pfam数据库，NR数据库以及从NCBI收集的Cas蛋白对CRISPR富集大分子蛋白质家族进行注释，得到新的CRISPR/Cas蛋白质家族。利用Mafft对每个CRISPR/Cas家族蛋白进行多重序列比对，然后用JPred和HHpred进行保守结构域分析，鉴定含有RuvC结构域的蛋白质家族。
在此基础上，本发明人获得了一种全新的Cas效应蛋白，即Cas12j，以其22种活性同源物序列，分别命名为Cas12j.3(SEQ ID NO:1)、Cas12j.4(SEQ ID NO:2)，Cas12j.5(SEQID NO:3)、Cas12j.6(SEQ ID NO:4)，Cas12j.7(SEQ ID NO:5)、Cas12j.8(SEQ ID NO:6)，Cas12j.9(SEQ ID NO:7)、Cas12j.10(SEQ ID NO:8)，Cas12j.11(SEQ ID NO:9)、Cas12j.12(SEQ ID NO:10)，Cas12j.13(SEQ ID NO:11)、Cas12j.14(SEQ ID NO:12)，Cas12j.15(SEQID NO:13)、Cas12j.16(SEQ ID NO:14)，Cas12j.17(SEQ ID NO:15)、Cas12j.18(SEQ IDNO:16)，Cas12j.19(SEQ ID NO:17)，Cas12j.20(SEQ ID NO:18)，Cas12j.21(SEQ ID NO:19)，Cas12j.22(SEQ ID NO:20)，Cas12j.1(SEQ ID NO:107)，Cas12j.2(SEQ ID NO:108)，20种同源物的编码DNA分别如SEQ ID NOs:21-40所示。Cas12j.3、Cas12j.4、Cas12j.5、Cas12j.6、Cas12j.7、Cas12j.8、Cas12j.9、Cas12j.10、Cas12j.11、Cas12j.12、Cas12j.13、Cas12j.14、Cas12j.15、Cas12j.16、Cas12j.17、Cas12j.18、Cas12j.19、Cas12j.20所对应的原型同向重复序列(pre-crRNA中所含有的repeat序列)分别如SEQ ID NOs:41-60所示。
实施例2.Cas12j基因对pre-crRNA的加工
1、Cas12j蛋白的体外表达及纯化
Cas12j蛋白的体外表达及纯化的步骤具体如下：
1、人工合成编码带有核定位信号的Cas12j蛋白(SEQ ID NO:82-101)的DNA序列。
2、将步骤1合成的双链DNA分子与原核表达载体pET-30a(+)连接，得到重组质粒pET-30a-CRISPR/Cas12j。
3、将重组质粒pET-30a-CRISPR/Cas12j导入大肠杆菌EC100，得到重组菌，将该重组菌命名为EC100-CRISPR/Cas12j。
取EC100-CRISPR/Cas12j的单克隆，接种至100mL LB液体培养基(含50μg/mL氨苄霉素)，37℃、200rpm振荡培养12h，得到培养菌液。
4、取培养菌液，按体积比为1:100接种至50mL LB液体培养基(含50μg/mL氨苄霉素)，37℃、200rpm振荡培养至OD600nm值为0.6，然后加入IPTG并使其浓度为1mM，28℃、220rpm振荡培养4h，4℃、10000rpm离心10min，收集菌体沉淀。
5、取菌体沉淀，加入100mL pH 8.0、100mM的Tris-HCl缓冲液，重悬后超声破碎(超声波功率600W，循环程序为：破碎4s，停6s，共20min)，然后4℃、10000rpm离心10min，收集上清液甲。
6、取上清液甲，4℃、12000rpm离心10min，收集上清液乙。
7、采用GE公司生产的镍柱对上清液乙进行纯化(纯化的具体步骤参考镍柱的说明书)，然后采用赛默飞世尔公司生产的蛋白定量试剂盒对Cas12j蛋白进行定量。
二、Cas12j蛋白导向RNA的转录及纯化：
1、设计导向RNA转录的模板，转录模板的结构为：T7启动子+Cas12j的原型的repeat(SEQ ID NO:41-60)+spacer(SEQ ID NO:104)，引物的设计使用Primer5.0软件，保证正向引物和反向引物有至少18bp的重叠序列。
2、配置如下反应体系，轻轻吹打混匀后短暂离心，置于PCR仪中缓慢退火：
PCR扩增反应
引物退火PCR反应程序
3、使用MinElute PCR Purifcation Kit进行模板的纯化，步骤如下：
1)向PCR产物中加入5倍体积的PB，将一个MinElute柱子放至2ml收集管上，室温静置2min，12000g/2min；
2)弃废液，加入750μl Buffer PE(用之前记得加乙醇)，12000g/2min；
3)弃废液，加入350μl Buffer PE,12000g/2min，弃废液，12000g，空离2min；
4)将MinElute柱子换至新的1.5ml离心管上，开盖，65℃静置2min；
5)加入20μl预热的EB溶液，静置2min后，12000g/2min，为了提高回收率，可将离心管内容物过2-3遍MinElute离心柱；
6)用Nanodrop测定浓度，冻存-20℃备用。
4、导向RNA的纯化：酚：氯仿：异戊醇(25：24：1)抽提去除体系内的DNAseI
1)向转录后的反应体系中加入80μl RNA free H2O，调整体积至100μl；
2)取出2ml的Phase Lock Gel(PLG)Heavy，15000g，离心2min，加入100μl酚：氯仿：异戊醇(25：24：1)、100μl经过DNAseI消化的RNA，用手轻轻弹Phase-Lock tube 5-10次，使其混合均匀，之后15℃/16000g离心12min；
3)取一个新的RNA-free的1.5ml离心管，将上步离心的上清吸出至离心管中，注意不要吸到凝胶，加入与上清等体积的异丙醇以及十分之一体积的醋酸钠溶液，用枪头吸打混匀后放入-20℃冰箱1h或过夜静置；
4)4℃/16000g，离心30min，弃上清，加入75％预冷的乙醇，将沉淀吸打混匀，4℃/16000g，离心12min，弃上清，在通风橱静置2-3min，晾干RNA表面的乙醇，加入100μl的RNAfree H2O，吸打混匀。
5)用Nanodrop测定纯化后的crRNA浓度，并统一稀释至250ng/μl，分装至200μl的PCR离心管中，冻存-80℃备用。
4、Cas12f的precrRNA转录采用NEB的HiScribe T7高效RNA合成试剂盒，反应体系如下表所示：
DNA转录体系
设置PCR反应程序为：37℃/3h或31℃/forever，加入DNAseI，37℃/45min
5、precrRNA的纯化：
(1)酚：氯仿：异戊醇(25：24：1)抽提去除体系内的DNAseI
1)向转录后的反应体系中加入80μl RNA free H2O，调整体积至100μl；
2)取出2ml的Phase Lock Gel(PLG)Heavy，15000g，离心2min，加入100μl酚：氯仿：异戊醇(25：24：1)、100μl经过DNAseI消化的RNA，用手轻轻弹Phase-Lock tube 5-10次，使其混合均匀，之后15℃/16000g离心12min；
3)取一个新的RNA-free的1.5ml离心管，将②步离心的上清吸出至离心管中，注意不要吸到凝胶，加入与上清等体积的异丙醇以及十分之一体积的醋酸钠溶液，用枪头吸打混匀后放入-20℃冰箱1h或过夜静置；
4)4℃/16000g，离心30min，弃上清，加入75％预冷的乙醇，将沉淀吸打混匀，4℃/16000g，离心12min，弃上清，在通风橱静置2-3min，晾干RNA表面的乙醇，加入100μl的RNAfree H2O，吸打混匀。
(2)跑胶并从聚丙烯酰胺凝胶中纯化precrRNA，使用ZYMO RESEARCH的ZR Small-RNATM PAGE Recovery Kit试剂盒纯化回收precrRNA。步骤如下：
1)precrRNA条带大小为90bp左右，切割相应条带的RNA片段，转移至1.5ml RNA-free的离心管中；
2)使用SquisherTM-single将胶完全捣碎，加入400μl的RNA Recovery Buffer，65℃水浴锅加热15min；
3)液氮速冻5min，立即取出放入65℃水浴锅加热5min；
4)取出Zymo-SpinTM IV的柱子于收集管上，然后将溶解后的凝胶加入其中，12000g离心5min，并保留收集管中液体；
5)取出Zymo-SpinTM IIIC的柱子于新的收集管上，将上步收集的液体加入其中，2000g离心2min，保留收集管中液体；
6)估算收集管中液体体积，加入2倍体积的RNA MAX Buffer，上下颠倒混匀；
7)取出Zymo-SpinTM IC的柱子于新的收集管中，将⑥步收集管中的液体加入其中，静置2min后，12000g离心2min；
8)加入800μl RNA Wash Buffer(注意用之前按照说明书加入一定体积的无水乙醇)，12000g离心2min，弃收集管中液体；
9)加入400μl RNA Wash Buffer,12000g离心2min，弃收集管中液体，再空离2min；
10)65℃烘箱静置1min，加入20μl RNA-free H2O，用nanodrop测定所收集的precrRNA的浓度，并统一调整浓度至200ng/μl，分装在PCR离心管中，负80℃冻存备用。
6、建立体外pre-crRNA酶切体系
(1)配置如下反应体系，轻轻吹打混匀后短暂离心。置于37℃，1hour；
体外pre-crRNA酶切体系
(2)向以上反应体系中加入10μl 2×RNA loading dye，置于98℃，3min。反应结束后立即置于冰上2min；
(3)10％TBE-Urea聚丙烯酰胺凝胶上样孔中上样10μl，150V/40min；
(4)在1×TBE电泳缓冲液中加入SYBR Gold nucleic acid gel stain dye，置入凝胶，室温染色10-15min后扫胶。
扫胶结果如图1所示，结果显示，Cas12j.1、Cas12j.4、Cas12j.18、Cas12j.19、Cas12j.21、Cas12j.22在体外具有pre-crRNA切割活性。
实施例3.Cas12j蛋白的PAM结构域鉴定
1.构建重组质粒pACYC-Duet-1+CRISPR/Cas12j并测序。根据测序结果，对重组质粒pACYC-Duet-1+CRISPR/Cas12j进行结构描述如下：将载体pACYC-Duet-1的限制性内切酶Pml I和Kpn I识别序列间的小片段替换为Cas12j基因(SEQ ID NO:21-40所示的序列中自5’端起第1位至3’末端最后一位所示的双链DNA分子)。重组质粒pACYC-Duet-1+CRISPR/Cas12j表达Cas12j蛋白(SEQ ID NO:1-20、107、108)和SEQ ID NO:104所示的Cas12j导向RNA。
2.重组质粒pACYC-Duet-1+CRISPR/Cas12j中含有表达盒，该表达盒的核苷酸序列由Cas12j基因分别与SEQ ID NO:104连接构成。例如SEQ ID NO:102所示。SEQ ID NO:102所示的序列中，自5’末端起第1至44位为pLacZ启动子的核苷酸序列，第45至3056位为Cas12j.3基因的核苷酸序列，第3057至3143位为rrnB T1终止子的核苷酸序列(用于终止转录)。自5’末端起第3144至3178位为J23119启动子的核苷酸序列，第3179至3241位为CRISPR阵列的核苷酸序列，第3244至3268位为rrnB-T2终止子的核苷酸序列(用于终止转录)。
3.重组大肠杆菌的获得：将重组质粒pACYC-Duet-1+CRISPR/Cas12j导入大肠杆菌EC100中，得到重组大肠杆菌，命名为EC100/pACYC-Duet-1+CRISPR/Cas12j。将重组质粒pACYC-Duet-1导入大肠杆菌EC100中，得到重组大肠杆菌，命名为EC100/pACYC-Duet-1。
4.PAM文库的构建：人工合成SEQ ID NO:103所示的序列，并连接到pUC19载体，其中SEQ ID NO:103所示的序列包括5’端八个随机碱基和靶序列。对PAM文库的靶标序列5’端前面设计了8个随机碱基构建质粒文库。将质粒分别转入到含有Cas12j基因座的大肠杆菌中和不含有Cas.12j基因座的大肠杆菌中。在37℃下处理1小时后，我们对质粒进行提取，并对PAM区域序列进行PCR扩增和测序。
5.PAM文库结构域的获得：分别统计实验组和对照组中65,536种组合的PAM序列出现次数，并用各自组所有的PAM序列数目进行标准化。对于任意一条PAM序列，当log2(对照组标准化值/实验组标准化值)大于3.5时，我们认为这条PAM被显著消耗。我们用Weblogo对显著消耗的PAM序列进行预测，发现各个蛋白的PAM结构域，其中，Cas12j.1为5’-TTVW，Cas12j.4、Cas12j.12为5’-TTN，Cas12j.18为5’-AYR，Cas12j.19为5’-ATG，Cas12j.21为5’-VTTG，Cas12j.22为5’-KTR。PAM结构域分析结果见附图2A-2B。
实施例4.CRISPR/Cas12j系统的DNA切割方式的鉴定
一、Cas12j蛋白的体外表达及纯化
Cas12j蛋白的体外表达及纯化的步骤具体如下：
1、人工合成编码带有核定位信号的Cas12j蛋白(SEQ ID NO:82-101)的DNA序列。
2、将步骤1合成的双链DNA分子与原核表达载体pET-30a(+)连接，得到重组质粒pET-30a-CRISPR/Cas12j。
3、将重组质粒pET-30a-CRISPR/Cas12j导入大肠杆菌EC100，得到重组菌，将该重组菌命名为EC100-CRISPR/Cas12j。
取EC100-CRISPR/Cas12j的单克隆，接种至100mL LB液体培养基(含50μg/mL氨苄霉素)，37℃、200rpm振荡培养12h，得到培养菌液。
4、取培养菌液，按体积比为1:100接种至50mL LB液体培养基(含50μg/mL氨苄霉素)，37℃、200rpm振荡培养至OD600nm值为0.6，然后加入IPTG并使其浓度为1mM，28℃、220rpm振荡培养4h，4℃、10000rpm离心10min，收集菌体沉淀。
5、取菌体沉淀，加入100mL pH 8.0、100mM的Tris-HCl缓冲液，重悬后超声破碎(超声波功率600W，循环程序为：破碎4s，停6s，共20min)，然后4℃、10000rpm离心10min，收集上清液甲。
6、取上清液甲，4℃、12000rpm离心10min，收集上清液乙。
7、采用GE公司生产的镍柱对上清液乙进行纯化(纯化的具体步骤参考镍柱的说明书)，然后采用赛默飞世尔公司生产的蛋白定量试剂盒对Cas12j蛋白进行定量。
二、Cas12j蛋白导向RNA的转录及纯化：
1、设计导向RNA转录的模板，转录模板的结构为：T7启动子+Cas12j的原型repeat(SEQ ID NO:41-60)+spacer(SEQ ID NO:105)，引物的设计使用Primer5.0软件，保证正向引物和反向引物有至少18bp的重叠序列。
2、配置如下反应体系，轻轻吹打混匀后短暂离心，置于PCR仪中缓慢退火：
PCR扩增反应
3、使用MinElute PCR Purifcation Kit进行模板的纯化，步骤如下：
1)向PCR产物中加入5倍体积的PB，将一个MinElute柱子放至2ml收集管上，室温静置2min，12000g/2min；
2)弃废液，加入750μl Buffer PE(用之前记得加乙醇)，12000g/2min；
3)弃废液，加入350μl Buffer PE,12000g/2min，弃废液，12000g，空离2min；
4)将MinElute柱子换至新的1.5ml离心管上，开盖，65℃静置2min；
5)加入20μl预热的EB溶液，静置2min后，12000g/2min，为了提高回收率，可将离心管内容物过2-3遍MinElute离心柱；
6)用Nanodrop测定浓度，冻存-20℃备用。
4、导向RNA的纯化：酚：氯仿：异戊醇(25：24：1)抽提去除体系内的DNAseI
1)向转录后的反应体系中加入80μl RNA free H2O，调整体积至100μl；
2)取出2ml的Phase Lock Gel(PLG)Heavy，15000g，离心2min，加入100μl酚：氯仿：异戊醇(25：24：1)、100μl经过DNAseI消化的RNA，用手轻轻弹Phase-Lock tube 5-10次，使其混合均匀，之后15℃/16000g离心12min；
3)取一个新的RNA-free的1.5ml离心管，将上步离心的上清吸出至离心管中，注意不要吸到凝胶，加入与上清等体积的异丙醇以及十分之一体积的醋酸钠溶液，用枪头吸打混匀后放入-20℃冰箱1h或过夜静置；
4)4℃/16000g，离心30min，弃上清，加入75％预冷的乙醇，将沉淀吸打混匀，4℃/16000g，离心12min，弃上清，在通风橱静置2-3min，晾干RNA表面的乙醇，加入100μl的RNAfree H2O，吸打混匀。
5、用Nanodrop测定纯化后的crRNA浓度，并统一稀释至250ng/μl，分装至200μl的PCR离心管中，冻存-80℃备用。
6、双链DNA酶切体系的建立：
(1)配置如下反应体系，轻轻吹打混匀后短暂离心。置于37℃，15min；
DNA切割反应体系
(2)加入300ng底物DNA(SEQ ID NO：106)(100ng/μl)，3μL，轻轻吹打混匀后短暂离心。置于37℃，8hour；
(3)加入RNase，置于37℃，15min，充分消化体系中的RNA杂质；
(4)加入蛋白酶K，置于58℃，15min，消化Cas12j蛋白；
(5)琼脂糖跑胶检测。
跑胶结果如图3所示，Cas12j.4，Cas12j.19以及Cas12j.22均能够有效的切割双链DNA，但Cas12j.22的切割活性很弱。
三、Cas12j.4、Cas12j.19、Cas12j.22体外切割位点分析的结果
接下来我们对这三个具有DNA双链切割活性的蛋白的体外切割活性位点进行了分析。我们将上一步的切割后的条带进行回收，并送公司进行Sanger测序。测序结果用seqman软件进行比对，比对结果如附图4所示，由峰图我们可以看出：Cas12j.4，Cas12j.19，Cas12j.22具有不同的切割方式，Cas12j.4和Cas12j.22的切割位点位于PAM末端18nt和25nt处，切割后形成了7nt的粘性末端，而Cas12j.19在距PAM远端25nt处有一个切割位点，形成约1nt的末端。
实施例5.Cas12j.19在不同温度下体外酶切活性检测的结果
将Cas12j.19(SEQ ID NO:17)和导向RNA(SEQ ID NO:105)在25℃下孵育15分钟，形成RNA和蛋白的混合物，通常称为RNP，之后向反应体系中加入双链DNA(SEQ ID NO：106)，并分别置于设置的不同温度中，设置的温度有：17℃，22℃，27℃，32℃，37℃，42℃，47℃，52℃，62℃，67℃，72℃，反应8h，反应结束后加入RNase，37℃消化15分钟RNA，以及蛋白酶K，58℃反应15分钟消化蛋白，通过琼脂糖凝胶电泳检测其对于DNA消耗的结果。结果如图5所示，结果显示，发现Cas12j.19在27℃～42℃之间都具有双链DNA切割活性。
实施例6.Cas12j.19不同spacer长度对酶切活性影响的结果
由于Cas12j.19的切割位点在靶序列之外，我们进一步测试了Cas12j.19导向RNA(SEQ ID NO:105)含有靶位点的序列，通常也称为spacer序列的长度对切割活性的影响。将导向RNA含有靶位点的序列进行截短(14～28nt)获得图6中所示的截短体，将Cas12j.19和截短的导向RNA在25℃下孵育15分钟，形成RNP，之后向反应体系中加入双链DNA(SEQ IDNO：106)，37℃，反应8h。反应结束后加入RNase，37℃消化15分钟RNA，以及蛋白酶K，58℃反应15分钟消化蛋白，通过琼脂糖凝胶电泳检测酶切结果。结果如图6所示，结果显示，Cas12j.19发挥切割活性所需的spacer长度至少为14nt。
实施例7.Cas12j.19不同repeat长度对酶切活性影响的结果
同样我们测试了导向RNA的同向重复序列repeat的长度对于Cas12j.19双链DNA切割活性的影响。我们将导向RNA(SEQ ID NO:105)中的同向重复序列进行截短为24～34nt获得图7中的截短体，将Cas12j.19与相应的不同repeat长度的导向RNA在25℃下孵育15分钟，形成RNP，之后向反应体系中加入双链DNA，37℃，反应8h。反应结束后加入RNase，37℃消化15分钟RNA，以及蛋白酶K，58℃反应15分钟消化蛋白，通过琼脂糖凝胶电泳检测酶切结果。结果如图7所示，结果显示，检测所需的最短的同向重复序列repeat长度为32nt。
实施例8.Cas12j.19对于spacer错配容忍的结果
导向RNA中含有靶位点的序列与原始的靶向序列的互补配对对于DNA的重组和切割具有重要的意义。将导向RNA(SEQ ID NO:105)中含有靶序列部分依次进行点突变(即spacer 5’端开始的1，3，5，7，9，11，13，15，17位点的碱基)，以获得图8中的突变体，从而与靶序列形成错配。通过将Cas12j.19与相应的含有突变位点的导向RNA在25℃下孵育15分钟，形成RNP，之后向反应体系中加入双链DNA(SEQ ID NO：106)，37℃，反应8h。反应结束后加入RNase，37℃消化15分钟RNA，以及蛋白酶K，58℃反应15分钟消化蛋白，通过琼脂糖凝胶电泳检测酶切结果。结果如图8所示，结果显示，spacer序列5’端前5nt内，靶序列碱基的突变对于Cas12j.19双链DNA的切割具有重要的影响。另外，第13nt靶序列的错配对Cas12j.19双链DNA的切割活性影响很大。Cas12j.19严格的错配容忍度使其可能具有更低的脱靶率。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 中国农业大学
<120> CRISPR-Cas12j酶和系统
<130> IDC210019
<150> CN201811355943.0
<151> 2018-11-15
<160> 108
<170> PatentIn version 3.5
<210> 1
<211> 1003
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.3的氨基酸序列
<400> 1
Met Thr Lys Glu Lys Ile Lys Lys Thr Lys Lys Ala Lys Val Glu Lys
1 5 10 15
Asp Ser Val Thr Arg Ala Gly Ile Leu Arg Ile Leu Leu Asn Pro Asp
20 25 30
Gln His Gln Glu Leu Asp Thr Leu Ile Ser Asp His Gln Glu Ala Ala
35 40 45
Arg Glu Ile Gln Thr Ala Thr Tyr Lys Leu Ser Gly Leu Lys Leu Tyr
50 55 60
Asp Lys Thr Asn Asn Met Val Val Asp Gly Ser Lys Ala Thr Pro Glu
65 70 75 80
Glu Gln Glu Ala Tyr Tyr Lys Ile Ile Asn Trp Glu Gly Gln Pro Ile
85 90 95
Ser Ile Ser Asn Pro Met Val Arg Ala Thr Phe Lys Ser Ile Ala Lys
100 105 110
Val Lys Glu Asp Ile Arg Arg Lys Gln Glu Glu Tyr Ala Lys Leu Glu
115 120 125
Glu Ala Asp Leu Thr Lys Met Ser Thr Gly Asp Val Lys Lys His Lys
130 135 140
Asn Glu Leu Arg Lys Ala Ala Asn Arg Ile Lys His Ser Glu Glu Ile
145 150 155 160
Leu Gln Phe Ala Lys Trp Arg Leu Ala Asp Ile Phe Pro Leu Pro Leu
165 170 175
Ser His Asn Ser Gln Leu His Leu Lys Asn Asn Tyr His Gln Asn Val
180 185 190
Phe Ser Gly Phe His Ala Arg Val Lys Gly Trp Asn Ala Cys Asp Ile
195 200 205
Ala Ala Gln Ala Asn Tyr Ala Glu Ile Asp Asn Arg Leu Thr Glu Leu
210 215 220
Ser Ser Glu Leu Ser Gly Asp Tyr Gly Ser Glu Val Ile Thr Asp Leu
225 230 235 240
Met Gly Leu Leu Gln Tyr Thr Lys Glu Leu Gly Glu Gly Tyr Thr Asp
245 250 255
Thr Ser Tyr Leu Asn Tyr Lys Phe Leu Ser Phe Phe Lys Glu Cys Trp
260 265 270
Arg Pro Asn Ala Ile Ala Asn Asn Thr Gly Leu Leu Glu Gly Phe Trp
275 280 285
Leu Ala Asn Asn Lys His Thr Asn Lys Lys Asn Gln Val Ala Tyr Ser
290 295 300
Phe Asn Pro Lys Ile Ser Glu Glu Leu Phe Arg Arg Arg Ser Leu Trp
305 310 315 320
Glu Ser Asp Lys Cys Leu Leu Ser Asp Pro Arg Phe Glu Lys Tyr Val
325 330 335
Glu Leu Phe Asp Lys His Gly Arg Tyr Arg Lys Gly Ala Ser Leu Thr
340 345 350
Leu Ile Ser Lys Glu Ser Pro Ile Pro Ile Gly Phe Ser Met Asp Arg
355 360 365
Asn Ala Ala Lys Leu Val Arg Ile Asp Asn Asp Thr Ala Asn Arg Gln
370 375 380
Leu Thr Ile Thr Ile Glu Leu Pro Asn Lys Glu Glu Arg Ser Tyr Val
385 390 395 400
Ala Ala Tyr Gly Arg Lys His Glu Thr Lys Cys Tyr Tyr Asn Gly Leu
405 410 415
Thr Thr Arg Leu Pro Arg Ser Glu Lys Glu Leu Leu Ala Leu Ala Lys
420 425 430
Ala Glu Asn Arg Glu Leu Thr Asp Lys Glu Ile His Glu Ala Ser Leu
435 440 445
Glu Lys Cys Tyr Ile Phe Glu Tyr Ala Arg Ala Gly Lys Ile Pro Val
450 455 460
Phe Ala Val Val Lys Thr Leu Tyr Phe Arg Arg Asn Pro Ser Asn Gly
465 470 475 480
Glu Tyr Tyr Val Ile Leu Pro Thr Asn Ile Phe Val Glu Tyr His Ala
485 490 495
Asn Asn Glu Phe Asn Ser Lys Glu Leu Phe Lys Ile Arg Ser Glu Leu
500 505 510
Gln Lys Ala Trp Asp Glu Val Arg Thr Pro Lys Arg Asn Val Gln Ser
515 520 525
Cys Val Leu Asp Lys Asp Leu Ser Lys Arg Phe Ala Gly Arg Thr Leu
530 535 540
Lys Tyr Ala Gly Ile Asp Leu Gly Tyr Ser Asn Pro Tyr Thr Val Ser
545 550 555 560
Tyr Tyr Asn Val Val Gly Thr Glu Glu Gly Ile Gln Ile Lys Glu Thr
565 570 575
Gly Asn Glu Ile Val Ser Thr Val Phe Asn Glu Gln Tyr Ile Gln Leu
580 585 590
Lys Gly Asn Ile Tyr Gln Leu Ile Asn Ile Ile Arg Ala Ser Arg Arg
595 600 605
Tyr Leu Gln Glu Ser Gly Glu Leu Lys Leu Ser Lys Asp Asp Ile Lys
610 615 620
Ser Phe Asp Gln Leu Met Glu Leu Leu Pro Ser Glu Gln Arg Ile Thr
625 630 635 640
Ile Asp Gln Phe Ile Lys Asp Ile Lys Lys Ala Lys Gln Glu Gly Lys
645 650 655
Leu Ile Arg Asp Ile Lys Gly Lys Leu Pro Val Glu Gly Lys Lys Lys
660 665 670
Glu Tyr Trp Val Ile Ser Asn Leu Met Tyr Val Ile Thr Gln Thr Met
675 680 685
Asn Gly Ile Arg Gly Asn Arg Asp Ser Asn Asn His Leu Thr Glu Lys
690 695 700
Lys Asn Trp Leu Ser Ala Pro Pro Leu Ile Glu Leu Ile Asp Ala Tyr
705 710 715 720
Tyr Asn Leu Lys Lys Thr Phe Asn Asp Ser Gly Asp Gly Ile Lys Met
725 730 735
Leu Pro Lys Asp His Val Tyr Ala Glu Gly Glu Lys Gln Arg Cys Thr
740 745 750
Leu Arg Glu Glu Asn Phe Cys Lys Gly Ile Leu Glu Trp Arg Asp Asn
755 760 765
Val Lys Asp Tyr Phe Ile Lys Lys Leu Phe Ser Gln Ile Ala His Arg
770 775 780
Cys Tyr Glu Leu Gly Ile Gly Ile Val Ala Met Glu Asn Leu Asp Ile
785 790 795 800
Met Gly Ser Ser Lys Asn Thr Lys Gln Ser Asn Arg Met Phe Asn Ile
805 810 815
Trp Pro Arg Gly Gln Met Lys Lys Ser Ala Glu Asp Ala Phe Ser Tyr
820 825 830
Met Gly Ile Leu Ile Gln Tyr Val Asp Glu Asn Gly Thr Ser Arg His
835 840 845
Asp Ala Asp Ser Gly Ile Tyr Gly Cys Arg Asp Gly Ala Asn Leu Trp
850 855 860
Leu Pro Asn Lys Lys Leu His Ala Asp Val Asn Ala Ser Arg Met Ile
865 870 875 880
Ala Leu Arg Gly Leu Thr His His Thr Asn Leu Tyr Cys Arg Ser Leu
885 890 895
Thr Glu Ile Glu Asn Gly Lys Tyr Val Asn Thr Tyr Glu Leu Phe Asp
900 905 910
Thr Thr Lys Asn Asp Gln Ser Gly Ala Ala Lys Arg Leu Arg Gly Ala
915 920 925
Glu Thr Leu Leu His Gly Tyr Ser Ala Thr Val Tyr Gln Ile His Thr
930 935 940
Thr Asn Thr Gly Ala Gly Val Ala Leu Leu Pro Asp Leu Thr Ala Thr
945 950 955 960
Asp Val Ile Lys Asn Lys Lys Ile Thr Ala Thr Lys Glu Asn Thr Ala
965 970 975
Lys Tyr Tyr Lys Leu Asp Asn Thr Asn Thr Tyr Tyr Pro Trp Ser Val
980 985 990
Cys Glu Lys Leu His Lys Asn Trp Lys Leu Ser
995 1000
<210> 2
<211> 874
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.4的氨基酸序列
<400> 2
Met Lys Lys Lys Lys Asn Phe Ser Val Ser Ala Thr Gly Val Phe Ser
1 5 10 15
Phe Pro Thr Thr Glu Ala Lys Met Asp Phe Phe His Arg Phe Ile Glu
20 25 30
Leu Asn Gly Leu Ala Ala Glu Ile Glu Thr His Phe Leu Asn Leu Lys
35 40 45
Asn Asp Lys Asn Gly Glu Ser Val Tyr Asn Lys Val Leu Ser Asn Ser
50 55 60
Asn His Ser Arg Pro Phe Ser Thr Pro Leu Leu Gly Thr Met Thr Gly
65 70 75 80
Ser Thr Lys Val Thr Asp Lys Asn Ala Leu Tyr Gly Asn Asp Leu Asp
85 90 95
His Cys Arg Lys Lys Lys Ile Val Pro Phe Ser Ser Ser Ser Pro Leu
100 105 110
Ser Ser Gln Glu Lys Phe Phe Cys Ile Glu Ala Val Phe Arg Arg Ala
115 120 125
Lys Ser His Met Glu Cys Lys Lys Leu Phe Gln Asp Glu Thr Asn Arg
130 135 140
Met Asp Ser Gln Ile Asn Gly Ile Leu Asn Glu Leu Pro Tyr Gly Val
145 150 155 160
Glu Leu Ser Asn Met Leu Ser Glu Leu Ile Ala Ile Pro Phe Ala Ile
165 170 175
Gly Trp Lys Leu Glu Gly Tyr Leu Gly Gln Val Phe Phe Pro Ser Ile
180 185 190
Ala Glu Gly Leu Thr Pro Pro Lys Ser Ala Lys Ile Lys Gly Arg Arg
195 200 205
Arg Ser Ile Asp Tyr Ser Val Thr Asp Glu Ala Tyr Asp Ile Leu Met
210 215 220
Lys Tyr Ser Asn Leu His Ser Ser Phe Glu Thr Gly Leu Lys Met Ser
225 230 235 240
Asn Leu Phe Ser Ala Phe Tyr Lys Lys Ser Asn Arg Lys Asp Glu Ile
245 250 255
Gln Phe Thr Pro Ile Ser Met Glu Ser Arg Cys Asp Leu Leu Leu Gly
260 265 270
Lys Asn Phe Leu Lys Phe Asp Leu Lys Asn Cys Asp His Arg Ser Gly
275 280 285
Ser Leu Met Leu Thr Ile Asn Asp Lys Asn Arg Leu Asn Gly Asp Tyr
290 295 300
Glu Ile Arg Val Gly Ser Asp Lys Lys Asp Ser Tyr Leu Thr Gly Val
305 310 315 320
Asn Val Thr Asn Leu Gly Asp Asn Val Phe Asn Leu Asn Tyr Lys Val
325 330 335
Asn Gly Lys Arg Glu Tyr Asn Met Leu Leu Lys Glu Pro Ser Ile His
340 345 350
Ile Lys Met His Arg Met Arg Asp Asp Gly Asn Tyr Leu Ser Ser Asp
355 360 365
Phe Asp Phe Tyr Met Ile Phe Ser Met Ser Ser Glu Lys Asp Glu Glu
370 375 380
Lys Leu Ala Arg Ser Trp Asp Met Arg Ala Ala Met Ser Thr Ala Tyr
385 390 395 400
Gly Thr Asp Ile Lys Lys Tyr His Ser Ser Phe Pro Cys Arg Ile Leu
405 410 415
Ala Cys Asp Leu Gly Val Lys His Pro Tyr Ser Ala Ala Val Met Asp
420 425 430
Ile Gly Gln Leu Asn Glu Asn Gly Met Pro Val Ser Val Asp Lys Val
435 440 445
His Cys Met His Ser Glu Gly Val Ser Glu Ile Gly Gln Gly Tyr Asn
450 455 460
His Leu Ile Gln Lys Ile Leu Ala Leu Asn Tyr Ile Leu Ala Tyr Cys
465 470 475 480
Arg Glu Phe Val Ser Gly Thr Val Asp Asp Phe Asp Lys Ile Asp Tyr
485 490 495
Lys Leu Ser Gln Leu Ser Tyr Lys Gln Glu Asp Leu Leu Ile Asn Leu
500 505 510
Gln Glu Met Lys Asp His Phe Gly Asn Asp Met Gln Ala Trp Lys Lys
515 520 525
Ser Arg Thr Trp Val Val Ser Thr Leu Phe Phe Glu Leu Arg Gln Glu
530 535 540
Phe Asn Gln Leu Arg Asn Gln Arg Pro Gly Lys Lys Thr Val Ser Leu
545 550 555 560
Ala Asp Glu Phe Gln Tyr Ile Asp Met Arg Arg Lys Phe Ile Ser Leu
565 570 575
Ser Arg Ser Tyr Thr Asn Val Gly Arg Gln Ser Ser Lys His Arg His
580 585 590
Asp Ser Tyr Gln Thr His Tyr Asp Val Ile Asn Arg Cys Lys Lys Asn
595 600 605
Leu Leu Arg Asn Ile Cys Arg Arg Met Ile Asp Met Ala Val Gln Asn
610 615 620
Lys Cys Asp Ile Ile Val Val Glu Asp Leu Ser Phe Gln Leu Ser Ser
625 630 635 640
His Asn Ser Arg Arg Asp Asn Val Phe Asn Ala Leu Trp Ser Cys Lys
645 650 655
Ser Ile Lys Asn Met Leu Gly Ile Met Ala Glu Gln His Asn Ile Ile
660 665 670
Ile Ser Glu Val Asp Pro Asn His Thr Ser Lys Ile Asp Cys Glu Thr
675 680 685
Gly Asn Phe Gly Tyr Arg Tyr Ser Ser Asp Phe Tyr Ser Val Ile Asp
690 695 700
Gly Gln Leu Val Arg Arg His Ala Asp Glu Asn Ala Ala Ile Asn Ile
705 710 715 720
Gly Asn Arg Trp Ala Ser Arg His Thr Asp Leu Lys Ser Phe Asn Cys
725 730 735
Arg Gln Ile Ser Ile Asp Gly Arg Lys Val Ala Phe Pro Tyr Ala Lys
740 745 750
Gly Lys Arg Lys Ser Ala Leu Phe Gly Tyr Leu Phe Gly Asn Cys Lys
755 760 765
Thr Val Phe Val Ser Asp Asp Gly Asp Ser Tyr Thr Pro Ile Pro Tyr
770 775 780
Ser Lys Phe Arg Lys Ser Ile Ser Lys Asp Asp His Asp Val Val Asn
785 790 795 800
Tyr Leu His Asp Leu Thr Met Asn Lys Asn Val Ile Arg Val Glu Tyr
805 810 815
Asn Lys Ser Ile Lys Ser Ala Ser Val Glu Leu Tyr Leu Asn Asp Asp
820 825 830
Arg Val Ile Ser Arg Ser Leu Arg Asp Lys Glu Val Asp Ala Ile Glu
835 840 845
Lys Leu Val Ser Arg Gly Ser Leu Ile Asn Glu Ser Gly Pro Ser Leu
850 855 860
Glu His Asp Glu Val Lys Ser Val Thr His
865 870
<210> 3
<211> 870
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.5的氨基酸序列
<400> 3
Met Lys Val His Glu Ile Pro Arg Ser Gln Leu Leu Lys Ile Lys Gln
1 5 10 15
Tyr Glu Gly Ser Phe Val Glu Trp Tyr Arg Asp Leu Gln Glu Asp Arg
20 25 30
Lys Lys Phe Ala Ser Leu Leu Phe Arg Trp Ala Ala Phe Gly Tyr Ala
35 40 45
Ala Arg Glu Asp Asp Gly Ala Thr Tyr Ile Ser Pro Ser Gln Ala Leu
50 55 60
Leu Glu Arg Arg Leu Leu Leu Gly Asp Ala Glu Asp Val Ala Ile Lys
65 70 75 80
Phe Leu Asp Val Leu Phe Lys Gly Gly Ala Pro Ser Ser Ser Cys Tyr
85 90 95
Ser Leu Phe Tyr Glu Asp Phe Ala Leu Arg Asp Lys Ala Lys Tyr Ser
100 105 110
Gly Ala Lys Arg Glu Phe Ile Glu Gly Leu Ala Thr Met Pro Leu Asp
115 120 125
Lys Ile Ile Glu Arg Ile Arg Gln Asp Glu Gln Leu Ser Lys Ile Pro
130 135 140
Ala Glu Glu Trp Leu Ile Leu Gly Ala Glu Tyr Ser Pro Glu Glu Ile
145 150 155 160
Trp Glu Gln Val Ala Pro Arg Ile Val Asn Val Asp Arg Ser Leu Gly
165 170 175
Lys Gln Leu Arg Glu Arg Leu Gly Ile Lys Cys Arg Arg Pro His Asp
180 185 190
Ala Gly Tyr Cys Lys Ile Leu Met Glu Val Val Ala Arg Gln Leu Arg
195 200 205
Ser His Asn Glu Thr Tyr His Glu Tyr Leu Asn Gln Thr His Glu Met
210 215 220
Lys Thr Lys Val Ala Asn Asn Leu Thr Asn Glu Phe Asp Leu Val Cys
225 230 235 240
Glu Phe Ala Glu Val Leu Glu Glu Lys Asn Tyr Gly Leu Gly Trp Tyr
245 250 255
Val Leu Trp Gln Gly Val Lys Gln Ala Leu Lys Glu Gln Lys Lys Pro
260 265 270
Thr Lys Ile Gln Ile Ala Val Asp Gln Leu Arg Gln Pro Lys Phe Ala
275 280 285
Gly Leu Leu Thr Ala Lys Trp Arg Ala Leu Lys Gly Ala Tyr Asp Thr
290 295 300
Trp Lys Leu Lys Lys Arg Leu Glu Lys Arg Lys Ala Phe Pro Tyr Met
305 310 315 320
Pro Asn Trp Asp Asn Asp Tyr Gln Ile Pro Val Gly Leu Thr Gly Leu
325 330 335
Gly Val Phe Thr Leu Glu Val Lys Arg Thr Glu Val Val Val Asp Leu
340 345 350
Lys Glu His Gly Lys Leu Phe Cys Ser His Ser His Tyr Phe Gly Asp
355 360 365
Leu Thr Ala Glu Lys His Pro Ser Arg Tyr His Leu Lys Phe Arg His
370 375 380
Lys Leu Lys Leu Arg Lys Arg Asp Ser Arg Val Glu Pro Thr Ile Gly
385 390 395 400
Pro Trp Ile Glu Ala Ala Leu Arg Glu Ile Thr Ile Gln Lys Lys Pro
405 410 415
Asn Gly Val Phe Tyr Leu Gly Leu Pro Tyr Ala Leu Ser His Gly Ile
420 425 430
Asp Asn Phe Gln Ile Ala Lys Arg Phe Phe Ser Ala Ala Lys Pro Asp
435 440 445
Lys Glu Val Ile Asn Gly Leu Pro Ser Glu Met Val Val Gly Ala Ala
450 455 460
Asp Leu Asn Leu Ser Asn Ile Val Ala Pro Val Lys Ala Arg Ile Gly
465 470 475 480
Lys Gly Leu Glu Gly Pro Leu His Ala Leu Asp Tyr Gly Tyr Gly Glu
485 490 495
Leu Ile Asp Gly Pro Lys Ile Leu Thr Pro Asp Gly Pro Arg Cys Gly
500 505 510
Glu Leu Ile Ser Leu Lys Arg Asp Ile Val Glu Ile Lys Ser Ala Ile
515 520 525
Lys Glu Phe Lys Ala Cys Gln Arg Glu Gly Leu Thr Met Ser Glu Glu
530 535 540
Thr Thr Thr Trp Leu Ser Glu Val Glu Ser Pro Ser Asp Ser Pro Arg
545 550 555 560
Cys Met Ile Gln Ser Arg Ile Ala Asp Thr Ser Arg Arg Leu Asn Ser
565 570 575
Phe Lys Tyr Gln Met Asn Lys Glu Gly Tyr Gln Asp Leu Ala Glu Ala
580 585 590
Leu Arg Leu Leu Asp Ala Met Asp Ser Tyr Asn Ser Leu Leu Glu Ser
595 600 605
Tyr Gln Arg Met His Leu Ser Pro Gly Glu Gln Ser Pro Lys Glu Ala
610 615 620
Lys Phe Asp Thr Lys Arg Ala Ser Phe Arg Asp Leu Leu Arg Arg Arg
625 630 635 640
Val Ala His Thr Ile Val Glu Tyr Phe Asp Asp Cys Asp Ile Val Phe
645 650 655
Phe Glu Asp Leu Asp Gly Pro Ser Asp Ser Asp Ser Arg Asn Asn Ala
660 665 670
Leu Val Lys Leu Leu Ser Pro Arg Thr Leu Leu Leu Tyr Ile Arg Gln
675 680 685
Ala Leu Glu Lys Arg Gly Ile Gly Met Val Glu Val Ala Lys Asp Gly
690 695 700
Thr Ser Gln Asn Asn Pro Ile Ser Gly His Val Gly Trp Arg Asn Lys
705 710 715 720
Gln Asn Lys Ser Glu Ile Tyr Phe Tyr Glu Asp Lys Glu Leu Leu Val
725 730 735
Met Asp Ala Asp Glu Val Gly Ala Met Asn Ile Leu Cys Arg Gly Leu
740 745 750
Asn His Ser Val Cys Pro Tyr Ser Phe Val Thr Lys Ala Pro Glu Lys
755 760 765
Lys Asn Asp Glu Lys Lys Glu Gly Asp Tyr Gly Lys Arg Val Lys Arg
770 775 780
Phe Leu Lys Asp Arg Tyr Gly Ser Ser Asn Val Arg Phe Leu Val Ala
785 790 795 800
Ser Met Gly Phe Val Thr Val Thr Thr Lys Arg Pro Lys Asp Ala Leu
805 810 815
Val Gly Lys Arg Leu Tyr Tyr His Gly Gly Glu Leu Val Thr His Asp
820 825 830
Leu His Asn Arg Met Lys Asp Glu Ile Lys Tyr Leu Val Glu Lys Glu
835 840 845
Val Leu Ala Arg Arg Val Ser Leu Ser Asp Ser Thr Ile Lys Ser Tyr
850 855 860
Lys Ser Phe Ala His Val
865 870
<210> 4
<211> 964
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.6的氨基酸序列
<400> 4
Met Ser Ala Asn Arg Val Ser Ala Asn Ser Gln Phe Glu Leu Gly Tyr
1 5 10 15
Pro Met Ser Leu Ser Leu Arg Gly Lys Val Phe Asn Ser Arg Glu Met
20 25 30
Met Lys Glu Ile Leu Pro Val Met Asn Asn Ile Val His Tyr Gln Asn
35 40 45
Asn Leu Leu Lys Leu Met Leu Ile Leu Arg Gly Glu Lys Tyr Thr Leu
50 55 60
Asp Gly Gln Phe Phe Ser Gln Lys Asp Val Asp Arg Gln Phe Gly Asp
65 70 75 80
Leu Cys Lys Glu His Asn Ile Lys Gly Ser Ile Cys Ser Leu Lys Glu
85 90 95
Lys Ser Arg Lys Leu Tyr Glu Val Phe Ser Cys Tyr Ile Asp Lys Lys
100 105 110
Gly Asn Leu Lys Thr Asn Ser Lys Ala Arg Ser Phe Ala Gly Val Leu
115 120 125
Leu Asn Pro Lys Asp Val Lys Leu Pro Pro Gln Ile Asp Ser Ile Ser
130 135 140
Ser Phe Val Val Glu Leu Arg Ala Lys Gly Val Leu Pro Ile Lys His
145 150 155 160
Glu Gly Asn Tyr Leu Ser Gly His Pro Ser Leu Lys Tyr Ser Val Ala
165 170 175
Gln Asn Val Leu Val Lys Leu Thr Ser Met Glu Lys Leu Gln Lys Ile
180 185 190
Tyr Ser Asp Glu Lys Ala Gly Trp Glu Asn Ile Val Ser Glu Val Arg
195 200 205
Ser Asp Leu Pro Lys Ile Glu Arg Tyr Glu Arg Met Leu Leu Ser Ile
210 215 220
Lys Ala Val Lys Glu Met Glu Lys Phe Gly Ile Asn Asn Tyr Arg His
225 230 235 240
Leu Leu Asn Asn Trp Arg Asp Glu Val Asp Lys Asp Ser Gly Lys Val
245 250 255
Leu Lys Gln Gly Met Arg Thr Tyr Phe Val Asn Met Leu Glu Ser Lys
260 265 270
Lys Asp Tyr Arg Phe Glu Glu Ser Asp Arg Tyr Leu Phe Gly Tyr Ala
275 280 285
Pro Glu Val Met Asn Leu Val Tyr His Asp Phe Arg Asp Leu Trp Gln
290 295 300
Gly Glu Asp Ile Ile Gly Ser Gln Ser Pro Glu Lys Lys Asp Arg Asp
305 310 315 320
Tyr Val Asp Val Ile Phe Asn Tyr Phe Asn Trp Arg Lys Glu Ser Ile
325 330 335
Asn Ile Ser Ser Phe Asp Ser Tyr Gly Lys Thr Ala Gln Ile Lys Leu
340 345 350
Gly Asp Asn Tyr Val Pro Phe Ser Asn Phe Gln Tyr Asp Lys Ile Leu
355 360 365
Asp Ala Trp Thr Leu Glu Ile Ala Asn Val Ser Gly Glu Gly Asp Asn
370 375 380
His Lys Leu Val Ile Ala Arg Ser Pro Gln Phe Asp Ser His Ser Ser
385 390 395 400
Val Lys Asp Ile Val Met Lys Asn Leu Lys Gly Lys Glu Ala Ser Lys
405 410 415
Thr Thr Leu Glu Phe Arg Tyr Ser Gly Asp Ser Lys Lys Ser Thr Trp
420 425 430
Tyr Arg Gly Thr Leu Lys Glu Pro Thr Leu Arg Tyr Ser Ser Ser Lys
435 440 445
Asn Cys Leu Tyr Val Asp Phe Ala Leu Ser Asn His Ile Val Glu Gly
450 455 460
Leu Ile Ser Asp Asn Leu Gly Ile Ser Asp Lys Met Tyr Lys Phe Arg
465 470 475 480
Gly Glu Phe Met Lys Ala Ser Pro Ser Ser Gly Lys Gln Ser Asn Ser
485 490 495
Ile Asn Leu Pro Ile Lys Lys Leu Arg Ala Met Gly Val Asp Phe Asn
500 505 510
Leu Arg Arg Pro Phe Gln Ala Ser Ile Tyr Asp Val Glu Asn Lys Asn
515 520 525
Gly Asn Leu Glu Phe Ser Phe Val Lys His Val Gln Ser Phe Ser Asn
530 535 540
Glu Asn Asp Glu Glu Arg Ala Lys Glu Leu Leu Asn Ile Glu Arg Asn
545 550 555 560
Ile Leu Ala Leu Lys Ile Leu Ile Trp Gln Thr Val Gly Tyr Val Thr
565 570 575
Gly Lys Asn Asp Thr Ile Asp Gly Val Val Thr Arg Lys Asn Asn Ala
580 585 590
Val Asp Ile Glu Lys Thr Leu Gly Ile Asn Met Lys Glu Tyr Met Ala
595 600 605
Tyr Leu Asn Gln Phe Arg Ser Tyr Glu Asp Lys Asn Lys Ala Phe Met
610 615 620
Asp Leu Arg Lys Arg Glu Tyr Ala Trp Ile Val Pro Pro Leu Ile Phe
625 630 635 640
Gln Cys Arg Ser Arg Leu Ile Ser Phe Arg Ser Glu Tyr Phe Asn Thr
645 650 655
Pro Lys Asp Glu Lys Ser His Tyr Cys Gln His Arg Asn Phe Val Asp
660 665 670
Tyr Ser Thr Phe Leu Lys Lys Asn Val Val Lys Lys Met Met Glu Leu
675 680 685
Arg Arg Ser Tyr Ser Thr Phe Gly Met Ser Ser Glu Gln Ser Ile Trp
690 695 700
Val Thr Asn Asn Asp His Ala Lys Asp Gly Ser Lys Lys Asn Gly Asn
705 710 715 720
Met Phe Asp Asp Asp Leu His Gln Trp Tyr Asn Gly Leu Val Arg Lys
725 730 735
Cys Ser Ser Leu Ala Ser Ser Ile Ile Asn Val Ala Arg Asp Asn Gly
740 745 750
Ala Ile Leu Val Phe Ile Glu Asp Leu Asp Cys His Pro Ser Ala Phe
755 760 765
Asp Ser Glu Glu Asp Asn Ser Leu Lys Ser Ile Trp Gly Trp Gly Ser
770 775 780
Ile Lys Ala Ser Leu Ala His Gln Ala Arg Lys His Asn Ile Ala Val
785 790 795 800
Val Ala Asn Asp Pro His Leu Thr Ser Leu Val Ser Ser Thr Thr Gly
805 810 815
Glu Leu Gly Ile Ala Lys Gly Arg Asp Val Leu Phe Phe Asp Ser Lys
820 825 830
Gly Lys Leu Thr Ser Lys Val Asn Arg Asp Glu Asn Ala Ala Gln Asn
835 840 845
Ile Ala Ile Arg Gly Phe Val Arg His Ser Asp Leu Arg Glu Phe Val
850 855 860
Ala Glu Lys Ile Glu Glu Asn Arg Tyr Arg Val Val Val Asn Lys Thr
865 870 875 880
His Lys Arg Lys Ala Gly Ala Ile Tyr Arg His Ile Gly Ser Thr Glu
885 890 895
Cys Ile Met Ser Lys Gln Ala Asp Gly Ser Leu Lys Ile Asp Lys Thr
900 905 910
Glu Leu Thr Pro Leu Glu Ile Lys Met Glu Lys Lys Asn Asp Lys Lys
915 920 925
Met Tyr Val Ile Leu His Gly Lys Thr Trp Arg Leu Arg His Glu Leu
930 935 940
Asn Glu Lys Leu Glu Lys Asp Leu Asp Asn His Leu Lys Ser Lys Ser
945 950 955 960
Ser Val Ile Ser
<210> 5
<211> 962
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.7的氨基酸序列
<400> 5
Met Ser Ser Ala Asn Asp Gln Leu Gly Leu Gly Tyr Pro Leu Thr Leu
1 5 10 15
Thr Leu Arg Gly Lys Val Tyr Asn His Asp Thr Ala Met Glu Ala Phe
20 25 30
Ala Pro Val Met Lys Gly Met Val Pro Tyr Ala Asn Asn Leu Met Arg
35 40 45
Ile Leu Leu Thr Leu Arg Leu Glu Lys Tyr Thr Leu Asp Gly Ile His
50 55 60
His Thr Lys Glu Glu Val Glu Lys Asp Leu Arg Gly Leu Met Lys Glu
65 70 75 80
Tyr Gly Ile Asn Leu Ser Phe Ala Lys Phe Ser Glu Met Ala Gly Glu
85 90 95
Val Tyr Arg Val Phe Val Cys Tyr Val Asp Ala Lys Gly Lys Leu Lys
100 105 110
Val Asn Gly Lys Ala Arg Gly Phe Ala Asn Val Phe Phe Ser Glu Asp
115 120 125
Asp Ala Thr Ile Pro Glu Asn Cys Pro Ser Met Glu Leu Leu Arg Lys
130 135 140
Lys Gly Met Phe Pro Ile Leu Val Asp Gly Lys Pro Ile Ser Ser Ile
145 150 155 160
Ser Arg Glu Lys Thr Pro Leu Lys Tyr Ser Val Ala Gln Asp Val Leu
165 170 175
Thr Lys Leu Thr Ser Met Glu Glu Ile Ser Lys Glu Tyr Glu Lys Ala
180 185 190
Lys Thr Asp Trp Glu Asn Glu Cys Gln Lys Val Ile Ser Gln Leu Pro
195 200 205
Leu Ile Gly Arg Tyr Glu Ala Leu Leu Thr Thr Ile Pro Leu Ile Pro
210 215 220
Glu Met Arg Gly Phe Asp Gly Asp Asn Tyr Arg Lys Met Leu Asn Arg
225 230 235 240
Trp Arg Asp Tyr Val Asn Glu Asp Gly Glu Leu Val Arg Gly Gly Met
245 250 255
Lys Thr Tyr Phe Leu Asp Leu Leu Ser Lys Asp Thr Ser His Lys Phe
260 265 270
Asn Glu Glu Glu Arg Tyr Leu Phe Gly Tyr Cys Pro Glu Phe Met Asn
275 280 285
Leu Ile Tyr His Asp Phe Arg Asp Leu Trp Ser Lys Glu Asp Ile Ile
290 295 300
Gly Ser Gln Arg Lys Gly Lys Gly Leu Lys Gly Lys Asp Tyr Val Asp
305 310 315 320
Val Ile Phe Asn Cys Phe His Trp Arg Arg Glu Ser Ile Asn Ile Ser
325 330 335
Ser Phe Gly Asn Asn Asp Lys Val Met Asn Ile His Leu Gly Asp Asn
340 345 350
Phe Val Pro Phe Glu Leu Lys Ser Gln Asn Gly Ile Trp Glu Val His
355 360 365
Val Gln Asn Leu His Gly Gln Asn Asp Pro His Arg Val Ile Val Cys
370 375 380
Arg Cys Pro Gln Phe Asn Glu Asp Ser Ser Met Lys Met Val His Pro
385 390 395 400
Leu Ala Lys Asn Gly Glu Glu Ser Asp Lys Glu Asn Ile Glu Phe Arg
405 410 415
Tyr Ser Gly Asp Ser Lys Arg Glu Thr Trp Tyr Thr Gly Leu Leu Lys
420 425 430
Glu Pro Thr Leu Arg Tyr Asp Val Glu Arg Lys Ser Leu Tyr Val Asp
435 440 445
Phe Ile Leu Ser Asn His Arg Val Glu Gly Val Val Thr Asn Glu Tyr
450 455 460
Leu Lys Asp Pro Arg Asp Leu Phe Gly Val Arg Gly Tyr Phe Leu Ser
465 470 475 480
Ser Ser Val Ser Asn Pro Arg Gln Lys Asp Lys Thr Ser Leu Pro Asp
485 490 495
Gly Lys Phe Asn Val Met Gly Val Asp Leu Gly Leu Lys Cys Pro Tyr
500 505 510
Glu Cys Ala Ile Tyr Gly Ile Thr Val Lys Asn Gly Lys Met Gln His
515 520 525
Lys Trp Ser His Asn Val Ser Ala Glu Asp Asn Asn Asn Val Ser Glu
530 535 540
Arg Leu Ala Asn Leu Lys Lys Ile Asp Glu Lys Ile Leu Ala Thr Gln
545 550 555 560
Val Leu Ile Ser Leu Thr Lys Met Cys Val Val Lys Asp Glu Glu Ile
565 570 575
Pro Asp Ser Tyr Thr Leu Arg Glu His Arg Val Asp Ile Ala Lys Ser
580 585 590
Leu Asp Leu Asp Met Asp Lys Tyr Arg Arg Tyr Val Glu Lys Cys Lys
595 600 605
Lys Asn Pro Asp Lys Ile Gln Ala Leu Lys Asp Ile Arg Lys Ser Glu
610 615 620
Asn Asn Trp Ile Val Ala Glu Lys Ile Asn Glu Ile Arg Ser Leu Ile
625 630 635 640
Ser Glu Ile Arg Ser Glu Tyr Tyr Ala Ser Lys Asp Lys Arg Asn Tyr
645 650 655
Cys Arg Asn Leu Asn Gly Val Asp Leu Ser Val Phe Leu Lys Lys Lys
660 665 670
Val Val Lys Asn Trp Ile Ser Leu Leu Arg Ser Phe Ser Thr Phe Gly
675 680 685
Met Thr Pro Gln Glu Ser Ala Tyr Ile Arg Lys Asp Phe Ala Lys Asn
690 695 700
Leu Ser Lys Trp Tyr Lys Gly Leu Val Arg Lys Cys Gly Ser Ile Ala
705 710 715 720
Ala His Ile Val Asn Ile Ala Arg Asp Asn Lys Val Met Val Ile Phe
725 730 735
Ile Glu Asp Leu Asp Ala Arg Thr Ser Ala Phe Asp Ser Lys Glu Asp
740 745 750
Asn Glu Leu Lys Ile Leu Trp Gly Trp Gly Glu Ile Lys Lys Trp Ile
755 760 765
Gly His Gln Ala Arg Lys His Asn Ile Ala Val Val Ala Val Asp Pro
770 775 780
His Leu Thr Ser Leu Val Asn His Glu Ser Gly Leu Leu Gly Ile Ala
785 790 795 800
Gly Ser Gly Asn Asp Arg Asn Ile Tyr Thr Phe Gln Lys Asn Lys Lys
805 810 815
Tyr Val Val Ile Asn Arg Asp Asn Asn Ala Ala His Asn Ile Ala Leu
820 825 830
Arg Gly Leu Ser Lys His Thr Asp Ile Arg Glu Phe Tyr Val Glu Gln
835 840 845
Ile Asp Val Asp His Tyr Arg Leu Met Tyr Gly Pro Glu Ala Glu Asn
850 855 860
Gly Lys Arg Arg Ser Gly Ala Ile Tyr Lys His Ile Gly Ser Thr Glu
865 870 875 880
Cys Val Phe Ser Lys Gln Lys Asn Gly Thr Leu Lys Val Glu Lys Thr
885 890 895
Ser Leu Thr Lys Asp Glu Lys Glu Met Pro Lys Ile Asn Gly Lys Gly
900 905 910
Val Tyr Ala Ile Leu His Gly Asn Glu Trp Arg Leu Arg His Glu Leu
915 920 925
Asn Glu Glu Leu Gly Ala Lys Leu Asp Gly Ile Ser Val Lys Arg Val
930 935 940
Val Ser Glu Pro Asn Lys Val Lys Thr Ser Leu Val Lys Gly Ser Val
945 950 955 960
Arg Ala
<210> 6
<211> 907
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.8的氨基酸序列
<400> 6
Met Lys Lys Gln Thr Ile Val Lys Lys Asp Ser Lys Ala Glu Thr Lys
1 5 10 15
Glu Asn Lys Met Tyr Pro Asp Lys Asp Thr Asp Phe Pro Val Asn Ser
20 25 30
Gln Phe Ser Arg Ser Ile Ser Ile Arg Ala Asn Val Asp Pro Lys Asp
35 40 45
Leu Leu Val Leu Lys Arg Thr Phe Glu Glu Thr Thr Lys Ile Ser Asp
50 55 60
Glu Leu Leu Ser Thr Leu Leu Met Leu Arg Gly Lys Asp Tyr Cys Leu
65 70 75 80
Asp Asn Val Val Cys Lys Gly Glu Glu Val Leu Glu Asn Leu Tyr Lys
85 90 95
Lys Leu Ser Lys Asn Ala Thr Val Asn Arg Asp Lys Phe Ile Ser Thr
100 105 110
Ala Lys Ala Phe Tyr Glu Tyr Phe His Gly Cys Ser Tyr His Lys Gly
115 120 125
Phe Lys Ser Phe Phe Phe Ser Ser Lys Glu Ile Asp Ser Ile Gln Ser
130 135 140
Glu Lys Phe Gly Tyr Leu Arg Glu Ile Gly Leu Phe Pro Ile Lys Ile
145 150 155 160
Asp Ala Gln Ile Ser Asn Asp Leu Gln Tyr Ser Ile Val Ala Ser Asn
165 170 175
His Ala Lys Ile Lys Gly Phe Glu Lys Ile Asp Lys Glu Tyr Gln Ala
180 185 190
Asn Lys Glu Lys Trp Asn Lys Thr Ile Gly Glu Ser Thr Leu Lys His
195 200 205
Leu Asn Arg Tyr Gly Glu Met Leu Lys Gly Leu Ser Asp Leu Gly Thr
210 215 220
Met Gly Asn Phe Asn Gly Lys Lys Tyr Asp Arg Phe Met Gly His Trp
225 230 235 240
Arg Asn Glu Gln Lys Ile Pro Asp His Ile Ser Met Leu Asp Phe Phe
245 250 255
Arg Lys Ile Tyr Gln Glu Lys Gly Lys Ser His Arg Phe Thr Ala Ile
260 265 270
Asp Asn Phe Thr Tyr Gly Tyr Glu Ser Glu Phe Met Asn His Ile Tyr
275 280 285
Leu Asn Phe Ser Asp Leu Trp Leu Lys Glu Asp Val Ile Gly Asp Glu
290 295 300
Glu Tyr Val Ser Leu Ile Arg Gly Ala Tyr His Trp Gln Lys Asp Val
305 310 315 320
Val Gly Ile Ala Ser Phe Ser Gly Tyr Asn Lys Tyr Glu Lys Leu Phe
325 330 335
Met Gly Asp Asn Lys Ile Asn Tyr Ala Leu Asp Phe Ser Asn Lys Asp
340 345 350
Gln Trp Leu Met Lys Phe Asn Asn Val Ile Ser Lys Glu Pro Glu Thr
355 360 365
Ile Thr Leu Arg Leu Cys Lys Asn Gly Tyr Phe Asn Asn Leu Ser Val
370 375 380
Leu Glu Lys Asn Asp Glu Asn Gly Arg Tyr Lys Ile Arg Phe Ser Thr
385 390 395 400
Glu Lys Gln Gly Lys Tyr Phe Tyr Glu Ala Phe Ile Arg Glu Pro Phe
405 410 415
Leu Arg Tyr Asn Lys Asp Asn Asp Lys Ile Tyr Val His Phe Cys Leu
420 425 430
Ser Glu Glu Ile Lys Glu Asn Cys Pro Asn His Leu Asp Thr Arg Ser
435 440 445
Asp Lys Tyr Leu Phe Lys Ser Ala Leu Leu Thr Asn Ser Arg Gln Lys
450 455 460
Leu Gly Lys Leu His Tyr Arg Asp Phe His Ile Val Gly Val Asp Leu
465 470 475 480
Gly Ile Asn Pro Val Ala Lys Ile Thr Val Cys Lys Val His Val Asp
485 490 495
Lys Asn Glu Asn Leu Lys Ile Thr Lys Ile Ile Thr Glu Glu Thr Arg
500 505 510
Lys Asn Ile Asp Thr Asn Tyr Leu Asp Gln Leu Asn Leu Leu Tyr Lys
515 520 525
Lys Ile Val Ser Leu Lys Arg Leu Ile Arg Ala Thr Val Ala Phe Lys
530 535 540
Lys Asp Gly Glu Glu Ile Pro Lys Met Phe Lys Met Gly Lys Lys Ser
545 550 555 560
Pro Tyr Phe Leu Asn Trp Thr Glu Val Leu Asn Val Asn Tyr Asp Asp
565 570 575
Tyr Ile Lys Glu Ile Ser Thr Phe Ser Val Asp Arg Leu Ser Gly Leu
580 585 590
Thr Leu Pro Met Gln Trp Ala Arg Ser Gln Asn Lys Trp Val Val Lys
595 600 605
Asp Leu Thr Lys Met Val Arg Lys Gly Ile Ser Asp Leu Ile Tyr Ala
610 615 620
Arg Tyr Phe Asn Cys Ser Asp Lys Thr Gln Tyr Val Thr Glu Asn Asn
625 630 635 640
Ala Val Asp Ile Thr Thr Phe Lys Lys His Asp Ile Ile Ser Glu Ile
645 650 655
Ile Gly Leu Gln Lys Met Phe Ser Gly Gly Gly Lys Asp Val Ala Lys
660 665 670
Lys Asp Tyr Leu Tyr Leu Arg Gly Leu Arg Lys His Ile Gly Asn Tyr
675 680 685
Thr Ala Ser Ala Ile Val Ser Ile Ala Gln Lys Tyr Asn Ala Val Phe
690 695 700
Ile Phe Ile Glu Asp Leu Asp Leu Lys Ile Ser Gly Met Asn Gly Lys
705 710 715 720
Lys Glu Asn Lys Val Lys Ile Leu Trp Gly Val Gly Gln Leu Lys Lys
725 730 735
Arg Leu Ser Glu Lys Ala Glu Lys Phe Gly Ile Gly Ile Val Pro Val
740 745 750
Asn Pro Glu Leu Thr Ser Gln Met Asp Arg Glu Thr Phe Leu Leu Gly
755 760 765
Tyr Arg Asn Pro Thr Asn Lys Lys Glu Leu Tyr Val Lys Arg Asp Asp
770 775 780
Lys Ile Glu Ile Leu Asp Ala Asp Glu Thr Ala Ser Tyr Asn Val Ala
785 790 795 800
Leu Arg Gly Leu Gly His His Ala Asn Leu Ile Gln Phe Arg Ala Asp
805 810 815
Lys Met Pro Asn Gly Cys Phe Arg Val Met Pro Asp Arg Lys Tyr Lys
820 825 830
Gln Gly Ala Leu Tyr Gly Tyr Leu Asn Ser Thr Ala Val Leu Phe Lys
835 840 845
Asp Lys Gly Asp Gly Val Leu Thr Ile His Lys Ser Lys Leu Thr Lys
850 855 860
Lys Glu Arg Asp Ser Arg Pro Ile Lys Gly Lys Lys Thr Phe Val Val
865 870 875 880
Lys Asn Gly Lys Arg Trp Ile Leu Arg His Val Leu Asp Glu Glu Val
885 890 895
Lys Lys Tyr Pro Glu Met Tyr Asn Ser Gln Asn
900 905
<210> 7
<211> 912
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.9的氨基酸序列
<400> 7
Met Ser Asp Tyr Lys Phe Ser Asn Asn Gly Val Thr Asn Thr Gly Ser
1 5 10 15
Ala His Ile Gly Leu Ser Pro Glu Asn Ser Ser Thr Val Met Asp Met
20 25 30
Phe Lys Val Ile Thr Lys Asp Ala Asp Phe Leu Leu Lys Asn Leu Leu
35 40 45
Ile Met Glu Gly Gly Glu Tyr Met Leu Asn Arg Glu Ile His Asn Gly
50 55 60
Asp Lys Glu Phe Asp Lys Ile Ile Ser Lys Leu Gly Leu Ser Lys Lys
65 70 75 80
Glu Lys Glu Asn Leu Lys Met Lys Cys Lys Asp Phe Phe Phe Asp Phe
85 90 95
Val Lys Leu Gln Asn Gly Arg Ser Leu Ala Asn Ile Leu Phe Glu Thr
100 105 110
Lys Gly Thr Thr Leu Ile Gly Cys Gly Lys Asp Lys Lys Gly Glu Lys
115 120 125
Val Asp Gly Glu Tyr Pro Thr Ile Tyr His Asp His Glu Thr Leu Arg
130 135 140
Ser Thr Gly Leu Leu Pro Leu Lys Phe Ser Lys Asn Ile Asp Asp Val
145 150 155 160
Asp Tyr Lys Tyr Leu Ile Cys Tyr Leu Val His Asn Val Leu Ser Ser
165 170 175
Phe Ile Glu Lys Arg Asp Ala Tyr Asn Asp Asn Lys Lys Glu Trp Glu
180 185 190
Ser Lys Leu Ser Asn Ser Asn Leu Pro Gln Leu Glu Arg Met Ser Glu
195 200 205
Phe Leu Asn Gly Ile Asn His Leu Gly Asn Ile Ile Gly Trp Asn Gly
210 215 220
Lys Lys Tyr Ile Gly Phe Ile Lys Lys Trp Thr Asp Glu Glu Ser Ser
225 230 235 240
Met Tyr Asp Phe Phe Val Gln Lys Leu Gln Asp Asn Pro Lys Tyr Lys
245 250 255
Phe Gly Lys Lys Asp Gln Phe Leu Tyr Gly Tyr Glu Pro Glu Phe Leu
260 265 270
Asn Tyr Leu Phe His Asp Phe Arg Asp Leu Trp His Pro Asp Asn Leu
275 280 285
Ile Gly Lys Asp Glu Tyr Val Asp Leu Ile Ser Gly Lys Asn Asn Thr
290 295 300
Asp Ala Glu Thr Ala Asn Lys Gly Ala Tyr His Trp Leu Lys Asp Phe
305 310 315 320
Ile Asn Ile Ser Ser Phe Asp Ala Tyr Gly Lys Met Ala Thr Ile Gly
325 330 335
Met Gly Asn Asn Leu Ile Asn Tyr Ser Met Asn Ile Asp Lys Asp Gly
340 345 350
Lys Ile Ile Val Asn Met Asp Asn Ile Phe Asp Arg Ser Lys Pro Ile
355 360 365
Val Phe Asn Val Tyr Arg Asn Ser Tyr Phe Arg Asn Phe Lys Ile Ile
370 375 380
Glu Ser Asp Asp Lys Lys Gly Ile Tyr Lys Val Glu Phe Ser Thr Ser
385 390 395 400
Asn Asn Gly Val Ile Tyr Glu Gly Tyr Ile Lys Ser Pro Ser Leu Arg
405 410 415
Phe Ala Thr Lys Gly Gly Thr Ile Lys Ile Asp Phe Pro Ile Ser Asp
420 425 430
Lys Arg Ile Lys Gly Gly Arg Glu Met Asn Thr Asp Leu Met Trp Phe
435 440 445
Leu Asn Arg Ala Ser Pro Cys Ser Thr Lys Asn Lys Glu Val Asn Ser
450 455 460
Phe Ile Gly Lys Asn Phe Val Gly Leu Ala Ile Asp Arg Gly Ile Asn
465 470 475 480
Pro Leu Met Ala Trp Tyr Val Ala Glu Trp Thr Tyr Asp Lys Asp Gly
485 490 495
Lys Ala Lys Ile Val Arg Ser Ile Ala Asn Gly Arg Val Asp Ser Gly
500 505 510
His Asn Glu Ser Glu Val Lys Phe Val Arg Glu Thr Thr Asn Arg Ile
515 520 525
Val Gly Ile Lys Ser Leu Val Trp Asn Thr Val Lys Tyr Arg Thr Gly
530 535 540
Gly Ser Glu Gly Ile Asp Arg Cys Arg Lys Ser Gln Asn Gly Gln Val
545 550 555 560
Asp Leu Phe Glu Met Phe Asp Ile Asp Tyr Asn Asn Tyr Leu Lys Glu
565 570 575
Val Asn Asn Leu Pro Tyr Asp Pro Asn Ser Glu Arg Ser Ile Ile Gln
580 585 590
Thr Trp Val Ser Ser Pro Trp Lys Val Lys Asp Leu Val Lys Asp Ala
595 600 605
Lys Asn Arg Met Val Gln Ile Lys Thr Gln Tyr His Asn Ala Lys Asp
610 615 620
Lys Glu Lys Tyr Ile Thr Thr Gln Asn Arg Ala Gly Phe Tyr Asp Phe
625 630 635 640
Leu Lys Ile Glu Met Glu Lys Gln Phe Thr Ser Leu Gln Arg Met Phe
645 650 655
Ser Gly Gly Gln Lys Asp Ile Cys Lys Asn Asn Glu Glu Tyr Arg Arg
660 665 670
Gly Leu Arg Arg Arg Ile Asn Leu Tyr Thr Ser Ser Val Ile Met Ser
675 680 685
Leu Ala Arg Lys Phe Asn Val Asp Cys Ile Phe Leu Glu Asp Leu Asp
690 695 700
Ser Ser Lys Ser Ser Trp Asp Asp Ala Lys Lys Asn Ser Leu Lys Asp
705 710 715 720
Leu Trp Ser Thr Gly Gly Ala Asp Asp Ile Leu Gly Lys Met Ala Asn
725 730 735
Lys Tyr Lys Tyr Pro Ile Val Lys Val Asn Ser His Leu Thr Ser Leu
740 745 750
Val Asp Asn Lys Thr Gly Lys Ile Gly Tyr Arg Asp Pro Lys Lys Lys
755 760 765
Ser Asn Leu Tyr Val Glu Arg Gly Lys Lys Ile Glu Ile Ile Asp Ser
770 775 780
Asp Glu Asn Ala Ala Ile Asn Ile Leu Lys Arg Gly Ile Ser Lys His
785 790 795 800
Ile Asp Ile Arg Glu Phe Phe Ala Glu Lys Ile Glu Val Ser Gly Lys
805 810 815
Thr Leu Tyr Arg Ile Ser Asn Lys Leu Gly Lys Gln Arg Met Gly Ser
820 825 830
Leu Tyr Tyr Leu Glu Gly Asn Lys Glu Ile Leu Phe Gly Leu Gly Lys
835 840 845
Asn Gly Glu Pro Ile Val Cys Lys Arg Gly Leu Cys Lys Lys Glu Arg
850 855 860
Leu Ala Pro Arg Ile Ala Glu Lys Lys Ser Thr Tyr Leu Ile Met Asn
865 870 875 880
Gly Ser Lys Trp Met Phe Arg His Glu Ala Lys Lys Ile Val Glu Thr
885 890 895
Tyr Lys Asp Arg Tyr Cys Ala Asn His Lys Val Ala Ser Lys Asp Gly
900 905 910
<210> 8
<211> 1119
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.10的氨基酸序列
<400> 8
Met Met Asn Ile Asn Glu Met Val Lys Leu Met Lys Ser Glu Tyr Leu
1 5 10 15
Phe Glu Asp Asp Gly Ile Val Thr Lys Asn Lys Ile Gln Glu Arg Leu
20 25 30
Arg Asn Gly Phe Ser Asp Ile Gly Val Asp Pro Ser Leu Val Ser Tyr
35 40 45
Ala Ser Lys Phe Leu Asp Ser Met Phe Ile Cys Phe Ser Arg Val Lys
50 55 60
Gly Glu Lys Asn Phe Lys Ala Lys Asn Val Arg Lys Asn Met Ser Ser
65 70 75 80
Ala Glu Lys Lys Ala Gln Lys Lys Lys Glu Tyr Gln Glu Tyr Tyr Gln
85 90 95
Gly Val Met Ala Gln Gln Asp Ala Tyr Ala Gln Leu Leu Ser Asp Pro
100 105 110
Thr Gln Glu Asn Leu Asp Lys Leu Asn Glu Leu Ile Ser Met Ser Val
115 120 125
Asn Gly Ser Leu Val Glu Asp Phe Phe Pro Ala Leu Lys Asn Met Ile
130 135 140
Gln Lys Ala Asp Tyr Ser Ile Asp Lys Lys Gly Leu Leu Asp Phe Ser
145 150 155 160
Cys Cys Met Met Asp Arg Tyr Glu Asp Arg Ser Leu Thr Arg Ala Ile
165 170 175
Ser Ile Ser Ala Phe Asn Ile His Ser Gly Gly Leu Arg Lys Ala Leu
180 185 190
Ser Asp Ile Ser Glu Lys Val Gln Asp Leu Ser Asn Thr Leu Leu Ile
195 200 205
Arg Ile Leu Tyr Met Lys Gly Glu Glu Leu Ser Ile Asp Gly Glu Lys
210 215 220
Ile Ser Lys Glu Glu Val Gln Arg Gln Leu Lys Ala Asp Tyr Glu Glu
225 230 235 240
His Lys Glu Tyr Phe Glu Asp Phe Glu Asp Phe Ala Lys Lys Cys Arg
245 250 255
Phe Phe Tyr Asn Lys Phe Ser Lys Lys Lys Lys Thr Arg Gly Phe Gly
260 265 270
Thr Tyr Phe Phe Gly Asp Lys Lys Lys Glu Ile Ser Ser Ala Glu Tyr
275 280 285
Lys Ala His Lys Glu Leu Arg Asp Ser Gly Tyr Leu Trp Phe Asp Ile
290 295 300
Gly Trp Ser Glu Ser Ser Asp Phe Lys Tyr Val Ile Val Gly Asn Val
305 310 315 320
Ser Gly Lys Leu Lys Ser Phe Glu Glu Thr Ser Glu Glu Tyr Gln Lys
325 330 335
Ser Lys Asn Cys Trp Glu Ala Glu Arg Val Lys Leu Tyr Glu Gln Asp
340 345 350
Ser Asp Phe Val Leu Phe Val Glu Asp Met Ile Glu Ser Lys Tyr Gly
355 360 365
Pro Ile Glu Lys Met Lys Leu Arg Thr Phe Lys Thr Ile Val Lys Lys
370 375 380
Leu Asp Lys Glu Phe Gly Lys Arg Gly Asp Lys Thr Pro Ser Ile His
385 390 395 400
Asp Tyr Phe Glu Ser Leu Asp Pro Asn His Thr Phe Ser Gln Ser Glu
405 410 415
Gln Phe Met Tyr Gly Leu Asp Val Thr Leu Met Gln Phe Leu Phe Asn
420 425 430
Asn Lys Lys Gln Phe Tyr Lys Leu Cys Lys Asp His Asp Gly Lys Arg
435 440 445
Thr Phe Ala Lys Val Val Glu Glu Ser Tyr His Trp Gly Lys Asn Ser
450 455 460
Ile Asn Val Ser Thr Phe Gln Asn Ser Thr Ser Ile Leu Leu Gly Gly
465 470 475 480
Asn Tyr Leu Asn Tyr Ser Met Ser Ile Glu Gly Glu Gly Leu Val Ile
485 490 495
Lys Phe Asp Asn Pro Leu Ser Gly Lys Glu Val His Phe Val Val Cys
500 505 510
Asn Asn Lys Tyr Leu Ser Asp Leu Glu Ile Leu Ser Gly Asn Pro Asn
515 520 525
Arg Lys Asp Asn Asn Tyr Thr Ile Ser Tyr Ser Thr Gly Gly Lys Ala
530 535 540
Arg Phe Ile Ala Lys Ser Lys Glu Pro Arg Ile Phe Phe Asn Arg Lys
545 550 555 560
Thr Lys Lys Trp Glu Ile Ala Phe Gln Leu Ser Asp Val Ser Pro Leu
565 570 575
Asn Gly Lys Phe Gly Lys Gln Gly Glu Phe Leu Ser Asn Leu Arg Lys
580 585 590
Phe Val Tyr Asn His Val Ala Lys Ser Pro Ser Lys Leu Asn Ile Ser
595 600 605
Asp Asn Asn Cys Arg Ala Val Ala Tyr Asp Leu Gly Ile Arg Asn Val
610 615 620
Gly Ala Trp Ser Ser Phe Asp Phe Ser Tyr Lys Asp Gly Val Leu Gly
625 630 635 640
Gly Tyr Lys Tyr Leu Thr Ser Gly Ser Leu Arg Ser Lys Ser Glu Ser
645 650 655
Ser Glu Met Asp Gln Gly Tyr Tyr Phe Val Leu Asn Leu Lys Lys Ile
660 665 670
Val Lys Leu Ile Pro Val Val Lys Lys Ser Ile Ile Asp Asp Pro Glu
675 680 685
Leu Lys Arg Gln Phe Ile Gly Val Leu Asn Glu Asn Gly Asn Thr Val
690 695 700
Gly Leu Gly Asn Ile Gly Lys Leu Asp Ile Ala Ser Arg Lys Ala Val
705 710 715 720
Gln Ser Phe His Asn Cys Ile Gln Gln Ile Asn Tyr Tyr Val Asp Thr
725 730 735
Tyr Ala Asp His Ile Asp Lys Ile Ser Ala Lys Asp Phe Val Asp Asp
740 745 750
Ile Asp Gly Ile Lys Val Leu Asp Glu Asp Asp Pro Tyr Val Val Lys
755 760 765
Ile Leu Ser His Leu Pro Glu Asp Val Glu Gly Asn Gln Asp Asp Ile
770 775 780
Leu Asn Ile Ser Leu Leu Lys Trp Lys Thr Ser Asn Ala Gln Phe Val
785 790 795 800
Pro Pro Leu Ile Gln Glu Ala Lys Ala Ile Met Ser Arg Ile Lys Arg
805 810 815
Glu Asn Leu Asp Asn Ile Arg Gly Lys Lys Thr Gln Val Val Thr Gln
820 825 830
Lys Thr Phe His Lys Ile Lys Phe Ala Lys Ala Leu Leu Ser Leu Met
835 840 845
Lys Ser Trp Ser Ser Ile Gly Thr Val Arg Val Val Lys Thr Asp Gln
850 855 860
Ile Tyr Gly Lys Lys Ile Trp Asp Tyr Ile Asn Gly Leu Arg Arg Asn
865 870 875 880
Val Leu Thr Tyr Leu Ser Ser Ala Ile Val Asn Asn Ala Leu Asp Leu
885 890 895
Gly Ala His Met Ile Ile Leu Glu Asp Leu Asp Ser Ser Val Ser Lys
900 905 910
Tyr Arg Glu Lys Asp Lys Asn Ala Ile Gln Ser Leu Trp Gly Ser Gly
915 920 925
Glu Leu Lys Lys Arg Ile Glu Glu Lys Ala Glu Lys His Arg Val Val
930 935 940
Val Gln Tyr Val Ser Pro Tyr Leu Thr Ser Gln Leu Asp Asn Glu Thr
945 950 955 960
Lys Asp Ile Gly Tyr Arg Lys Gly Gly Arg Leu Tyr Val Val Arg Asn
965 970 975
Gly Lys Ile Lys Ser Ile Asp Ala Asp Ile Asn Ala Ser Lys Asn Ile
980 985 990
Gly Glu Arg Phe Phe Asp Arg Asp Leu Ile Gln Thr Leu Ser Gly Val
995 1000 1005
Val Val Glu Asp Gln Ser Thr Val Tyr Ile Leu Gln Lys Arg Asn
1010 1015 1020
Val Ser Ser Asp Asn Arg Lys Arg Phe Tyr Lys Lys Phe Leu Glu
1025 1030 1035
Asp Val Gly Gly Lys Ser Lys Lys Asp Ala Val Leu Lys Met Gly
1040 1045 1050
Asp His Gly Glu Leu Glu Val Glu Arg Leu Ile Asp Gly Lys Lys
1055 1060 1065
Leu Asp Ile Asp Gly Lys Lys Ile Leu Val Asp Gly Glu Lys Val
1070 1075 1080
Pro Phe Arg Asn Thr Ser Val Tyr Tyr Ser Pro Lys Lys Lys Lys
1085 1090 1095
Trp Val Ser Lys Glu Leu Arg Cys Asn His Ile Lys Leu Thr Val
1100 1105 1110
Glu Glu Gln Asp Ile Lys
1115
<210> 9
<211> 1135
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.11的氨基酸序列
<400> 9
Met Asn Asn Tyr Asp Asn Tyr Leu Ser Asp Tyr Leu Ala Met Leu Pro
1 5 10 15
His Thr Lys Arg Thr Glu Ile Lys Lys Thr Ala Ser Lys Ile Ser Arg
20 25 30
Lys Leu Asn Gln Lys Glu Val Lys Lys Gln Ile Glu Arg Ser Glu Tyr
35 40 45
Ile Arg Ser Asn Cys Gly Tyr Ile Asn Ile Glu Arg Pro Gln Lys Ser
50 55 60
Leu Ser Phe Leu Ser Tyr Ser Thr Ile Lys Ser Ala Cys Met Ser Val
65 70 75 80
Asn Phe Arg Ala Phe Gln Asn Pro Ile Asn Asp Tyr Glu Thr Ala Ile
85 90 95
Cys Asn Gly Ile Asn Glu Cys Glu Arg Phe Phe Tyr Gln Gln Ile Asp
100 105 110
Ser Ile Tyr Met Ser Gln Ile Ile Glu Gln Leu Phe Asp Phe Tyr Ile
115 120 125
Ala Ser Arg Gln His Asp Met Phe Ile Asn Asn Thr Val Val Pro Tyr
130 135 140
Asp Val Asn Lys Leu Lys Ser Tyr Tyr Thr Ala Asn Glu Lys Tyr Ser
145 150 155 160
Phe Glu Gln Phe Cys Asp Asp Ile Lys Glu Phe Thr Asn Lys Gly Phe
165 170 175
Thr Ser Gly Gly Val Ser Cys Ile Leu Asn Leu Phe Tyr Lys Gly Ser
180 185 190
Val Lys Asp Ser Lys Asn Lys Lys Asp Tyr Ile Lys Ser Val Lys Arg
195 200 205
Leu Glu Thr Asn Gly Leu Phe Lys Lys Leu Asn Ile Phe Glu Lys Asn
210 215 220
Gly Ile Ser Lys Tyr Phe Ala Ala Ser Thr Leu Ser Thr Phe Phe Ala
225 230 235 240
Thr Ile Ser Ser Trp Lys Lys Gln Asn Asp Asp Trp Thr Gly Val Ala
245 250 255
Lys Asp Gly Thr Ser Leu Leu Ser Lys Leu Glu Asn Lys Thr Ile Thr
260 265 270
Leu Gln Ser Ile Ile Lys His His Arg Val Ile Asn Glu Leu Ala Val
275 280 285
Leu Ile Val Lys Ala Tyr Lys Asp Pro Val Lys Thr Leu Asn Asn Leu
290 295 300
Phe Glu Glu Arg Ser Asp Asn Asn Asn Asp Phe Lys Tyr Thr Cys Ser
305 310 315 320
Asp Asp Glu Asp Lys Tyr Pro Met Tyr Ile Lys Arg Glu Ile Ala Glu
325 330 335
Phe Val Lys Lys His Lys Thr Val Trp Glu Glu Ile Arg Tyr Phe Asp
340 345 350
Glu Ser Asp Thr Lys Lys Lys Lys Arg Asp Lys Lys Glu Ser Ser Ser
355 360 365
Asp Asp Lys Ser Tyr Leu Cys Cys Gly Asp Ser Trp Asp Tyr Leu Lys
370 375 380
Thr Trp Val Arg Leu Tyr Gly Glu Tyr Tyr Phe Phe Asp Asn Ala Leu
385 390 395 400
Asn Gln Phe Leu Arg Lys Pro Ser Ala Ser Met His Leu Tyr Thr Ser
405 410 415
Leu Asp Trp Ile Asn Lys Lys Thr Ile Cys Ile Val Gly Ala Asn Tyr
420 425 430
Tyr Lys Ile Gly Lys Val Glu Val Val Glu Arg Asn Asn Gln Arg Phe
435 440 445
Leu Leu Val Tyr Val Ser Val Pro Glu Met Glu Asn Tyr Ile Ile Ile
450 455 460
Pro Leu Gln Leu Asn Lys Tyr Phe Gly Asn Phe Gln Cys Lys Ile Phe
465 470 475 480
Glu Gly Arg Leu Gln Ala Ile Phe Lys Arg Tyr Ala Asn Phe Asn Ala
485 490 495
Leu Lys Asn Asn Lys Pro Gln Pro Ser Pro Asn Ile Ser Val Arg Ile
500 505 510
Asn Glu Phe His Phe Ala Leu Arg Ser Tyr Arg Lys Gln Gln Ile Ser
515 520 525
Ala Glu Asp Phe Ser Lys Gly Arg Phe Ser Leu Ile Ser Lys Ile Gly
530 535 540
Phe Gln Met Thr Asn Asp Glu Val Phe Gly Arg Thr Pro Arg Glu Ile
545 550 555 560
Ala Leu Val Lys Asp His Leu Ser Lys Gly Tyr Val His Phe Gly Ser
565 570 575
Gln Ile Ile Glu Asp Ser Arg Lys Glu Val Glu Gln Val Leu Lys Lys
580 585 590
Pro Met Ile Leu Met Gly Val Asp Phe Gly Tyr Ser Pro Leu Ala Ser
595 600 605
Tyr Asn Ile Lys Pro Leu Gln Thr Gly Lys Pro Ala Thr Asp Trp Val
610 615 620
Lys Asn Leu His Gly Asn Phe Leu Cys Gln Asn Val Ser Leu Gly Glu
625 630 635 640
Thr Ile Thr Glu Gly Glu Ile Gly Asp Val Pro Thr Asp Thr Tyr Thr
645 650 655
Ser Ser Asn Glu Ile Tyr Ser Ile Ala Thr Leu Thr Phe Arg Asn Ala
660 665 670
Asp Gly Lys Leu Glu Asn Arg Ser Phe Ser Arg Phe Tyr His Glu Leu
675 680 685
Asn Asn Thr Leu Asn Ile Ile Glu Gln Ile Lys Gly Thr Phe Asn Phe
690 695 700
Ile His Ser Ile Asn Thr Gln Phe Lys Glu Ile Lys Ala Leu Lys Thr
705 710 715 720
Thr Glu Glu Phe Ser Ser Tyr Val Ser Thr Leu Thr Trp Asp Gln Phe
725 730 735
Ile Glu Asp Ser Arg Lys Thr Ala Arg Tyr Ser Lys Tyr Trp Ile His
740 745 750
Ile Ile Asn Glu Asn Pro Lys Arg Arg Thr Ile Ala Thr Leu Asn Glu
755 760 765
Thr Leu Lys Leu Val Asp Glu Lys His Arg Phe Thr Val Thr Ile Gln
770 775 780
Glu Ile Phe Asp Leu Val Lys Tyr Cys Gln Gln His Gly Tyr Tyr Pro
785 790 795 800
Lys Ser Asn Val Met Ser Lys Leu Arg Asn Leu Ala Ile Lys Leu Ile
805 810 815
Asn Asp Leu Ile Arg Tyr Gln Lys Ile Gly Ile His Ser Cys Tyr Leu
820 825 830
Asp Phe Cys Val Leu Ile Lys Asn His Ile Ala Leu Leu Asn Ser Ser
835 840 845
Thr Ala Phe Ile Ile Asn Phe Ser Arg Asn Lys Glu Asn Ile Ile Arg
850 855 860
Asn Asn Thr Ser Lys Ile His Ser Leu Trp Val Tyr Arg Asp Asn Phe
865 870 875 880
Arg Arg Gln Met Ile Lys Asn Leu Cys Ser Gln Ile Leu Lys Ile Ala
885 890 895
Ala Lys Asn Lys Val His Ile Val Val Val Glu Lys Leu Asn Asn Met
900 905 910
Arg Thr Asn Asn Arg Asn Asn Glu Asp Lys Asn Asn Met Ile Asp Leu
915 920 925
Leu Ala Thr Gly Gln Phe Arg Lys Gln Leu Ser Asp Gln Ala Lys Trp
930 935 940
Tyr Gly Ile Ala Val Val Asp Thr Ala Glu Tyr Asn Thr Ser Lys Val
945 950 955 960
Asp Phe Met Thr Gly Glu Tyr Gly Tyr Arg Asp Glu Asn Asn Lys Arg
965 970 975
His Phe Tyr Cys Arg Lys Gln Asp Lys Thr Val Leu Leu Asp Cys Asp
980 985 990
Lys Lys Ala Ser Glu Asn Ile Leu Leu Ala Phe Val Thr Gln Ser Leu
995 1000 1005
Leu Leu Asn His Leu Lys Val Leu Ile Thr Glu Asp Gly Lys Thr
1010 1015 1020
Ala Val Ile Asp Leu Ser Glu Arg Thr Thr Glu Pro Gln Lys Ile
1025 1030 1035
Arg Ser Lys Ile Trp Thr Asn Ser Asp Val Gln Lys Ile Ile Phe
1040 1045 1050
Cys Lys Gln Glu Asn Gly Ser Tyr Val Leu Lys Lys Gly Ser Thr
1055 1060 1065
Asp Ile Lys Glu Lys Met His Lys Ala Val Leu His Arg His Gly
1070 1075 1080
Ser Leu Trp Tyr Asp Tyr Leu Asn His Lys Asn Met Ile Glu Asp
1085 1090 1095
Ile Lys Asn Leu His Leu Ser Asn Cys Ser Leu Thr Thr Ser Thr
1100 1105 1110
Asn Ser Asp Val Ile Asn Ser His Ser Gly Ser Ser Arg Ser Leu
1115 1120 1125
Asp Lys Thr Lys Thr Tyr Ala
1130 1135
<210> 10
<211> 1013
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.12的氨基酸序列
<400> 10
Met Ala Ser Ser Asp Ala Gln Lys Phe Pro Gln Thr His Asn Lys Val
1 5 10 15
Met Ser Phe Arg Leu Thr Ala Ser Asn Ile Gly Ser Val Leu Ser Leu
20 25 30
His Ser Asn Leu His Asp Ala Ala Glu Ile Gly Ile Asn Glu Cys Arg
35 40 45
Trp Trp Ile Gly Asp Gly Glu Ile Tyr Glu Arg Asp Pro Ala Cys Arg
50 55 60
Ser Ile Lys Lys Gly Asn Asp Ile Arg Thr Val Thr Ser Glu Lys Ile
65 70 75 80
Lys Glu Leu Trp Thr Lys His Thr Asp His Ser Val Pro Leu Val Asp
85 90 95
Phe Ile Asp Met Leu Lys Phe Val Ala Gln Cys Ala Ile Tyr Gly Asp
100 105 110
Ser Arg Ala Leu Ala Ser Thr Leu Phe Gly Lys Ser Lys Ala Glu Thr
115 120 125
Arg Gly Val Ser Thr Glu Asp Met Thr Val Ile Arg Ala Trp Ile Ala
130 135 140
Glu Thr Asp Ala Val Leu Ala Ser Gly Leu Ser Pro Lys Lys Lys Lys
145 150 155 160
Lys Lys Glu Lys Glu Ala Gly Lys Lys Glu Arg Lys Pro Asp Val Lys
165 170 175
Met Glu Met Cys Arg Arg Ile Arg Cys Thr Met Val Gln Cys Gly Tyr
180 185 190
Phe Arg Arg Phe Pro Phe Glu Ala Lys Ile Asp Asn Gly Gly Glu Arg
195 200 205
Gly Lys Met Asp Ser Glu Leu Ser Tyr Val Ser Ala Arg Asn Leu Leu
210 215 220
Arg Cys Leu Ser Thr Trp Arg Ala Ser Ser Val Met Arg Arg Asp Ser
225 230 235 240
Tyr Leu Ile Glu Glu Glu Arg Ile Lys Glu Ala Glu Ser Lys Met Thr
245 250 255
Pro Glu Ile Ile Asp Gly Leu Arg Arg Leu Tyr Arg Tyr Cys Ala Val
260 265 270
Asp His Asp Phe Leu Lys Trp Phe Gly Gly Arg Ile Ile Arg His Ile
275 280 285
Asp Ser Cys Leu Ala Pro Ala Ile Ala Gly Asn Thr Gly Arg Pro Thr
290 295 300
Gly Gly Glu Ser Phe Thr Val Ile Tyr Asp Arg Arg Lys Lys Arg Asp
305 310 315 320
Val Lys Ile Thr Tyr Ser Val Pro Glu Glu Ile Tyr Gly Tyr Leu Ser
325 330 335
Ser His Pro Glu Leu Val Ala Ile Gly Lys Asp Gly Met Thr Pro Ile
340 345 350
Ser Arg His Ala Asp Tyr Leu Glu Met Ile Ala Ser His Glu Lys His
355 360 365
Arg Trp Tyr Ala Thr Phe Pro Thr Val Gly Lys Glu Asp Gly Tyr Arg
370 375 380
Thr Ser Val Leu Leu Gly Lys Asn Tyr Leu Thr Tyr Asp Leu Ser Tyr
385 390 395 400
Asp Gly Glu Ser Val Pro Asp Lys Lys Ile Asn Val Ile Ser Lys Gly
405 410 415
Gln Pro Val Cys Leu Asp Leu His Asp Gly Arg Arg Val Ser Ser Leu
420 425 430
Tyr Leu Thr Val Gly Glu Ser Ala Ala Tyr Asp Ile Ala Val Arg Lys
435 440 445
Asn Lys Arg His His Gly Lys Pro Ala Asp Tyr Cys Arg Met Arg Val
450 455 460
His Leu Thr Gln Glu Arg Glu Asp Lys Thr Tyr Asn Asp Pro Tyr Phe
465 470 475 480
Ser Asn Met Glu Ile Trp Arg Ala Gly Asp Gln Val Tyr Ala Ile Glu
485 490 495
Phe Asp Arg His Gly Ala Arg Tyr Thr Ala Ile Val Lys Glu Pro Ser
500 505 510
Val Glu Tyr Arg Asn Lys Lys Leu Tyr Leu Arg Val Asn Met Val Leu
515 520 525
Asp Ser Pro Ser Arg Gln Asp Asp Lys Asp Met Tyr Tyr Ala Tyr Met
530 535 540
Thr Ala Tyr Pro Ser Ser Asn Pro Pro Val Glu Thr Ser Asp Asn Lys
545 550 555 560
Lys Arg Phe Glu Arg Leu Gly Pro Gly Arg Arg Ala Ile Gly Gly Ile
565 570 575
Asp Ile Gly Ile Gly Arg Pro Tyr Val Ala Val Val Ala Ser Tyr Glu
580 585 590
Val Gly Pro Ala Gly Thr Glu Gln Lys Phe Gln Ile Glu Asp Arg Leu
595 600 605
Ile Glu Asp Asp Gly Ser Ser Pro Tyr Asp Ser Leu Tyr Asn Asp Phe
610 615 620
Leu Thr Asp Ile Arg Thr Val Ser Arg Ile Ile Glu Ala Ala Lys Lys
625 630 635 640
Ile Ser Glu Gly Asp Leu Glu Asp Ile Pro Ser Asp Met Ser Val Asp
645 650 655
Glu Asp Gly Ser Ile Ala Ala Thr Met Lys Arg Met Ser Ala Arg Ile
660 665 670
Ala Glu Arg His His Leu Tyr Gly Glu Arg Lys Ser Glu Ala Tyr Ala
675 680 685
Thr Phe Leu Lys Met Asn His Lys Gln Arg Leu Asp Ile Leu Leu Thr
690 695 700
Gln Lys Ala Ser Asn Ala Thr Leu Lys Gln Leu Val Glu Glu Asp Pro
705 710 715 720
Ser Phe Leu Pro Arg Ile Cys Val Tyr Tyr Val Ile Ser Val Glu Arg
725 730 735
Glu Leu Lys Asn Lys His Arg Asn Ala Tyr Leu Asp Gly Leu Thr Val
740 745 750
Asp Glu Lys Tyr Ser Gly Glu Thr Lys Arg Gly Tyr Ala Gln Lys Arg
755 760 765
Leu Asn Ser Met Leu Arg Ala Tyr Ser Ala Leu Gly Glu Glu Glu Thr
770 775 780
Asp Glu Val Arg Thr Phe Ser Thr Arg Ser Glu Lys Val Arg Asn Met
785 790 795 800
Ala Lys Asn Ala Ile Lys Arg Asn Ala Arg Lys Leu Val Asn Phe Tyr
805 810 815
Val Gly Lys Gly Ile Arg Thr Ile Val Ala Glu Asp Thr Asp Pro Thr
820 825 830
Lys Ser Arg Asn Asp Gly Lys Lys Ser Asn Arg Ile Lys Ala Ala Trp
835 840 845
Ser Pro Lys Gln Phe Leu Ala Ala Val Lys Asn Ala Ala Gln Trp His
850 855 860
Gly Leu Glu Ile Ala Glu Val Asp Pro Arg Met Thr Ser Gln Val His
865 870 875 880
Pro Glu Thr Gly Leu Ile Gly Tyr Arg Asp Gly Asp Thr Leu His Cys
885 890 895
Pro Asp Gly Ser Lys Ile Asp Ala Asp Val Ala Gly Ala Ala Asn Val
900 905 910
Cys Arg Val Phe Ala Gly Arg Gly Leu Trp Arg Phe Ser Ile Asn Thr
915 920 925
Asn Ile Asp Ile Ser Asn Lys Asp Glu Lys Lys Arg Leu Arg Ala Tyr
930 935 940
Ile Val His His Phe Gly Ser Glu Ser Asn Trp Glu Lys Phe Arg Lys
945 950 955 960
Gln Tyr Pro Ser Gly Thr Thr Leu Tyr Leu His Gly Arg Glu Trp Leu
965 970 975
Thr Ala Glu Glu His Lys Ser Ala Ile Asp Arg Ile Arg Asp Asp Val
980 985 990
Gly Arg Asp Ala Glu Asn Asp His Val Ala Ile Val Thr Ala Ala Glu
995 1000 1005
Lys Val Glu Ile Phe
1010
<210> 11
<211> 1052
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.13的氨基酸序列
<400> 11
Met Ser His Asp Leu Lys Pro Gln Arg Leu Ile Arg Ser Asn Ile Thr
1 5 10 15
Lys Thr His Ser Asp Gln Asn Ala Lys Gln Val Ala Glu Glu Val Lys
20 25 30
Lys Glu His Leu Asn Tyr Leu Leu Ile Lys Asn Glu Met Leu Ile Ser
35 40 45
Ile Val Pro Glu Ala Lys Asp Asp Asp Gly Asn Asp Ile Asp Phe Lys
50 55 60
Lys Gln Leu Lys Ser Leu Tyr Lys Glu Thr Asp Gln Ser Val Ser Phe
65 70 75 80
Ser Val Phe Cys Gln Met Met Lys Phe Arg Asn Ile Ala Leu Leu Tyr
85 90 95
Ala Lys Gly Gln Ser Arg Trp Ala Val Ser Ser Tyr Phe Thr Gly Asn
100 105 110
Arg Arg Lys Asp Asp Tyr Ala Lys Asp Leu Ser Leu Leu Asp Glu Ala
115 120 125
Ile Glu Leu Leu Glu Cys Lys Arg Arg Lys Lys Ala Glu Glu Glu Asn
130 135 140
Glu Glu Glu Asn Glu Thr Pro Lys Lys Lys Glu Asp Asn Pro Ser Asn
145 150 155 160
Ile Ser Glu Glu Gln Ile Met Lys Leu Phe Tyr Ala Val Asn Lys Lys
165 170 175
Leu Lys Glu Ile Gly Tyr Leu Asp Arg Tyr Ser His Ile Glu Lys Gln
180 185 190
Glu Gln Tyr Ala Ile Ile Gly Val Thr Ser Arg Thr Val Lys Ala Trp
195 200 205
Asp Tyr Ala Asn Phe Ala Thr Arg Asn His Tyr Gln Ser Val Gln Asn
210 215 220
Glu Tyr Gln Lys Lys Leu Lys Ala Leu Pro Gly Thr Lys Lys Asp Lys
225 230 235 240
Val Cys Leu Glu Lys Phe Phe Asp His Leu Asn Glu Asn Asn Ile Ala
245 250 255
Ala Asp Trp Asp Lys Trp Arg Leu Lys Lys His Ile Leu Gln Cys Ile
260 265 270
Ile Pro Ala Ala Lys Ile Gly Leu Lys Glu Leu Lys Gln Ser Phe Tyr
275 280 285
Val Asp Asn Lys Gly Asn Lys His Asn Tyr Phe Val Asn Gly Leu Tyr
290 295 300
Glu Glu Ile Leu Lys Arg Pro Phe Leu Tyr Ser Ala Glu Asp Pro Glu
305 310 315 320
Glu Ser Ile Leu Tyr Leu Gly Val Glu Val Ala Ser Leu His Ser Lys
325 330 335
Leu Asn His Leu Arg Ser Glu Ala Arg Phe Ser Phe Glu Thr Pro Asp
340 345 350
Asp Ile Cys Lys Tyr Met Thr Ile Cys Gly Asp Asn Tyr His Asn Phe
355 360 365
Thr Met Ser Ala Ile Gly Glu Asp Val Glu Asp Ile Glu Val Glu Val
370 375 380
Tyr Asp Tyr Asn His Ser Lys Lys Tyr Glu Thr Met Arg Phe Ile Asn
385 390 395 400
Gly Lys Arg Thr Thr Asp Leu Ser Leu Asn Phe Lys Gly Ile Pro Val
405 410 415
Arg Leu Cys Leu Glu Gly Lys Arg Asn Asn Ser Tyr Phe Ala Asp Ala
420 425 430
Ile Val Trp Glu Leu Asp Asn Lys Asp Lys Thr Gly Tyr Leu Ile Glu
435 440 445
Tyr Gly Lys Ser Asn Asn Arg Leu Tyr Met Leu Val Lys Glu Pro Leu
450 455 460
Ile Gly Cys Arg Arg Lys Phe Gly Lys Asp Val Leu Phe Val Ser Leu
465 470 475 480
Ser Gly Thr Leu Val Asn Lys Tyr Ile Glu Asp Asp Ile Val Ser Ala
485 490 495
Arg Tyr Leu Met Gln Thr Ala Ala Pro Ile Phe Lys Thr Ser Arg Ala
500 505 510
Lys Lys Gln Asp Lys Ile Gly Asp Lys Trp Phe Glu His Cys Gln Gly
515 520 525
Ser Thr Ile Lys Ile Ala Gly Ile Asp Ile Gly Ile Asn Pro Ile Ala
530 535 540
Ala Ile Thr Val Ala Asn Val Thr Phe Asp Arg Ala Leu Gly Asn Lys
545 550 555 560
Ile Lys Asn Gln Lys Gln Ile Val Ile Asp Cys Tyr Ala Glu Asp Tyr
565 570 575
Lys Ile Asp Pro Val Val Val Lys Arg Met Glu Asp Ile Arg His Ile
580 585 590
Lys Tyr Thr Ile Asn Ser Trp Tyr His Leu Ala Asp Cys Cys Arg Leu
595 600 605
Lys Ala Ala Asn Lys Glu Tyr Val Val Asn Glu Arg Lys Gln Gly Phe
610 615 620
Phe Arg Glu Asn Ile Glu Tyr Leu Lys Glu Val Ala Lys Lys Ala Ile
625 630 635 640
Thr Glu Ser Asp Gln Gln Ile Lys Glu Gln Lys Ala Ala Leu Lys Arg
645 650 655
Phe Asp Gly Glu Lys Lys Lys Glu Ile Gln Ala Thr Ile Asn Gly Phe
660 665 670
Asn Leu Lys Ile Lys Ile Leu Lys Lys Phe Val Arg Gln Ser Ala Lys
675 680 685
Lys Ile Phe Asp Ser Thr Leu Glu Thr Leu Glu Lys Tyr Asp Asn Asn
690 695 700
Ile Glu Gln Ala Lys Arg Asp Arg Glu Phe Gly Leu Lys Ile Ile Tyr
705 710 715 720
Asp Leu Ile Ile Lys Tyr Tyr Lys Arg Ser Lys Lys Glu Arg Glu Met
725 730 735
Asn Gln Arg Ile Tyr Val Asp Asp Tyr Asn Gln Glu Glu Ile Asp Thr
740 745 750
Glu Arg Thr Lys Lys Ile Arg Lys Glu Thr Ile Thr Phe Cys Asp Asn
755 760 765
Asp Trp Asn Ser Leu Thr Lys Arg Ile His Asp Leu Glu Lys Lys Met
770 775 780
Lys Lys Ile Gly Ile Ser Glu Pro Gly Arg Val Glu Gln Glu Ile Asn
785 790 795 800
Asp Arg Asp Tyr Tyr Asn Asn Ile Gln Asp Asn Thr Lys Lys Arg Gln
805 810 815
Ala Lys Ile Ile Val Asp Ala Leu Lys Glu Glu Gly Val Ser Ile Ile
820 825 830
Val Val Glu Asp Leu Thr Gly Gly Gly Ser Glu Asn Thr Lys Glu Ile
835 840 845
Asn Lys Ser Phe Asp Ala Phe Ala Pro Ile Arg Phe Leu Asn Ala Leu
850 855 860
Lys Asn Cys Ala Glu Thr Asn Gly Ile Gln Val Thr Glu Val Leu Ser
865 870 875 880
Pro Met Ser Ser Lys Met Val Pro Ser Thr Gly Glu Ile Gly His Arg
885 890 895
Asp Lys Arg Asp Lys Gln Leu Tyr Tyr Lys Asp Gly Glu Glu Leu Lys
900 905 910
Ser Ile Asp Gly Asp Ile Ser Ala Ser Glu Ile Leu Leu Arg Arg Gly
915 920 925
Val Ser Arg His Thr Glu Leu Ile Gly Thr Met Asn Val Glu Asp Val
930 935 940
Leu Asp Lys Asn Asn Asn Lys Asn Lys Cys Ile Lys Gly Tyr Val Cys
945 950 955 960
Asn Arg Trp Gly Asn Ile Gln Asn Phe Glu Lys Ile Leu Lys Glu Lys
965 970 975
Gly Ile Gly Glu Arg Glu Ile Ile Tyr Leu His Gly Asp Lys Ile Leu
980 985 990
Thr Met Asp Glu Lys Arg Thr Leu Gln Ala Ser Ile Arg Lys Glu Leu
995 1000 1005
Lys Glu Met Arg Glu Arg Glu Ser Gly Glu Glu Asn Ala Gly Thr
1010 1015 1020
Ala Arg Lys Lys Ser Lys Pro Lys Lys Lys Lys Lys Ile Lys Arg
1025 1030 1035
Asn Asn Asp Gln Asp Leu Ser Asn Asn Arg Pro Ala Ala Ser
1040 1045 1050
<210> 12
<211> 1045
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.14的氨基酸序列
<400> 12
Met Lys Glu Asn Lys Met Lys Glu Asn Gly Ser Met Thr Thr His Ser
1 5 10 15
Lys Val Ile Ala Leu Lys Met Lys Ser Glu Asn Val Glu Phe Asp Thr
20 25 30
Phe Tyr Lys Glu Ser Phe Glu Leu Phe Lys Gln Phe Thr Asn Glu Phe
35 40 45
Val Ala Trp Gly Asn Asp Glu Ile Tyr Gln Tyr Gly Ser Ser Lys Arg
50 55 60
Lys Lys Asp Asp Gln Lys Ile Ser Leu Ile Pro Val Ile Glu Asp Ile
65 70 75 80
Tyr Lys Ser Val Glu Lys Lys Ala Thr Ala Glu Gly Ile Ser Lys Thr
85 90 95
Asp Phe Arg Ala Val Leu Lys Tyr Leu Tyr His Gln Ile Ile Asn Val
100 105 110
Gly Asn Ser Gly Arg Ser Tyr Gly Thr Ser Leu Phe Gly Gly Cys Glu
115 120 125
Val Lys Glu Lys Leu Ser Lys Gln Asp Ile Ser Asn Ile Val Glu Cys
130 135 140
Val Lys Glu Leu Glu Leu Cys Lys Ser Lys Gln Glu Glu Ser Asp Ala
145 150 155 160
Tyr Asp Lys Ile Leu Leu Lys Glu Lys Ile Thr His Ile Val Lys Ser
165 170 175
Gly Glu Thr Ala Gly Asp Ile Thr Lys Lys Tyr Asn Gln Ala Thr Thr
180 185 190
Gly Arg Lys Thr Ser Ser Lys Gly Phe Phe Asp Lys Ser Thr Lys Thr
195 200 205
Glu Val Lys Tyr Lys Asp Ile Lys Asp Asp Thr Leu Leu Gln Asp Gly
210 215 220
Ser Thr Ile Phe Ile Lys Ser Ser Val Asp Leu Phe Val Lys Lys Val
225 230 235 240
Cys Asn Thr Leu Arg Glu Ile Asn Phe Phe Asp Arg Leu Pro Phe Lys
245 250 255
Asn Asn His Ser Asn Asn Tyr Gly Leu Leu Phe Ser Met Leu Ser Gln
260 265 270
Ile Glu Ser Trp Lys Thr Ile Ser Glu Thr Thr Lys Lys Ser His Glu
275 280 285
Glu His Gly Glu Lys Ile Ala Ser Met Val Lys Lys Leu Asp Leu Thr
290 295 300
Gln Thr Glu Leu Met Lys Asp Phe Ala Ala Phe Cys Ile Glu Asn Asn
305 310 315 320
Ile Thr Lys Lys Phe Asp His Lys Phe Lys Arg His Met Glu Asp Cys
325 330 335
Val Ile Pro Ser Phe Lys Asn Gly Lys Ile Pro Asp Lys Leu Phe Tyr
340 345 350
Phe Asn Ile Ile Leu Ala Lys Lys Thr Asp Glu Gln Ile Asp Tyr Ser
355 360 365
Leu Ser Ser Glu Phe Tyr Thr Lys Leu Phe Ser Met Pro Asn Leu Trp
370 375 380
Gln Glu Glu Glu Ala Phe Ile Val Lys Asn Ile Asn Leu Ile Glu Glu
385 390 395 400
Ile Thr Ile Phe Asn Lys Arg Arg Asn Tyr Ala Cys Cys Pro Leu Ile
405 410 415
Lys Glu Lys Glu Tyr Asp Arg Phe Gln Ile Gln Leu Asn Glu Thr Asn
420 425 430
Phe Leu Lys Phe Gln Phe Asp Pro Lys Asn Val Val Asn Ile Asp Glu
435 440 445
Asn Thr Thr Glu Ala Thr Val Gly Phe Asp Glu Lys Leu Lys Leu Val
450 455 460
Val Cys Ala Asp Lys Lys Tyr Ala Phe Ser Ile Phe Thr Gln Cys Lys
465 470 475 480
Tyr His Gly Asn Lys His Lys Pro Asn Thr Tyr Phe Asn Asn Leu Lys
485 490 495
Ile Ile Lys Val Ile Glu Ser Lys Ser Asn Ser Val Lys Ser Met Lys
500 505 510
Tyr Thr Phe Glu Phe Thr Lys Arg Asn Glu Leu Lys Arg Ala Glu Ile
515 520 525
Lys Gln Pro Ser Ile Val Tyr Lys Asn Asn Asn Tyr Tyr Ile Arg Ile
530 535 540
Asn Met Asn Val Ile Leu Asp Ala Asp Gln Thr Ser Tyr Lys Ile Ile
545 550 555 560
Asn Asn Asn Gln Thr Ala Ser Leu Pro Ser Tyr Phe Gln Ser Ser Leu
565 570 575
Pro Phe Glu Asn Asn Arg Gly Lys Ile His Asp Lys Gly Ile Val His
580 585 590
Trp Glu Lys Ile Lys Asn Arg Lys Ile Ile Ala Met Gly Val Asp Leu
595 600 605
Gly Val Arg Arg Pro Phe Ser Tyr Ala Ile Gly Asn Phe Thr Leu Asn
610 615 620
Lys Asp Ile Leu Asp Lys Asn Asp Val Asn Ile Val Ala Ser Gly Phe
625 630 635 640
Asn Leu Cys Ser Asp Ser Asp Val Tyr Phe Gln Val Phe Asn Gln Ile
645 650 655
Lys Thr Leu Ala Lys Phe Ile Gly Lys Leu Lys Ser His Asn Lys Gly
660 665 670
Leu Lys Val Asp Phe Glu Lys Asp Lys Lys Tyr Ile Phe Asp Leu Val
675 680 685
Asn Asp Ala Lys Ala Tyr Phe Lys Asp Met Ser Ala Lys Arg Ile Asn
690 695 700
Asp Thr Lys Asp Asn Ile Ser Asn Thr Val Thr Asn Lys Glu Arg Ile
705 710 715 720
Tyr Gly Ser Phe Val Ser Glu Ser Ala Glu Ser Ala Ile Gln Cys Ala
725 730 735
Ile Asp Arg Ser Glu Lys Glu Ser Gly Leu Thr Leu Lys Lys Asp Ile
740 745 750
Ser Trp Leu Val Asn Val Leu Ser Lys Tyr Leu Glu Arg Lys Phe Lys
755 760 765
Glu Val Lys Asn Asn Arg Lys Tyr Thr Asn Val Asn Lys Cys Asp Asn
770 775 780
Cys Phe Asn Trp Leu Arg Val Ile Glu Asn Ile Lys Arg Leu Lys Arg
785 790 795 800
Ser Ile Ser Tyr Leu Gly Glu Asp Leu Gln Lys Asn Pro Glu Leu Lys
805 810 815
Ile Glu Leu Lys Asn Leu Asn Glu Tyr Gly Asn Asn Val Lys Ser Asp
820 825 830
Phe Leu Lys Gln Ile Ala Ser Asn Ile Ile Lys Val Ala Ile Glu His
835 840 845
Lys Cys Asp Ile Val Phe Ile Glu Lys Leu Gly Lys Ala Asp Ser Arg
850 855 860
Ser Arg Lys Leu Asn Glu Met Phe Ser Phe Trp Ser Pro Lys Ala Ile
865 870 875 880
Lys Lys Ala Ile Glu Asn Ala Ala Ser Trp His Gly Ile Pro Val Val
885 890 895
Glu Val Asp Pro Ser Cys Thr Ser Lys Val His Tyr Glu Thr Asn Leu
900 905 910
Phe Gly His Arg Ile Gly Asn Asp Leu Tyr Tyr Val Glu Asp Gln Cys
915 920 925
Leu Lys Lys Val Asp Ala Asp Ile Asn Ala Ala Lys Gln Ile Leu Val
930 935 940
Arg Gly Ala Thr Arg His Gly Asn Ile Ser Ser Ile Asn Ile Lys Tyr
945 950 955 960
Leu Gln Ala Lys Ile Ala Glu Leu Asn Ser Glu Ala Asn Ser Glu Glu
965 970 975
Asp Lys Glu Glu Ile Lys Gln Gly Gly Lys Arg Ile Gln Gly Phe Leu
980 985 990
Trp Lys Lys Tyr Gly Asn Ile Thr Asn Ile Thr Asn Gln Leu Thr Ala
995 1000 1005
Ala His Lys Glu Arg Glu Ser Lys Phe Asp Tyr Ile Tyr Leu His
1010 1015 1020
Asn Asp Lys Trp Ile Ala Tyr Glu Asp Arg Asn Glu Ile Lys Lys
1025 1030 1035
Asp Ile Glu Lys Arg Leu Glu
1040 1045
<210> 13
<211> 895
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.15的氨基酸序列
<400> 13
Met Thr Ala Lys Lys Thr Ala Lys Lys Tyr Phe Pro Pro Lys Cys Leu
1 5 10 15
Arg Ser Ser His Phe Lys Ile Tyr Gly Ile Pro Thr Ala Ile Arg Ala
20 25 30
Leu Glu Glu Thr Asn Thr Phe Val Asn Lys Ala Ala Ala Asp Leu Met
35 40 45
Glu Met Phe Phe Leu Met Arg Gly Gln Pro Tyr Arg Arg Arg Ile Gly
50 55 60
Ser Glu Glu Lys Gln Val Thr Gln Glu His Ile Asp Ala Arg Leu Arg
65 70 75 80
Val Leu Val Gly Asp Tyr Ser Leu Asn Glu Val Lys Pro Leu Leu Arg
85 90 95
Gln Leu Tyr Asp Gly Ile Lys Ala Lys Gln Asn Tyr Ala Pro Thr His
100 105 110
Phe Val Arg Phe Phe Ile Gln Pro Thr Lys Gly Ala Ile Asp Lys Lys
115 120 125
Ser Pro Val Ser Gln Arg Ala Lys Lys Ala Gly Gln Lys Leu Gln Lys
130 135 140
Met Gly Val Leu Pro Ile Leu Pro Leu Ser Pro Gly Phe Lys Phe Trp
145 150 155 160
Thr Ala Ala Met Met Met Ala Cys Ser Arg Met Asn Ser Trp Glu Ala
165 170 175
Cys Asn Glu Lys Thr Ile Glu Asn His Lys Ala Phe Leu Glu Gly Ile
180 185 190
Glu Asn Tyr Lys Lys Glu Ile Arg Phe Glu Asp Leu Cys Glu Glu Trp
195 200 205
Ser Leu Phe Ser Asp Trp Leu Thr Glu Ala Glu Ser Asp Asn Glu Gly
210 215 220
Gly Cys Lys Phe Lys Leu Thr Pro Arg Phe Leu Gln Arg Trp Glu Arg
225 230 235 240
Ile Tyr Leu Lys Gln Met Arg Lys Gly Lys Ile Pro Ala Arg His Asn
245 250 255
Leu Gly Pro Val Met Glu Ala Leu Ala Gly Asp Lys Tyr Arg Gln Leu
260 265 270
Trp Asp Asn Gly Glu Glu Arg Asp Tyr Ile Thr Glu Leu Gly Asp Leu
275 280 285
Val Thr Ser Gln Arg Lys Ala Val Arg Leu Ser Arg Asp Ser Ala Val
290 295 300
Thr Phe Pro Asp Glu Glu Leu Ser Pro Val Gly Thr Glu Phe Gly His
305 310 315 320
Asn Tyr Met Ser Phe Ser Ile Asp Gln Glu Asn Ser His Leu Val Thr
325 330 335
Leu Glu Val Ile Gly Gly Lys Tyr Gln Phe Glu Ile Ser Lys Ser Asp
340 345 350
Tyr Phe Arg Asp Leu Ile Val Glu Glu Ala Gly Lys Gln Ser Lys Phe
355 360 365
Tyr Asn Val Ser Tyr Arg Lys Gly Asn Val Arg Glu Glu Asn Leu Ala
370 375 380
Gly Asp Phe Lys Glu Ala Thr Val Arg Asn Arg Arg Ser Leu Lys Thr
385 390 395 400
Gly Lys Arg Arg Leu Tyr Phe Tyr Met Ser His Ser Ile Pro Thr Arg
405 410 415
Phe Asp Asp Asp Leu Tyr Ala Gln Phe Thr Glu Lys Gly Gln Pro Asp
420 425 430
Phe Ser Lys Leu Tyr Lys Ala Val Thr Tyr Phe Gln Cys Ser Leu Gly
435 440 445
Asn Lys Lys Ala Asp Thr Tyr Arg Val Tyr Val Lys Met Gly Thr Arg
450 455 460
Phe Leu Gly Val Asp Ile Gly Val Ser Arg Leu Phe Gly Phe Ser Leu
465 470 475 480
Phe Glu Leu Arg Glu Glu Lys Pro Glu Lys Asn Pro Phe Phe Glu Leu
485 490 495
Pro Asp Asp Leu Gly Tyr Ala Val Cys Leu Glu Ser Trp Val Asp Gly
500 505 510
Val Glu Lys Asn His Lys Val Ala Gln Glu Met Lys Asp Trp Arg Arg
515 520 525
Glu Cys Leu Ala Ala Gln Arg Leu Ile His Tyr Ala Lys Phe Leu Lys
530 535 540
Lys Arg Asp Lys Asn Glu Glu Ile Asp Tyr Lys His Glu Glu Ser Leu
545 550 555 560
Glu Thr Ile Ala Gly Leu Leu Gly Ile Glu Ile Asp Pro Glu Gln Ile
565 570 575
Ile Asp Val Pro Leu Lys Leu Leu Asp Leu Val Gly Gln Ala Ile Gly
580 585 590
Ala Leu Arg Lys Lys Tyr Leu Val Leu Lys Lys Asn Glu Val Arg Gln
595 600 605
Gly Arg Ile Thr Ser Glu Leu Phe Leu Trp Pro Glu Cys Val Asp Thr
610 615 620
Tyr Ile Arg Leu Leu Lys Ser Trp Thr Tyr Lys Asp Lys Lys Pro Tyr
625 630 635 640
Gln Lys Gly Glu Thr Asn Lys Asp Ala Phe Lys Lys Leu Lys Gly Tyr
645 650 655
Leu Ala Arg Leu Arg Lys Asp Leu Ala Pro Lys Tyr Ala Ala Val Ile
660 665 670
Ala Asp Ala Ala Ile Arg His Lys Val His Val Val Val Ala Glu Asn
675 680 685
Leu Glu Gln Phe Gly Leu Ser Met Lys Asn Glu Lys Asp Leu Asn Arg
690 695 700
Val Leu Ala His Trp Ser His Gln Lys Ile Trp Ser Met Val Glu Glu
705 710 715 720
Gln Leu Arg Pro Tyr Gly Ile Met Val Val Tyr Val Asp Pro Arg His
725 730 735
Thr Ser Lys Leu Asp Phe Ala Thr Asp Glu Phe Gly Gly Arg Cys Phe
740 745 750
Thr Ser Leu Tyr Val Met Arg Asp Gly Lys Lys Thr Thr Thr Asp Thr
755 760 765
Glu Lys Asn Ala Ser Gln Asn Ile Pro Lys Lys Phe Leu Thr Arg His
770 775 780
Arg Asn Val Ser Trp Leu Leu Ala Tyr Ala Val Asp Leu Ser Asp Ser
785 790 795 800
Gln Lys Lys Lys Leu Gly Ile Gly Asp Glu Lys Val Trp Leu Pro Asn
805 810 815
Met Gly Leu Met Ile Ser Gly Ala Leu Lys Ala Lys His Gly Lys Asn
820 825 830
Ser Ala Leu Leu Val Glu Asp Gly Glu Asn Tyr Arg Leu Leu Pro Ile
835 840 845
Thr Ala Ala Gln Ala Lys Lys Phe Val Val Lys Arg Lys Lys Glu Glu
850 855 860
Phe Tyr Arg His Gly Glu Ile Trp Leu Thr Lys Glu Ala His Lys Ala
865 870 875 880
Arg Ile Glu Tyr Leu Phe Pro Glu Ser Lys Lys Gly Arg Lys Ser
885 890 895
<210> 14
<211> 956
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.16的氨基酸序列
<400> 14
Met Lys Lys Thr Asn Tyr Lys Thr Ser His Leu Leu Ile Asp Asn Pro
1 5 10 15
Pro Gln Ser Ile Ile Asp Leu His Arg Asp Val Ile Glu Ile Gly Ser
20 25 30
Tyr Leu Thr Lys Phe Phe Leu Ala Cys Leu Gly Arg Pro Val Asp Ser
35 40 45
Thr Ile Leu Ser Glu Pro Ala Leu His Phe Gln Phe Val Asn Gly Ile
50 55 60
Leu Pro Val Lys Asn Gly Pro Gly Ala Asp Asp Ser Ser Trp Arg His
65 70 75 80
Ser Glu Asn Cys Tyr Ser Met Leu Phe Glu Lys Asn Ser Lys Ser Gly
85 90 95
Lys Ser Asp Gly Lys Val Arg Gln Val Arg Glu Leu Lys Val Ala Leu
100 105 110
Phe Gly Lys Lys Glu Lys Gly Lys Gly Ile Val Gly Lys Lys Thr Trp
115 120 125
Asp Glu Leu Lys Val Val Leu Glu Ala Leu Pro Glu Glu His Gln Ile
130 135 140
Leu Ser Leu Glu Ile Cys Gln Arg His Tyr Glu Ser Arg Asp Val Lys
145 150 155 160
Ala Phe Gly Lys Leu Ala Leu Ser Ser Lys Ser Arg Pro Ser Val Glu
165 170 175
Ala Gly Leu Lys Leu Arg Glu Leu Gly Leu Leu Pro Leu Asp Ser Arg
180 185 190
Gly Leu Asp Lys Asn Lys Leu Leu Gly Ile Leu Ala Ala Val Thr Gly
195 200 205
Arg Leu Lys Ser Trp Arg Asp Arg Asp Cys Ala Cys Lys Ala Asp Lys
210 215 220
Gln Ala Leu Arg Val Lys Phe Glu Glu Arg Leu Ser Lys Val Asp Gln
225 230 235 240
Ser Ala Tyr Gln Gln Phe Lys Gln Phe Ala Asp Glu Leu Leu Thr Gln
245 250 255
Glu Gly Tyr Arg Ile Ser Gly Arg Val Leu Arg Ala Val Glu Lys Lys
260 265 270
Asp Ser Asp Tyr Ser Pro Val Leu Thr Val Leu Ala Lys Tyr Pro Asp
275 280 285
Leu Gln Asp Asn Phe Glu Glu Leu Cys Arg Ala Cys Leu Ala Glu Gln
290 295 300
Ala Phe Asn Lys Lys Lys Ala Asp Ala Arg Val Thr Val Cys Ser Glu
305 310 315 320
Thr Ser Pro Leu Gln Phe Pro Phe Gly Met Thr Gly Asn Gly Tyr Pro
325 330 335
Phe Thr Leu Ser Ala Cys Glu Gly Arg Ile Asn Ala Thr Ile His Phe
340 345 350
Pro Gly Gly Asp Leu Pro Leu Arg Leu Arg Lys Ser Lys Tyr Phe Gln
355 360 365
Asn Pro Glu Ile Leu Pro Val Lys Asp Gly Phe Gln Ile Thr Phe Thr
370 375 380
Arg Gly Lys Thr Pro Leu Val Gly Thr Ile Lys Glu Pro Ser Leu Leu
385 390 395 400
Lys Lys Asn Asn His Tyr Tyr Leu Ser Leu Arg Val Asn Val Pro Ser
405 410 415
Val Lys Ile Pro Lys Glu Val Arg Asp Thr Arg Ala Tyr Tyr Ser Ser
420 425 430
Ala Val Gly Gly Asp Glu Thr Thr Pro Val Pro Val Lys Ala Val Ala
435 440 445
Ile Asp Leu Gly Val Thr Thr Leu Ala Asp Tyr Ser Ile Ile Asp Thr
450 455 460
Cys Leu Pro Gly Asp Cys Lys Val Phe Gly Gly Glu Thr Ala Ala Phe
465 470 475 480
Thr Ala His Gly Lys Ile Gly Gln Cys Ala Asn Lys Ser Leu Arg Asp
485 490 495
Arg Leu Tyr Lys Asn Thr Glu Glu Ala Leu Phe Leu Gly Lys Phe Ile
500 505 510
Arg Leu Ser Lys Lys Leu Arg Asp Gly Glu Gly Leu Asn Arg Trp Glu
515 520 525
Val Glu Lys Leu Pro Gly Tyr Ala Glu Arg Leu Gly Ile Thr Gln His
530 535 540
Leu Asp Asn Ala Tyr Thr Arg Lys Asp Glu Ile Ala Arg Lys Phe Lys
545 550 555 560
Gln Ile Lys Gly Asn Phe Asp Lys Leu Val Ser Glu Phe Ala Leu Arg
565 570 575
Asp His Pro Ser Lys Lys Gly Glu Ser Trp Glu Thr Ile Ser Ala Glu
580 585 590
Thr Ile Gln Val Leu Ala Ala Leu Lys Arg Ile Gln Ser Leu Leu Lys
595 600 605
Ser Trp Thr Tyr Tyr Ser Trp Thr Ala Glu Asp Tyr Val Leu Ala Leu
610 615 620
Thr Ala Asp Gly Pro Val Cys Ile Asp Gly Glu His Val Lys Ala Val
625 630 635 640
Thr Ala Thr Ser Arg Arg Ser Phe Ala Pro Cys Gly Lys Ala Ala Leu
645 650 655
Leu Arg Leu Ile Glu Ser Gly Glu Ile Val Glu Thr Gly Gly Gln Tyr
660 665 670
Gln Leu Ala Thr Gly Val Lys His Arg Asn His Pro Val Asn Phe Leu
675 680 685
Ser Ser Tyr Ile Lys His Phe Asn Gly Leu Arg Arg Asp Leu Thr Asn
690 695 700
Lys Leu Val Arg Ala Ile Val Asn Lys Ala Gln Glu Tyr Arg Val Gln
705 710 715 720
Ile Val Ile Val Glu Asp Phe Gly Ile Ala Asp Leu Glu Asp Arg Ile
725 730 735
Lys Asp Ala Tyr Glu Asn Tyr Arg Trp Asn Leu Phe Ala Pro Ala Thr
740 745 750
Ile Val Lys Lys Leu Glu Ala Ala Leu Leu Glu Val Gly Ile Ala Met
755 760 765
Ala Gln Val Asp Pro Arg His Thr Ser Gln Ile Ala Pro Thr Gly Ala
770 775 780
Phe Gly Phe Arg Asp His Ala Phe Leu Tyr Tyr Gln Asp Asp Gly Leu
785 790 795 800
Cys Arg Ile Asp Ala Asn Thr Asn Ala Ser Met Arg Ile Ala Glu Arg
805 810 815
Phe Phe Met Arg His Ser Val Leu Thr Gln Leu Arg Ala Ala Lys Ile
820 825 830
Gly Glu Thr Glu Tyr Leu Ile Pro Glu Ser Ala Ser Lys Arg Leu Asn
835 840 845
Ala Phe Val Lys Leu Gln Thr Gly Lys Pro Phe Ala Lys Leu Ile Met
850 855 860
Asn Cys Ser Gly Phe Val Leu Glu Gly Leu Thr Lys Lys Gln Tyr Ala
865 870 875 880
Lys Leu Ala Glu Thr Ala Gly Lys Lys Glu Ser Phe Tyr Gln Tyr Asp
885 890 895
Asp Arg Trp Phe Asp Lys Gly His His Phe Ala Cys Arg Ala Thr Leu
900 905 910
Glu Asn Lys Val Gln Val Cys Leu Asn Gly Gly Gly Arg Ile Lys Asp
915 920 925
Thr Thr Pro Asp Phe Asn Pro Lys Ser Leu Leu Arg Ser Asp Leu Gln
930 935 940
Thr Pro Leu Asp Gln Leu Phe Gly Asn Ser Gly Ala
945 950 955
<210> 15
<211> 946
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.17的氨基酸序列
<400> 15
Met Ser Asn Thr Thr Tyr Lys Thr Ser His Leu Leu Ile Asp Leu Pro
1 5 10 15
Gln Gln Glu Leu Ile Asp Leu His Arg Asp Ser Asn Glu Met Gly Ser
20 25 30
Tyr Leu Thr Lys Phe Phe Leu Ala Ala Leu Gly Arg Pro Val Asp Asn
35 40 45
Ser Ile Val Leu Pro Pro Glu Leu Ala Asp Leu Tyr Phe Gln Phe Ala
50 55 60
Asn Gly Ile Leu Pro Val Asp Lys Gly Pro Gly Ser Asp Asp Pro Ser
65 70 75 80
Trp Leu His Ser Glu Asn Cys Tyr Ser Met Phe Phe Glu Lys Asp Ser
85 90 95
Met Ser Gly Asn Cys Thr Asn Lys Ile Lys Gln Tyr Gln Glu Leu Lys
100 105 110
Thr Ala Leu Cys Gly Gln Lys Val Lys Gly Gln Lys Gly Leu Val Gly
115 120 125
Lys Lys Thr Trp Ala Gln Leu Lys Lys Val Leu Thr Ala Leu Pro Gln
130 135 140
Lys Tyr Gln Ile Leu Ser Pro Lys Ile Cys Gln Lys Tyr Phe Lys Ser
145 150 155 160
Gly Asn Leu Glu Gly Phe Gly Lys Leu Ala Leu Ala Gly Lys Asn Arg
165 170 175
Pro Ser Met Ser Ala Gly Leu Gln Leu Arg Glu Leu Gly Leu Leu Pro
180 185 190
Leu Asp Ser Arg Gly Ile Asp Lys Asn Lys Leu Leu Gly Ile Leu Val
195 200 205
Gly Ile Thr Gly Arg Leu Lys Ser Trp Arg Asp Arg Asp Trp Ala Cys
210 215 220
Lys Thr Val Lys Glu Glu Leu Arg Val Thr Phe Glu Lys Gly Leu Gly
225 230 235 240
Glu Val Asp Pro Thr Ala Tyr Pro Gln Phe Lys Gln Phe Ala Asp Gln
245 250 255
Leu Phe Lys Gln Glu Gly Tyr Lys Ile Ser Gly Arg Val Leu Arg Ala
260 265 270
Val Glu Gly Lys Asp Ala Asp Tyr Gln Pro Val Leu Ser Leu Leu Thr
275 280 285
Gln Tyr Pro Asp Leu Gln Gly Asp Phe Glu Glu Leu Gly Arg Val Tyr
290 295 300
Leu Ala Glu Ala Glu Tyr Leu Arg Lys Lys Val Asp Ala Arg Val Thr
305 310 315 320
Val Cys Asp Ala Glu Thr Ser Pro Leu Gln Phe Pro Phe Gly Leu Thr
325 330 335
Gly Asn Gly Tyr Ser Ile Thr Leu Thr Val Val Lys Gly Gln Ile Ala
340 345 350
Ala Thr Leu His Leu Pro Gly Gly Asp Ile Thr Pro Arg Leu Arg Arg
355 360 365
Ser Lys Tyr Phe Gln Asn Pro Glu Ile Ala Pro Val Lys Asp Gly Lys
370 375 380
Gly Lys Val Asn Gly Phe Gln Ile Ser Phe Lys Arg Gly Lys Thr Pro
385 390 395 400
Leu Val Gly Ile Ile Lys Glu Pro Lys Leu Leu Lys Lys Asn Gly Asn
405 410 415
Tyr Tyr Leu Ser Leu Ala Val Gly Ile Asn Lys Thr Glu Ile Pro Lys
420 425 430
Glu Ile Cys Asp Ala Arg Ala Tyr Tyr Ser Ser Thr Ser Arg Thr Asp
435 440 445
Thr Pro Pro Ala Val Lys Ala Met Ser Ile Asp Leu Gly Val Thr Thr
450 455 460
Leu Ala Asp Tyr Ser Ile Ile Asp Thr Gly Leu Pro Gly Asp Cys Gly
465 470 475 480
Val Phe Gly Gly Ser Thr Ala Ala Phe Thr Glu His Gly Lys Ile Gly
485 490 495
Arg Cys Gly Ser Lys Ser Leu Arg Asp Gly Leu Tyr Lys Asn Thr Glu
500 505 510
Ala Gly Tyr Phe Leu Ala Lys Tyr Ile Arg Leu Ser Lys Asn Leu Arg
515 520 525
Gly Gly Val Gly Leu Asn Lys Leu Glu Lys Glu Lys Leu Leu Glu His
530 535 540
Val Glu Arg Leu Gly Ile Glu His Cys Ala Asp Asp Phe Ala Arg Lys
545 550 555 560
Asp Glu Ile His Arg Lys Phe Ser Glu Ile Lys Ser Lys Leu Glu Lys
565 570 575
Ser Ile Ser Glu Phe Ala Leu Arg Asp Arg Pro Asp Lys Lys Gly Ala
580 585 590
Ser Trp Glu Gly Ile Cys Ala Glu Thr Val Gln Val Leu Gly Ala Val
595 600 605
Lys Arg Trp Gln Ser Leu Ala Lys Ser Trp Thr Tyr Tyr Ser Trp Thr
610 615 620
Ala Glu Asp Tyr Val Leu Ala Leu Thr Gly Glu Gly Arg Thr Arg Val
625 630 635 640
Ser Asp Glu His Val Glu Ser Val Val Lys Thr Gly Arg Arg Gln Phe
645 650 655
Ala Pro Cys Gly Lys Ala Ala Leu Leu Arg Leu Leu Glu Lys Gly Lys
660 665 670
Ile Val Glu Val Cys Pro Gly Gln Phe Gln Leu Ala Glu Gly Val Asp
675 680 685
Tyr Lys Arg His Pro Thr Glu Phe Leu Ala Ala His Ile Arg His Phe
690 695 700
Asn Gly Leu Arg Arg Asp Leu Thr Asn Lys Leu Val Arg Ala Ile Val
705 710 715 720
Glu Lys Ala Gln Gln His Arg Val Gln Ile Val Ile Val Glu Asp Phe
725 730 735
Gly Ile Pro Asp Ile Glu Gly Arg Ile Met Asp His Tyr Asp Asn Tyr
740 745 750
Arg Trp Asn Leu Phe Ala Pro Ala Lys Val Ile Glu Lys Leu Glu Glu
755 760 765
Ala Leu Ser Glu Val Gly Ile Ala Met Ala Glu Val Asp Pro Arg His
770 775 780
Thr Ser Gln Leu Ala Pro Thr Gly Asp Phe Gly Phe Arg Asp His Glu
785 790 795 800
Asn Leu Tyr Phe Trp Glu Lys Gly Leu Cys Arg Thr Asp Ala Asn Thr
805 810 815
Asn Ala Ser Met Arg Ile Ala Glu Arg Phe Phe Thr Arg His Ser Val
820 825 830
Leu Ser Gln Leu Arg Ala Val Lys Ile Ser Glu Thr Glu Phe Leu Ile
835 840 845
Pro Val Ser Thr Gly Lys Arg Glu Asn Ala Phe Ile Lys Ser Gln Thr
850 855 860
Gly Lys Leu Phe Ala Lys Leu Val Ala Asp Ser Asn Gly Phe Val Met
865 870 875 880
Val Gly Leu Thr Glu Lys Gln His Gly Ala Thr Val Thr Val Gly Lys
885 890 895
Lys Val Ser Phe Tyr Asn His Ala Gly Arg Trp Leu Gly Lys Ala His
900 905 910
His Ile Ala His Arg Asp Arg Ile Lys Asn Glu Val Asn Gln Val Leu
915 920 925
Thr Ser Gly Arg Gly Arg Ile Arg Asn Ile Ala Pro Glu Leu Ser Pro
930 935 940
Lys Thr
945
<210> 16
<211> 930
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.18的氨基酸序列
<400> 16
Met Thr Asn Gln Lys Pro Lys Phe Lys Ser Ser Asp Ile Gln Ile Lys
1 5 10 15
His Ile Ser Pro Thr Asp Lys Lys Arg Leu Lys Thr Phe Tyr His Gln
20 25 30
Leu Tyr Glu Gln Val Asn Phe Ile Leu Glu Arg Met Ile Val Met Arg
35 40 45
Gly Arg Pro Arg Thr Ile Arg Asn Ile Asp Gly Thr Glu Ile Phe Val
50 55 60
Ser Gln Glu Glu Ala Asp Gln Gln Leu Leu Ser Leu Ala Gly Gly Ser
65 70 75 80
His Glu Gly Val Lys Tyr Leu Lys Gln Tyr Tyr Glu Ser Cys Val Asp
85 90 95
Ala Gly Lys Pro Ala Lys Tyr Ala Ala Asn Met Phe Leu Thr Lys Thr
100 105 110
Ile Ser Gly Thr Asn Pro Leu Gln Cys His Thr Ala Val Tyr Lys Leu
115 120 125
Tyr Lys Lys Val Gln Ala Lys Gln Ile Thr Lys Lys Glu Phe Ile Asp
130 135 140
Lys Leu Tyr Ser Lys Thr Lys Lys Lys Lys Ser Leu Lys Pro Ala Tyr
145 150 155 160
Lys Val Phe Thr Glu Asn Glu His Ile Glu Phe Tyr His Lys Val Arg
165 170 175
Ser Gly Lys Leu Pro Ala Ser Glu Val Arg Leu Glu Glu Ser Arg Arg
180 185 190
Ala Pro Asp Val Gly Leu Glu Val Gly Leu Leu Leu Arg Glu Leu Gly
195 200 205
Ile Phe Pro Phe Asn Phe Pro His Phe Thr Glu Lys Lys Tyr Leu Asp
210 215 220
Leu Ala Trp Thr Ile Ala Ile Arg Trp Leu Lys Asn Trp Asn Glu Asn
225 230 235 240
Asn Lys Asn Thr Ala Lys Glu Lys Ala Lys Gln Lys Ala Ile Val Asp
245 250 255
Lys Leu Arg Thr Ser Leu Asp Gln Lys Glu Val Asp Leu Phe Glu Glu
260 265 270
Phe Ala Glu Glu Cys Ser Gln Glu Gln Phe Gly Ile Arg Glu Gly Phe
275 280 285
Val Lys Ala Lys Lys Arg Leu Lys Ser Phe Pro Lys Gly Ile Glu Lys
290 295 300
Ser Ser Tyr Lys Glu Gly Met Arg Ile Leu Val Gln Asn Lys His Gly
305 310 315 320
Ser Ile Trp Asp Asn Phe Glu Asn Leu Ala Tyr His His Ile Ala Leu
325 330 335
Asn Glu Tyr Asn Arg Leu Arg Asp Glu Ala Ser Phe Ser Phe Pro Asp
340 345 350
Pro Ile Tyr His Pro Ile Arg Ala Glu Phe Gly Leu Thr Ser Leu Pro
355 360 365
Lys Phe Asn Val Gly Leu Asn Asp Arg Gly Asn Tyr Glu Phe Thr Ile
370 375 380
Asn Leu Pro Asp Gly Pro Leu Met Met Leu Gly Lys Lys Ser Arg Tyr
385 390 395 400
Tyr Leu Lys Pro Ile Ile Gln Gly Pro Leu Asn Asn Ala Phe Ser Phe
405 410 415
Glu Phe Ile Lys Gly Asn Lys Lys Arg Pro Lys Ile Ser Ala Lys Leu
420 425 430
Lys Ser Ile Thr Val Val Phe Ala Lys Ser Ser Ile Tyr Val Gly Leu
435 440 445
Pro Tyr Arg Pro Ile Ser Ile Pro Ile Pro Gln Ala Val Thr Asn Ser
450 455 460
Thr Tyr Tyr Phe Lys Lys Asn Leu Ser Ser Thr Ser Lys Phe Asp Lys
465 470 475 480
Asp Val Phe Met Gly Leu Thr Ala Val Ser Val Asp Leu Gly Leu Asn
485 490 495
Pro Val Phe Ser Met Ser Ala Cys Arg Leu Asp Glu Met Lys Ala Asp
500 505 510
Glu His Tyr Ser Cys Glu Val Pro Gly Phe Gly Trp Ala Asn Gln Ile
515 520 525
Trp Ser Lys Arg Ala Gly Gly Val Trp Asn Arg Ser Phe Arg Asp Lys
530 535 540
Ile Arg Gly Phe Val Pro Gly Asn Leu Ser Asp Arg Ile Phe Cys Cys
545 550 555 560
Lys Lys Ser Ile Ile Val Ser Lys Lys Leu Arg Asp Glu Lys Pro Leu
565 570 575
Thr Gln Tyr Glu Glu Glu Asn Phe Glu Arg Trp Met Gln Val Val Gly
580 585 590
Val Asp Pro Asn Glu Asp His Tyr Lys Gln Leu Arg Ile Ala Ile Arg
595 600 605
Asp Ile Lys Thr Glu Tyr Glu Thr Val Arg Ser Glu Phe Ala Leu Arg
610 615 620
Asp His Pro Asn Asn Ser Asn Lys Thr Thr Glu Asn Ile Cys Thr Glu
625 630 635 640
Cys Phe Asp Met Leu Phe Val Ile Lys Asn Leu Ile Ser Leu Leu Lys
645 650 655
Ser Trp Asn Arg Trp His Arg Thr Thr Gly Asp Ile Glu Glu Arg Gly
660 665 670
Lys Asp Pro Asn Glu Cys Ser Thr Tyr Trp Arg His Tyr Asn Gly Leu
675 680 685
Lys Thr Asp Leu Leu Lys Lys Leu Thr Asn Ile Leu Ile Glu Ser Ala
690 695 700
Lys Ser Ile Gly Ala His Ile Ile Ile Leu Glu Asp Leu Thr Leu Ser
705 710 715 720
Gln Arg Ser Ser Arg Ser Arg Arg Glu Asn Ser Leu Val Ala Ile Phe
725 730 735
Gly Ala Gln Thr Ile Ile Lys Thr Ile Ser Glu Glu Ala Glu Ile Asn
740 745 750
Gly Ile Leu Val Tyr Leu Glu Asp Pro Arg His Ser Ser Gln Ile Ser
755 760 765
Ile Val Thr Asn Glu Phe Gly Tyr Arg Pro Lys Glu Asp Lys Ala Lys
770 775 780
Leu Tyr Phe Met Asp Glu Glu Thr Val Cys Val Thr Asn Cys Asp Asp
785 790 795 800
Ser Ala Ala Leu Met Leu Gln Gln Ser Phe Trp Ser Arg His Lys Asp
805 810 815
Val Val Lys Val Lys Gly Thr Lys Val Ser Asp Thr Glu Tyr Leu Val
820 825 830
Ser Ser Glu Asp Lys Asp Gly Thr Lys Met Arg Leu Arg Ser Tyr Leu
835 840 845
Lys Arg Asn Val Gly Thr Ala Asn Ala Ile Leu Gln Lys Asn Cys Asp
850 855 860
Gly Tyr Asp Leu Lys Lys Ile Ser Pro Gln Lys Lys Lys Lys Ile Glu
865 870 875 880
Glu Phe Gly Lys Asp Glu Tyr Phe Tyr Arg His Gly Glu Gln Trp Phe
885 890 895
Thr Ala Asp Ala His Phe Asp Lys Leu Arg Glu Phe Gly Asn Gln Val
900 905 910
Phe Leu Thr Pro Gln Ser Gln Ile Lys Arg Ile Asn Leu Gln Val Glu
915 920 925
Gly Thr
930
<210> 17
<211> 908
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.19的氨基酸序列
<400> 17
Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu
1 5 10 15
Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe
20 25 30
Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met
35 40 45
Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe
50 55 60
Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu
65 70 75 80
Arg Ser Phe Ala Leu Ala Val Leu His Gln Asp Asn Pro Lys Lys Arg
85 90 95
Ala Phe Leu Glu Ser Glu Asn Cys Val Ser Ile Leu Cys Leu Glu Lys
100 105 110
Ser Ala Ser Gly Thr Arg Tyr Tyr Lys Arg Pro Gly Tyr Gln Leu Leu
115 120 125
Lys Lys Ala Ile Glu Glu Glu Trp Gly Trp Asp Lys Phe Glu Ala Ser
130 135 140
Leu Leu Asp Glu Arg Thr Gly Glu Val Ala Glu Lys Phe Ala Ala Leu
145 150 155 160
Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp
165 170 175
Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala
180 185 190
Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu
195 200 205
His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu
210 215 220
Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys
225 230 235 240
Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro
245 250 255
Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala
260 265 270
Asp Tyr Asn Val Thr Lys Lys Leu Val Leu Ala Val Ser Ala Lys Phe
275 280 285
Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys
290 295 300
Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe
305 310 315 320
Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala
325 330 335
Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly
340 345 350
Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg
355 360 365
Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala
370 375 380
Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser
385 390 395 400
Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg
405 410 415
Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg
420 425 430
Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu Glu Val Ser Asp
435 440 445
Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser
450 455 460
Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly
465 470 475 480
Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln
485 490 495
Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp
500 505 510
Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu
515 520 525
Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg
530 535 540
Leu Ser Lys Lys Leu Arg Asp Arg Gly Ala Leu Asn Asp Ile Glu Ala
545 550 555 560
Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile
565 570 575
Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu
580 585 590
Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu Arg Tyr
595 600 605
His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe
610 615 620
Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp
625 630 635 640
Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn
645 650 655
Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met
660 665 670
Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu
675 680 685
His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg
690 695 700
Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp
705 710 715 720
Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu
725 730 735
Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser
740 745 750
Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr
755 760 765
Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val
770 775 780
Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser
785 790 795 800
Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val
805 810 815
Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly
820 825 830
Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu
835 840 845
Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys
850 855 860
Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr
865 870 875 880
Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu
885 890 895
Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr
900 905
<210> 18
<211> 821
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.20的氨基酸序列
<400> 18
Met Ala Phe Gln Ser Lys Arg Arg Ile Val Gly Asn Phe Val Lys Glu
1 5 10 15
Gln Cys Leu Lys Ala Val Asp Gly Lys Val Ile Leu Thr Asp Gln Glu
20 25 30
Lys Arg Glu Leu Ile Lys Arg Tyr Glu Leu His Leu Glu Pro His Lys
35 40 45
Trp Leu Leu Arg Leu Phe Leu Ser Gly Tyr Glu Gly Arg Asp Asp Gly
50 55 60
Phe Tyr Glu Glu Leu Gly Asn Thr Asn Leu Asp Lys Glu Lys Phe Phe
65 70 75 80
Glu Val Thr Ala Gly Leu Arg Asp Ala Leu Leu Arg Gln Ser Gly Ser
85 90 95
Ser Arg Ala Leu Lys Ser Ser Met Leu Gly Lys Cys Pro Pro Ser Ala
100 105 110
Ala Val Gly Lys Ala Ala Lys His Ile Gln Thr Leu Arg Asp Ala Gly
115 120 125
Ile Leu Pro Phe Lys Thr Gly Leu Thr Ser Gly Glu Asp Tyr Asn Val
130 135 140
Leu Gln Gln Ala Val Gln Gln Leu Arg Ser Trp Val Ala Cys Asp His
145 150 155 160
Arg Thr Arg Glu Ala Tyr Ala Glu Gln Gln Glu Lys Thr Ser Gln Ala
165 170 175
Glu Glu Ala Ala Lys Lys Ala Ala Asn Glu Val Lys Pro Glu Asp Ala
180 185 190
Lys Ser Leu Glu Arg His Glu Arg Val Leu Thr Lys Leu Arg Lys Gln
195 200 205
Glu Arg Arg Leu Glu Arg Met Lys Ser His Ala Gln Phe Ser Leu Asp
210 215 220
Glu Met Asp Cys Thr Gly Tyr Ser Leu Cys Met Gly Ala Asn Tyr Leu
225 230 235 240
Lys Asp Tyr Cys Leu Glu Lys Glu Gly Arg Gly Leu Arg Leu Thr Leu
245 250 255
Lys Asn Ser Thr Met Ala Gly Ser Tyr Tyr Val Ser Val Gly Asp Gly
260 265 270
Gln His Ala Gly Met Lys Asn Pro Gly Thr Pro Ala Gly Gly Ser Pro
275 280 285
Glu Lys Gly Arg Arg Arg Asn Ile Leu Phe Asp Phe Thr Val Glu Lys
290 295 300
Cys Gly Asp Asn Tyr Leu Phe Arg Tyr Asp Glu Asn Gly Lys Arg Pro
305 310 315 320
Arg Ala Gly Val Val Lys Glu Pro Arg Phe Cys Trp Arg Arg Lys Gly
325 330 335
Asn Ser Val Glu Leu Tyr Leu Ala Met Pro Ile Asn Ile Glu Asn Ser
340 345 350
Met Arg Asn Ile Phe Val Gly Lys Gln Lys Ser Gly Lys His Ser Ala
355 360 365
Phe Thr Arg Gln Trp Pro Lys Glu Val Glu Gly Leu Asp Glu Leu Arg
370 375 380
Asp Ala Val Val Leu Gly Val Asp Ile Gly Ile Asn Arg Ala Ala Phe
385 390 395 400
Cys Ala Ala Leu Lys Thr Ser Arg Phe Glu Asn Gly Leu Pro Ala Asp
405 410 415
Val Gln Val Met Asp Thr Thr Cys Asp Ala Leu Thr Glu Lys Gly Gln
420 425 430
Glu Tyr Arg Gln Leu Arg Lys Asp Ala Thr Cys Leu Ala Trp Leu Ile
435 440 445
Arg Thr Thr Arg Arg Phe Lys Ala Asp Pro Gly Asn Lys His Asn Gln
450 455 460
Ile Lys Glu Lys Asp Val Glu Arg Phe Asp Ser Ala Asp Gly Ala Tyr
465 470 475 480
Arg Arg Tyr Met Asp Ala Ile Ala Glu Met Pro Ser Asp Pro Leu Gln
485 490 495
Val Trp Glu Ala Ala Arg Ile Thr Gly Tyr Gly Glu Trp Ala Lys Glu
500 505 510
Ile Phe Ala Arg Phe Asn His Tyr Lys His Glu His Ala Cys Cys Ala
515 520 525
Val Ser Leu Ser Leu Ser Asp Arg Leu Val Trp Cys Arg Leu Ile Asp
530 535 540
Arg Ile Leu Ser Leu Lys Lys Cys Leu His Phe Gly Gly Tyr Glu Ser
545 550 555 560
Lys His Arg Lys Gly Phe Cys Lys Ser Leu Tyr Arg Leu Arg His Asn
565 570 575
Ala Arg Asn Asp Val Arg Lys Lys Leu Ala Arg Phe Ile Val Asp Ala
580 585 590
Ala Val Asp Ala Gly Ala Ser Val Ile Ala Met Glu Lys Leu Pro Ser
595 600 605
Ser Gly Gly Lys Gln Ser Lys Asp Asp Asn Arg Ile Trp Asp Leu Met
610 615 620
Ala Pro Asn Thr Leu Ala Thr Thr Val Cys Leu Met Ala Lys Val Glu
625 630 635 640
Gly Ile Gly Phe Val Gln Val Asp Pro Glu Phe Thr Ser Gln Trp Val
645 650 655
Phe Glu Gln Arg Val Ile Gly Asp Arg Glu Gly Arg Ile Val Ser Cys
660 665 670
Leu Asp Ala Glu Gly Val Arg Arg Asp Tyr Asp Ala Asp Glu Asn Ala
675 680 685
Ala Lys Asn Ile Ala Trp Leu Ala Leu Thr Arg Glu Ala Glu Pro Phe
690 695 700
Cys Met Ala Phe Glu Lys Arg Asn Gly Val Val Glu Pro Lys Gly Leu
705 710 715 720
Arg Phe Asp Ile Pro Glu Glu Pro Thr Arg Glu Gln Asp Glu Ser Asp
725 730 735
Gln Asp Phe Lys Lys Arg Leu Glu Glu Arg Asp Lys Leu Ile Glu Arg
740 745 750
Leu Gln Ala Lys Ala Asp Arg Met Gln Ala Ile Val Gln Arg Leu Phe
755 760 765
Gly Asp Arg Arg Pro Trp Asp Ala Phe Ala Asp Arg Ile Pro Glu Gly
770 775 780
Lys Ser Lys Arg Leu Phe Arg His Arg Asp Gly Leu Val Leu Asn Lys
785 790 795 800
Pro Phe Lys Gly Leu Cys Gly Ser Glu Asn Ser Glu Gln Lys Ala Ser
805 810 815
Ala Arg Asn Ser Arg
820
<210> 19
<211> 837
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.21的氨基酸序列
<400> 19
Met Gly Arg Phe Gly Lys Lys Lys Ile Ala Val Asn Gly Tyr Val Glu
1 5 10 15
Gln Asp Cys Ile Lys Thr Ile Ser Ala Lys Cys Leu Leu Thr Arg Ala
20 25 30
Gln Ile Asp Glu Leu Arg Ala Lys Tyr Asp Ala Val Leu Asp Thr Met
35 40 45
Arg Pro Leu Ile Arg Leu Ile Leu Ala Gly Tyr Glu Gly Arg Asp Asp
50 55 60
Gly Ile Tyr Glu Glu Ile Ala Pro Glu Met Ser Lys Lys Lys Phe Phe
65 70 75 80
Glu Ala Ala Thr Glu Trp Arg Glu Ser Ile Val Lys Asn Ala Ser Pro
85 90 95
Arg Ala Met Lys Ala Ser Val Phe Gly Asp Lys Glu Pro Cys Lys Ser
100 105 110
Thr Gly Gly Ala Arg Ala Val Ile Gly Lys Leu Arg Lys Ser Gly Val
115 120 125
Phe Pro Ile Glu Thr Gly Leu Ser Gly Gly Asp Glu Tyr Asn Leu Ile
130 135 140
Glu Gln Ala Ile Glu Tyr Ala Lys Ser Trp Leu Lys Ser Asp Glu Ala
145 150 155 160
Thr Arg Glu Ala Tyr Ala Asp Gln Gln Lys Asp Ile Lys Arg Leu Ile
165 170 175
Gly Glu Ala Lys Lys Leu Ala Leu Lys Ile Glu Lys Ala Glu Lys Lys
180 185 190
Leu Glu Ala Thr Asn Pro Gln Thr Lys Ser Trp Lys Lys Thr Thr Glu
195 200 205
Ile Ile Lys Lys Ser Lys Arg Glu Phe Gly Ser Val Thr Thr Lys Thr
210 215 220
Glu Lys Ala Glu Lys Arg Phe Glu Arg Met Lys Pro Phe Ser Lys Leu
225 230 235 240
Glu Leu Gln Asn Met Asp Cys Thr Lys Tyr Ser Thr Tyr Leu Gly Thr
245 250 255
Asn Tyr Ser Pro Phe Lys Leu Lys Lys Glu Gly Asp Leu Leu Gln Ile
260 265 270
Thr Val Thr Ser Ser Val Met Lys Gly Thr Tyr Leu Ala Ser Tyr Gly
275 280 285
Asp Gly Gln Tyr Gly Ser Arg Arg Asn Asn Gly Gln Ser Arg Arg Asp
290 295 300
Asp Phe Val Pro Asn Met Asn Gln Lys Arg Arg Arg Asn Leu Met Phe
305 310 315 320
Asp Cys Thr Val Glu Pro Phe Gly Asp Gly Ser Leu Leu Arg Tyr Glu
325 330 335
Glu Asn Gly Leu Arg Pro Arg Val Ala Glu Leu Lys Glu Pro Arg Leu
340 345 350
Cys Trp Arg Arg Arg Asn Gly Asn Tyr Glu Leu Tyr Leu Met Met Pro
355 360 365
Val Lys Met His Val Lys Ser Pro Glu Met Phe Ala Gly Asp His Leu
370 375 380
Ala Phe Ser Arg Tyr Trp Pro Lys Glu Val Glu Gly Leu Asp Ser Asp
385 390 395 400
Thr Lys Ile Thr Ala Leu Gly Val Asp Val Gly Ile Ile Arg Ser Ala
405 410 415
Tyr Cys Val Ala Val Thr Ala Glu Arg Phe Val Asp Gly Leu Pro Thr
420 425 430
Glu Met Thr Val Gly Lys Ala Ser Phe Asp Ala Gln Thr Glu Lys Gly
435 440 445
Arg Glu Tyr Phe Glu Leu Gly Arg Arg Ala Thr Met Leu Gly Trp Leu
450 455 460
Ile Lys Thr Thr Arg Arg Tyr Lys Lys Asp Pro Lys Asn Glu His Asn
465 470 475 480
Gln Ile Lys Glu Ser Asp Val Ala Ala Phe Asp Gly Ser Pro Gly Ala
485 490 495
Phe Glu His Tyr Ile Leu Ala Val Asp Glu Met Ser Asp Asp Pro Leu
500 505 510
Asp Val Trp Gly His Ala Asn Ile Thr Gly Tyr Gly Lys Trp Thr Lys
515 520 525
Gln Ile Phe Lys Glu Phe Asn Gln Leu Lys Arg Glu Arg Ala Glu Gly
530 535 540
Gln Val Glu Pro Asn Met Thr Asp Asp Leu Thr Trp Cys Ser Leu Ile
545 550 555 560
Asp Tyr Ile Ile Ser Leu Lys Lys Thr Leu His Phe Gly Gly Tyr Glu
565 570 575
Thr Lys Glu Arg Glu Ser Phe Cys Pro Ala Leu Tyr Asn Glu Arg Ala
580 585 590
Asn Cys Arg Asp Val Val Arg Lys Arg Leu Ala Arg Tyr Val Val Glu
595 600 605
Arg Ala Ile Ala Ala Glu Ala Gln Val Ile Ser Val Glu Asn Leu Ser
610 615 620
Lys Cys Arg Arg Asp Asp Lys Arg Lys Asn Arg Val Trp Asp Leu Met
625 630 635 640
Ser Gln Gln Ser Trp Ile Gly Val Leu Thr Asn Met Ala Arg Met Glu
645 650 655
Asn Ile Ala Val Val Ser Val Asn Pro Asp Leu Thr Ser Gln Trp Val
660 665 670
Glu Gln Cys Gly Ala Ile Gly Asp Arg Lys Ala Arg Thr Ile Ala Cys
675 680 685
Arg Asp Val Asn Gly Lys Phe Val Ser Leu Asp Ala Asp Leu Asn Ala
690 695 700
Ala Tyr Asn Ile Ala Ser Arg Ala Leu Thr Arg His Ala Glu Pro Phe
705 710 715 720
Ser Ile Thr Phe Lys Lys Lys Asp Gly Ile Leu Glu Gln Lys Asp Val
725 730 735
Cys Phe Asp Pro Gly Val Ile Pro Val Leu Glu Lys Asn Glu Asn Glu
740 745 750
Glu Lys Phe Arg Glu Arg Val Glu Lys Tyr Glu Lys Ser Leu Val Ile
755 760 765
Lys Gln Glu Arg Ala Val Arg Trp Arg Ala Ile Leu Gln His Leu Phe
770 775 780
Gly Asn Glu Arg Pro Trp Asp Glu Phe Thr Asp Glu Val Lys Glu Gly
785 790 795 800
Arg His Val Ser Leu Tyr Arg His His Gly Lys Leu Val Arg Thr Lys
805 810 815
Gln Tyr Ala Gly Leu Val Lys Glu Ala Asn Asn Glu Leu Val Pro Val
820 825 830
Cys Ala Val Ala Arg
835
<210> 20
<211> 968
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.22的氨基酸序列
<400> 20
Met Ser Lys Ala Thr Arg Lys Thr Lys Thr Thr Val Pro Glu Ser Thr
1 5 10 15
Asp Thr Glu Ser Pro Ala Ala Asp Thr Gln Val Arg Val His Trp Leu
20 25 30
Ala Ala Ser His Arg Ala Ser Pro Gly Leu Gln Gln Val Lys Glu Met
35 40 45
Ile Gln Gln His Ala Asp Val Ala Ser Val Leu Phe Gln Gly Leu Val
50 55 60
Arg Thr Ala Pro Ile Val Phe Arg Asn Asp Asp Gly Ser Pro Val Lys
65 70 75 80
Pro Leu Asp Leu Leu Leu Ala Ser Leu Arg Pro Thr Tyr Lys Val Gln
85 90 95
Arg Asp Thr Glu Thr Val Leu Val Thr Lys Asp Asp Val Ile Arg Cys
100 105 110
Leu Thr Leu Ala Thr Thr Ala Val Asn Gly Gly Gln Ala Thr Asn Val
115 120 125
Ala Val Phe Ala Ser Ala Asp Pro Ala Leu Ser Ala Pro Leu Ala Thr
130 135 140
Leu Leu Ala Gln Leu Arg Ala Leu Glu Ser Val Asp Ser Ser Trp Ser
145 150 155 160
Val Val Gly Lys Leu Asp Ile Asn Leu Arg Lys Phe Val Trp Leu Val
165 170 175
Leu Ser Ala Ala Gly Val Leu Pro Ala Leu Ala Asp Leu Glu Gly Tyr
180 185 190
Ala Ala Lys Ser Val Leu Ala Asn Val Gln Gly Lys Tyr Lys Ser Leu
195 200 205
Gln Ala Cys Ala Asp Thr His Ala Ala Leu Tyr Lys Gln His Gln Thr
210 215 220
Asn Lys Glu Gln Leu Glu Lys Leu Ile Ala Asp Pro Gly Phe Val Ala
225 230 235 240
Leu Cys Ser Ala Leu Leu Gln Asp Pro Asp Leu Arg Ser Val Asp Ser
245 250 255
Arg Arg Leu Ala Ala Leu Glu Glu Met Leu Gly Phe Val Ala Ala Asp
260 265 270
Lys Asn Tyr Ser Glu Tyr Thr Ser Thr Arg Lys Cys Asp Gly Trp Ala
275 280 285
Pro Pro Ala Asn Met Phe Asp Leu Leu Cys Glu His Lys Glu Ala Val
290 295 300
Arg Arg Asn Ile Val Val Asp Asn Ser Lys Cys Leu Ser Arg Arg Ile
305 310 315 320
Ser Leu Val Ala Asp Gly Asp Val Asn Glu Val Ser Val Phe Glu Leu
325 330 335
Leu Asn Glu Met Arg Trp Leu Ser Val His Ser Ser Gly Ile Arg Met
340 345 350
Pro Asn Tyr Pro Lys His Ala Tyr Ala Leu Lys Phe Gly Asp Asn Tyr
355 360 365
Ile Ser Val Lys Ser Phe Glu Thr Val Val Asp Gly Gly Cys Ser Leu
370 375 380
Leu Arg Met Thr Ala Arg Val Gly Lys Asn Asp Leu Val Cys Asp Phe
385 390 395 400
Val Leu Gly Arg Gly Asn Glu Tyr Trp Asn Asn Leu Lys Ile Thr Pro
405 410 415
Met Gly Lys Gly Ile Phe Ala Val Val Lys Thr Val Arg Arg Phe Thr
420 425 430
Ala Thr Gly Ala Lys Leu Val Glu Leu Arg Gly Val Cys Lys Glu Pro
435 440 445
Glu Ile Arg Tyr Glu Arg Gly Val Leu Gly Leu Arg Leu Pro Ile Ser
450 455 460
Phe Asp Val Tyr Gly Lys Val Glu Glu Asp Ser Ile Ala Phe Gly Lys
465 470 475 480
Asn Arg Val Ser Leu Arg Thr Thr Pro Phe Val Glu Lys Ala Asp Lys
485 490 495
Phe Gln Gly Leu Leu Asp Tyr Arg Asn Thr Thr Ala Arg Asp Gly Tyr
500 505 510
Ile Tyr Tyr Ala Gly Phe Asp Gln Gly Glu Asn Asp Gln Val Val Gly
515 520 525
Ile Tyr Arg Thr Arg Thr Tyr Lys Asn Ala Thr Met Leu Glu Phe Phe
530 535 540
Asn Val Ser Asp Thr Leu Glu Glu Val Ala Ser Cys Arg Phe Ser Asp
545 550 555 560
Tyr Gln Glu Arg Lys Arg Arg Leu Arg Gly Asp Thr Gly Val Leu Asp
565 570 575
Ile Asn Ser Ile Asn Val Leu Ala Asp Lys Val Gln Arg Leu Arg Arg
580 585 590
Leu Ile Ser Thr Leu Arg Ala Cys Ala Ser His Thr Asp Trp Tyr Pro
595 600 605
Lys Leu Lys Glu Arg Arg Arg Leu Glu Trp Ala Val Leu Ala Gln Gly
610 615 620
Val Gly Val Ser Asp Phe Asp Thr Glu Ile Glu Arg Ala Glu Thr Ala
625 630 635 640
Leu Ser Ala Val Ala Ala Val Asp Phe Val Arg Asp Pro Thr Cys Ile
645 650 655
Ile Asn Val Met Asp Lys His Ile Tyr Ala Gln Phe Lys Gln Leu Arg
660 665 670
Ser Glu Arg Asn Glu Lys Tyr Arg Ser Gln His Gln His Asp Tyr Lys
675 680 685
Trp Leu Gln Leu Val Asp Ser Val Ile Ser Leu Arg Lys Ser Ile Tyr
690 695 700
Arg Phe Gly Lys Ala Pro Glu Pro Arg Gly Ala Gly Glu Leu Tyr Pro
705 710 715 720
Gln Asn Leu Tyr Thr Tyr Arg Asp Asn Leu Met Gln Gln Tyr Arg Lys
725 730 735
Glu Val Ala Ala Phe Ile Arg Asp Val Cys Leu Glu His Gly Val Arg
740 745 750
Gln Leu Ala Val Glu Ala Leu Asn Pro Thr Ser Tyr Ile Gly Glu Asp
755 760 765
Ser Asp Ala Asn Arg Lys Arg Ala Leu Phe Ala Pro Ser Glu Leu His
770 775 780
Asn Asp Ile Val Leu Ala Cys Ser Leu His Ser Ile Ala Val Val Ala
785 790 795 800
Val Asp Glu Thr Met Thr Ser Arg Val Ala Pro Asn Asn Arg Leu Gly
805 810 815
Phe Arg Ser His Gly Asp Tyr Gln Lys Phe Ser Glu Thr Ala Gln Gly
820 825 830
Arg Phe Asn Trp Lys His Leu His Tyr Phe Gly Asp Asn Asp Val Ser
835 840 845
Glu His Cys Asp Ala Asp Glu Asn Ala Cys Arg Asn Ile Val Leu Arg
850 855 860
Ala Leu Thr Cys Gly Ala Ser Lys Pro Arg Phe Ser Arg Gln Ser Leu
865 870 875 880
Leu Gly Lys Ile Lys Gly Pro Val Leu Arg Thr Gln Leu Ala Tyr Leu
885 890 895
Ala His Lys Arg Gly Leu Leu Thr Ala Ser Thr Glu Pro Lys Lys Ala
900 905 910
Ala Glu Thr Gly Phe Glu Leu Val Glu Ala Asp Leu Gly Gly Ala Leu
915 920 925
Arg Val Gly Lys Gly Phe Ile Tyr Val Asp Ala Gly Ile Cys Ile Asn
930 935 940
Ala Thr Thr Arg Lys Glu Arg Ser His Lys Val Gly Glu Ala Val Val
945 950 955 960
Ser Arg Ser Leu Ala Ser Pro Phe
965
<210> 21
<211> 3012
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.3编码核酸序列
<400> 21
atgaccaagg agaagatcaa gaagaccaag aaggccaagg tggagaagga ctccgtgacc 60
agggccggca tcctgaggat cctgctgaac ccggaccagc accaggagct ggacaccctg 120
atctccgacc accaggaggc cgccagggag atccagaccg ccacctacaa gctgtccggc 180
ctgaagctgt acgacaagac caacaacatg gtggtggacg gctccaaggc caccccggag 240
gagcaggagg cctactacaa gatcatcaac tgggagggcc agccgatctc catctccaac 300
ccgatggtga gggccacctt caagtccatc gccaaggtga aggaggacat caggaggaag 360
caggaggagt acgccaagct ggaggaggcc gacctgacca agatgtccac cggcgacgtg 420
aagaagcaca agaacgagct gaggaaggcc gccaacagga tcaagcactc cgaggagatc 480
ctgcagttcg ccaagtggag gctggccgac atcttcccgc tgccgctgtc ccacaactcc 540
cagctgcacc tgaagaacaa ctaccaccag aacgtgttct ccggcttcca cgccagggtg 600
aagggctgga acgcctgcga catcgccgcc caggccaact acgccgagat cgacaacagg 660
ctgaccgagc tgtcctccga gctgtccggc gactacggct ccgaggtgat caccgacctg 720
atgggcctgc tgcagtacac caaggagctg ggcgagggct acaccgacac ctcctacctg 780
aactacaagt tcctgtcctt cttcaaggag tgctggaggc cgaacgccat cgccaacaac 840
accggcctgc tggagggctt ctggctggcc aacaacaagc acaccaacaa gaagaaccag 900
gtggcctact ccttcaaccc gaagatctcc gaggagctgt tcaggaggag gtccctgtgg 960
gagtccgaca agtgcctgct gtccgacccg aggttcgaga agtacgtgga gctgttcgac 1020
aagcacggca ggtacaggaa gggcgcctcc ctgaccctga tctccaagga gtccccgatc 1080
ccgatcggct tctccatgga caggaacgcc gccaagctgg tgaggatcga caacgacacc 1140
gccaacaggc agctgaccat caccatcgag ctgccgaaca aggaggagag gtcctacgtg 1200
gccgcctacg gcaggaagca cgagaccaag tgctactaca acggcctgac caccaggctg 1260
ccgaggtccg agaaggagct gctggccctg gccaaggccg agaacaggga gctgaccgac 1320
aaggagatcc acgaggcctc cctggagaag tgctacatct tcgagtacgc cagggccggc 1380
aagatcccgg tgttcgccgt ggtgaagacc ctgtacttca ggaggaaccc gtccaacggc 1440
gagtactacg tgatcctgcc gaccaacatc ttcgtggagt accacgccaa caacgagttc 1500
aactccaagg agctgttcaa gatcaggtcc gagctgcaga aggcctggga cgaggtgagg 1560
accccgaaga ggaacgtgca gtcctgcgtg ctggacaagg acctgtccaa gaggttcgcc 1620
ggcaggaccc tgaagtacgc cggcatcgac ctgggctact ccaacccgta caccgtgtcc 1680
tactacaacg tggtgggcac cgaggagggc atccagatca aggagaccgg caacgagatc 1740
gtgtccaccg tgttcaacga gcagtacatc cagctgaagg gcaacatcta ccagctgatc 1800
aacatcatca gggcctccag gaggtacctg caggagtccg gcgagctgaa gctgtccaag 1860
gacgacatca agtccttcga ccagctgatg gagctgctgc cgtccgagca gaggatcacc 1920
atcgaccagt tcatcaagga catcaagaag gccaagcagg agggcaagct gatcagggac 1980
atcaagggca agctgccggt ggagggcaag aagaaggagt actgggtgat ctccaacctg 2040
atgtacgtga tcacccagac catgaacggc atcaggggca acagggactc caacaaccac 2100
ctgaccgaga agaagaactg gctgtccgcc ccgccgctga tcgagctgat cgacgcctac 2160
tacaacctga agaagacctt caacgactcc ggcgacggca tcaagatgct gccgaaggac 2220
cacgtgtacg ccgagggcga gaagcagagg tgcaccctga gggaggagaa cttctgcaag 2280
ggcatcctgg agtggaggga caacgtgaag gactacttca tcaagaagct gttctcccag 2340
atcgcccaca ggtgctacga gctgggcatc ggcatcgtgg ccatggagaa cctggacatc 2400
atgggctcct ccaagaacac caagcagtcc aacaggatgt tcaacatctg gccgaggggc 2460
cagatgaaga agtccgccga ggacgccttc tcctacatgg gcatcctgat ccagtacgtg 2520
gacgagaacg gcacctccag gcacgacgcc gactccggca tctacggctg cagggacggc 2580
gccaacctgt ggctgccgaa caagaagctg cacgccgacg tgaacgcctc caggatgatc 2640
gccctgaggg gcctgaccca ccacaccaac ctgtactgca ggtccctgac cgagatcgag 2700
aacggcaagt acgtgaacac ctacgagctg ttcgacacca ccaagaacga ccagtccggc 2760
gccgccaaga ggctgagggg cgccgagacc ctgctgcacg gctactccgc caccgtgtac 2820
cagatccaca ccaccaacac cggcgccggc gtggccctgc tgccggacct gaccgccacc 2880
gacgtgatca agaacaagaa gatcaccgcc accaaggaga acaccgccaa gtactacaag 2940
ctggacaaca ccaacaccta ctacccgtgg tccgtgtgcg agaagctgca caagaactgg 3000
aagctgtcct ga 3012
<210> 22
<211> 2625
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.4编码核酸序列
<400> 22
atgaagaaga agaagaactt ctccgtgtcc gccaccggcg tgttctcctt cccgaccacc 60
gaggccaaga tggacttctt ccacaggttc atcgagctga acggcctggc cgccgagatc 120
gagacccact tcctgaacct gaagaacgac aagaacggcg agtccgtgta caacaaggtg 180
ctgtccaact ccaaccactc caggccgttc tccaccccgc tgctgggcac catgaccggc 240
tccaccaagg tgaccgacaa gaacgccctg tacggcaacg acctggacca ctgcaggaag 300
aagaagatcg tgccgttctc ctcctcctcc ccgctgtcct cccaggagaa gttcttctgc 360
atcgaggccg tgttcaggag ggccaagtcc cacatggagt gcaagaagct gttccaggac 420
gagaccaaca ggatggactc ccagatcaac ggcatcctga acgagctgcc gtacggcgtg 480
gagctgtcca acatgctgtc cgagctgatc gccatcccgt tcgccatcgg ctggaagctg 540
gagggctacc tgggccaggt gttcttcccg tccatcgccg agggcctgac cccgccgaag 600
tccgccaaga tcaagggcag gaggaggtcc atcgactact ccgtgaccga cgaggcctac 660
gacatcctga tgaagtactc caacctgcac tcctccttcg agaccggcct gaagatgtcc 720
aacctgttct ccgccttcta caagaagtcc aacaggaagg acgagatcca gttcaccccg 780
atctccatgg agtccaggtg cgacctgctg ctgggcaaga acttcctgaa gttcgacctg 840
aagaactgcg accacaggtc cggctccctg atgctgacca tcaacgacaa gaacaggctg 900
aacggcgact acgagatcag ggtgggctcc gacaagaagg actcctacct gaccggcgtg 960
aacgtgacca acctgggcga caacgtgttc aacctgaact acaaggtgaa cggcaagagg 1020
gagtacaaca tgctgctgaa ggagccgtcc atccacatca agatgcacag gatgagggac 1080
gacggcaact acctgtcctc cgacttcgac ttctacatga tcttctccat gtcctccgag 1140
aaggacgagg agaagctggc caggtcctgg gacatgaggg ccgccatgtc caccgcctac 1200
ggcaccgaca tcaagaagta ccactcctcc ttcccgtgca ggatcctggc ctgcgacctg 1260
ggcgtgaagc acccgtactc cgccgccgtg atggacatcg gccagctgaa cgagaacggc 1320
atgccggtgt ccgtggacaa ggtgcactgc atgcactccg agggcgtgtc cgagatcggc 1380
cagggctaca accacctgat ccagaagatc ctggccctga actacatcct ggcctactgc 1440
agggagttcg tgtccggcac cgtggacgac ttcgacaaga tcgactacaa gctgtcccag 1500
ctgtcctaca agcaggagga cctgctgatc aacctgcagg agatgaagga ccacttcggc 1560
aacgacatgc aggcctggaa gaagtccagg acctgggtgg tgtccaccct gttcttcgag 1620
ctgaggcagg agttcaacca gctgaggaac cagaggccgg gcaagaagac cgtgtccctg 1680
gccgacgagt tccagtacat cgacatgagg aggaagttca tctccctgtc caggtcctac 1740
accaacgtgg gcaggcagtc ctccaagcac aggcacgact cctaccagac ccactacgac 1800
gtgatcaaca ggtgcaagaa gaacctgctg aggaacatct gcaggaggat gatcgacatg 1860
gccgtgcaga acaagtgcga catcatcgtg gtggaggacc tgtccttcca gctgtcctcc 1920
cacaactcca ggagggacaa cgtgttcaac gccctgtggt cctgcaagtc catcaagaac 1980
atgctgggca tcatggccga gcagcacaac atcatcatct ccgaggtgga cccgaaccac 2040
acctccaaga tcgactgcga gaccggcaac ttcggctaca ggtactcctc cgacttctac 2100
tccgtgatcg acggccagct ggtgaggagg cacgccgacg agaacgccgc catcaacatc 2160
ggcaacaggt gggcctccag gcacaccgac ctgaagtcct tcaactgcag gcagatctcc 2220
atcgacggca ggaaggtggc cttcccgtac gccaagggca agaggaagtc cgccctgttc 2280
ggctacctgt tcggcaactg caagaccgtg ttcgtgtccg acgacggcga ctcctacacc 2340
ccgatcccgt actccaagtt caggaagtcc atctccaagg acgaccacga cgtggtgaac 2400
tacctgcacg acctgaccat gaacaagaac gtgatcaggg tggagtacaa caagtccatc 2460
aagtccgcct ccgtggagct gtacctgaac gacgacaggg tgatctccag gtccctgagg 2520
gacaaggagg tggacgccat cgagaagctg gtgtccaggg gctccctgat caacgagtcc 2580
ggcccgtccc tggagcacga cgaggtgaag tccgtgaccc actga 2625
<210> 23
<211> 2613
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.5编码核酸序列
<400> 23
atgaaggtgc acgagatccc gaggtcccag ctgctgaaga tcaagcagta cgagggctcc 60
ttcgtggagt ggtacaggga cctgcaggag gacaggaaga agttcgcctc cctgctgttc 120
aggtgggccg ccttcggcta cgccgccagg gaggacgacg gcgccaccta catctccccg 180
tcccaggccc tgctggagag gaggctgctg ctgggcgacg ccgaggacgt ggccatcaag 240
ttcctggacg tgctgttcaa gggcggcgcc ccgtcctcct cctgctactc cctgttctac 300
gaggacttcg ccctgaggga caaggccaag tactccggcg ccaagaggga gttcatcgag 360
ggcctggcca ccatgccgct ggacaagatc atcgagagga tcaggcagga cgagcagctg 420
tccaagatcc cggccgagga gtggctgatc ctgggcgccg agtactcccc ggaggagatc 480
tgggagcagg tggccccgag gatcgtgaac gtggacaggt ccctgggcaa gcagctgagg 540
gagaggctgg gcatcaagtg caggaggccg cacgacgccg gctactgcaa gatcctgatg 600
gaggtggtgg ccaggcagct gaggtcccac aacgagacct accacgagta cctgaaccag 660
acccacgaga tgaagaccaa ggtggccaac aacctgacca acgagttcga cctggtgtgc 720
gagttcgccg aggtgctgga ggagaagaac tacggcctgg gctggtacgt gctgtggcag 780
ggcgtgaagc aggccctgaa ggagcagaag aagccgacca agatccagat cgccgtggac 840
cagctgaggc agccgaagtt cgccggcctg ctgaccgcca agtggagggc cctgaagggc 900
gcctacgaca cctggaagct gaagaagagg ctggagaaga ggaaggcctt cccgtacatg 960
ccgaactggg acaacgacta ccagatcccg gtgggcctga ccggcctggg cgtgttcacc 1020
ctggaggtga agaggaccga ggtggtggtg gacctgaagg agcacggcaa gctgttctgc 1080
tcccactccc actacttcgg cgacctgacc gccgagaagc acccgtccag gtaccacctg 1140
aagttcaggc acaagctgaa gctgaggaag agggactcca gggtggagcc gaccatcggc 1200
ccgtggatcg aggccgccct gagggagatc accatccaga agaagccgaa cggcgtgttc 1260
tacctgggcc tgccgtacgc cctgtcccac ggcatcgaca acttccagat cgccaagagg 1320
ttcttctccg ccgccaagcc ggacaaggag gtgatcaacg gcctgccgtc cgagatggtg 1380
gtgggcgccg ccgacctgaa cctgtccaac atcgtggccc cggtgaaggc caggatcggc 1440
aagggcctgg agggcccgct gcacgccctg gactacggct acggcgagct gatcgacggc 1500
ccgaagatcc tgaccccgga cggcccgagg tgcggcgagc tgatctccct gaagagggac 1560
atcgtggaga tcaagtccgc catcaaggag ttcaaggcct gccagaggga gggcctgacc 1620
atgtccgagg agaccaccac ctggctgtcc gaggtggagt ccccgtccga ctccccgagg 1680
tgcatgatcc agtccaggat cgccgacacc tccaggaggc tgaactcctt caagtaccag 1740
atgaacaagg agggctacca ggacctggcc gaggccctga ggctgctgga cgccatggac 1800
tcctacaact ccctgctgga gtcctaccag aggatgcacc tgtccccggg cgagcagtcc 1860
ccgaaggagg ccaagttcga caccaagagg gcctccttca gggacctgct gaggaggagg 1920
gtggcccaca ccatcgtgga gtacttcgac gactgcgaca tcgtgttctt cgaggacctg 1980
gacggcccgt ccgactccga ctccaggaac aacgccctgg tgaagctgct gtccccgagg 2040
accctgctgc tgtacatcag gcaggccctg gagaagaggg gcatcggcat ggtggaggtg 2100
gccaaggacg gcacctccca gaacaacccg atctccggcc acgtgggctg gaggaacaag 2160
cagaacaagt ccgagatcta cttctacgag gacaaggagc tgctggtgat ggacgccgac 2220
gaggtgggcg ccatgaacat cctgtgcagg ggcctgaacc actccgtgtg cccgtactcc 2280
ttcgtgacca aggccccgga gaagaagaac gacgagaaga aggagggcga ctacggcaag 2340
agggtgaaga ggttcctgaa ggacaggtac ggctcctcca acgtgaggtt cctggtggcc 2400
tccatgggct tcgtgaccgt gaccaccaag aggccgaagg acgccctggt gggcaagagg 2460
ctgtactacc acggcggcga gctggtgacc cacgacctgc acaacaggat gaaggacgag 2520
atcaagtacc tggtggagaa ggaggtgctg gccaggaggg tgtccctgtc cgactccacc 2580
atcaagtcct acaagtcctt cgcccacgtg tga 2613
<210> 24
<211> 2895
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.6编码核酸序列
<400> 24
atgtccgcca acagggtgtc cgccaactcc cagttcgagc tgggctaccc gatgtccctg 60
tccctgaggg gcaaggtgtt caactccagg gagatgatga aggagatcct gccggtgatg 120
aacaacatcg tgcactacca gaacaacctg ctgaagctga tgctgatcct gaggggcgag 180
aagtacaccc tggacggcca gttcttctcc cagaaggacg tggacaggca gttcggcgac 240
ctgtgcaagg agcacaacat caagggctcc atctgctccc tgaaggagaa gtccaggaag 300
ctgtacgagg tgttctcctg ctacatcgac aagaagggca acctgaagac caactccaag 360
gccaggtcct tcgccggcgt gctgctgaac ccgaaggacg tgaagctgcc gccgcagatc 420
gactccatct cctccttcgt ggtggagctg agggccaagg gcgtgctgcc gatcaagcac 480
gagggcaact acctgtccgg ccacccgtcc ctgaagtact ccgtggccca gaacgtgctg 540
gtgaagctga cctccatgga gaagctgcag aagatctact ccgacgagaa ggccggctgg 600
gagaacatcg tgtccgaggt gaggtccgac ctgccgaaga tcgagaggta cgagaggatg 660
ctgctgtcca tcaaggccgt gaaggagatg gagaagttcg gcatcaacaa ctacaggcac 720
ctgctgaaca actggaggga cgaggtggac aaggactccg gcaaggtgct gaagcagggc 780
atgaggacct acttcgtgaa catgctggag tccaagaagg actacaggtt cgaggagtcc 840
gacaggtacc tgttcggcta cgccccggag gtgatgaacc tggtgtacca cgacttcagg 900
gacctgtggc agggcgagga catcatcggc tcccagtccc cggagaagaa ggacagggac 960
tacgtggacg tgatcttcaa ctacttcaac tggaggaagg agtccatcaa catctcctcc 1020
ttcgactcct acggcaagac cgcccagatc aagctgggcg acaactacgt gccgttctcc 1080
aacttccagt acgacaagat cctggacgcc tggaccctgg agatcgccaa cgtgtccggc 1140
gagggcgaca accacaagct ggtgatcgcc aggtccccgc agttcgactc ccactcctcc 1200
gtgaaggaca tcgtgatgaa gaacctgaag ggcaaggagg cctccaagac caccctggag 1260
ttcaggtact ccggcgactc caagaagtcc acctggtaca ggggcaccct gaaggagccg 1320
accctgaggt actcctcctc caagaactgc ctgtacgtgg acttcgccct gtccaaccac 1380
atcgtggagg gcctgatctc cgacaacctg ggcatctccg acaagatgta caagttcagg 1440
ggcgagttca tgaaggcctc cccgtcctcc ggcaagcagt ccaactccat caacctgccg 1500
atcaagaagc tgagggccat gggcgtggac ttcaacctga ggaggccgtt ccaggcctcc 1560
atctacgacg tggagaacaa gaacggcaac ctggagttct ccttcgtgaa gcacgtgcag 1620
tccttctcca acgagaacga cgaggagagg gccaaggagc tgctgaacat cgagaggaac 1680
atcctggccc tgaagatcct gatctggcag accgtgggct acgtgaccgg caagaacgac 1740
accatcgacg gcgtggtgac caggaagaac aacgccgtgg acatcgagaa gaccctgggc 1800
atcaacatga aggagtacat ggcctacctg aaccagttca ggtcctacga ggacaagaac 1860
aaggccttca tggacctgag gaagagggag tacgcctgga tcgtgccgcc gctgatcttc 1920
cagtgcaggt ccaggctgat ctccttcagg tccgagtact tcaacacccc gaaggacgag 1980
aagtcccact actgccagca caggaacttc gtggactact ccaccttcct gaagaagaac 2040
gtggtgaaga agatgatgga gctgaggagg tcctactcca ccttcggcat gtcctccgag 2100
cagtccatct gggtgaccaa caacgaccac gccaaggacg gctccaagaa gaacggcaac 2160
atgttcgacg acgacctgca ccagtggtac aacggcctgg tgaggaagtg ctcctccctg 2220
gcctcctcca tcatcaacgt ggccagggac aacggcgcca tcctggtgtt catcgaggac 2280
ctggactgcc acccgtccgc cttcgactcc gaggaggaca actccctgaa gtccatctgg 2340
ggctggggct ccatcaaggc ctccctggcc caccaggcca ggaagcacaa catcgccgtg 2400
gtggccaacg acccgcacct gacctccctg gtgtcctcca ccaccggcga gctgggcatc 2460
gccaagggca gggacgtgct gttcttcgac tccaagggca agctgacctc caaggtgaac 2520
agggacgaga acgccgccca gaacatcgcc atcaggggct tcgtgaggca ctccgacctg 2580
agggagttcg tggccgagaa gatcgaggag aacaggtaca gggtggtggt gaacaagacc 2640
cacaagagga aggccggcgc catctacagg cacatcggct ccaccgagtg catcatgtcc 2700
aagcaggccg acggctccct gaagatcgac aagaccgagc tgaccccgct ggagatcaag 2760
atggagaaga agaacgacaa gaagatgtac gtgatcctgc acggcaagac ctggaggctg 2820
aggcacgagc tgaacgagaa gctggagaag gacctggaca accacctgaa gtccaagtcc 2880
tccgtgatct cctga 2895
<210> 25
<211> 2889
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.7编码核酸序列
<400> 25
atgtcctccg ccaacgacca gctgggcctg ggctacccgc tgaccctgac cctgaggggc 60
aaggtgtaca accacgacac cgccatggag gccttcgccc cggtgatgaa gggcatggtg 120
ccgtacgcca acaacctgat gaggatcctg ctgaccctga ggctggagaa gtacaccctg 180
gacggcatcc accacaccaa ggaggaggtg gagaaggacc tgaggggcct gatgaaggag 240
tacggcatca acctgtcctt cgccaagttc tccgagatgg ccggcgaggt gtacagggtg 300
ttcgtgtgct acgtggacgc caagggcaag ctgaaggtga acggcaaggc caggggcttc 360
gccaacgtgt tcttctccga ggacgacgcc accatcccgg agaactgccc gtccatggag 420
ctgctgagga agaagggcat gttcccgatc ctggtggacg gcaagccgat ctcctccatc 480
tccagggaga agaccccgct gaagtactcc gtggcccagg acgtgctgac caagctgacc 540
tccatggagg agatctccaa ggagtacgag aaggccaaga ccgactggga gaacgagtgc 600
cagaaggtga tctcccagct gccgctgatc ggcaggtacg aggccctgct gaccaccatc 660
ccgctgatcc cggagatgag gggcttcgac ggcgacaact acaggaagat gctgaacagg 720
tggagggact acgtgaacga ggacggcgag ctggtgaggg gcggcatgaa gacctacttc 780
ctggacctgc tgtccaagga cacctcccac aagttcaacg aggaggagag gtacctgttc 840
ggctactgcc cggagttcat gaacctgatc taccacgact tcagggacct gtggtccaag 900
gaggacatca tcggctccca gaggaagggc aagggcctga agggcaagga ctacgtggac 960
gtgatcttca actgcttcca ctggaggagg gagtccatca acatctcctc cttcggcaac 1020
aacgacaagg tgatgaacat ccacctgggc gacaacttcg tgccgttcga gctgaagtcc 1080
cagaacggca tctgggaggt gcacgtgcag aacctgcacg gccagaacga cccgcacagg 1140
gtgatcgtgt gcaggtgccc gcagttcaac gaggactcct ccatgaagat ggtgcacccg 1200
ctggccaaga acggcgagga gtccgacaag gagaacatcg agttcaggta ctccggcgac 1260
tccaagaggg agacctggta caccggcctg ctgaaggagc cgaccctgag gtacgacgtg 1320
gagaggaagt ccctgtacgt ggacttcatc ctgtccaacc acagggtgga gggcgtggtg 1380
accaacgagt acctgaagga cccgagggac ctgttcggcg tgaggggcta cttcctgtcc 1440
tcctccgtgt ccaacccgag gcagaaggac aagacctccc tgccggacgg caagttcaac 1500
gtgatgggcg tggacctggg cctgaagtgc ccgtacgagt gcgccatcta cggcatcacc 1560
gtgaagaacg gcaagatgca gcacaagtgg tcccacaacg tgtccgccga ggacaacaac 1620
aacgtgtccg agaggctggc caacctgaag aagatcgacg agaagatcct ggccacccag 1680
gtgctgatct ccctgaccaa gatgtgcgtg gtgaaggacg aggagatccc ggactcctac 1740
accctgaggg agcacagggt ggacatcgcc aagtccctgg acctggacat ggacaagtac 1800
aggaggtacg tggagaagtg caagaagaac ccggacaaga tccaggccct gaaggacatc 1860
aggaagtccg agaacaactg gatcgtggcc gagaagatca acgagatcag gtccctgatc 1920
tccgagatca ggtccgagta ctacgcctcc aaggacaaga ggaactactg caggaacctg 1980
aacggcgtgg acctgtccgt gttcctgaag aagaaggtgg tgaagaactg gatctccctg 2040
ctgaggtcct tctccacctt cggcatgacc ccgcaggagt ccgcctacat caggaaggac 2100
ttcgccaaga acctgtccaa gtggtacaag ggcctggtga ggaagtgcgg ctccatcgcc 2160
gcccacatcg tgaacatcgc cagggacaac aaggtgatgg tgatcttcat cgaggacctg 2220
gacgccagga cctccgcctt cgactccaag gaggacaacg agctgaagat cctgtggggc 2280
tggggcgaga tcaagaagtg gatcggccac caggccagga agcacaacat cgccgtggtg 2340
gccgtggacc cgcacctgac ctccctggtg aaccacgagt ccggcctgct gggcatcgcc 2400
ggctccggca acgacaggaa catctacacc ttccagaaga acaagaagta cgtggtgatc 2460
aacagggaca acaacgccgc ccacaacatc gccctgaggg gcctgtccaa gcacaccgac 2520
atcagggagt tctacgtgga gcagatcgac gtggaccact acaggctgat gtacggcccg 2580
gaggccgaga acggcaagag gaggtccggc gccatctaca agcacatcgg ctccaccgag 2640
tgcgtgttct ccaagcagaa gaacggcacc ctgaaggtgg agaagacctc cctgaccaag 2700
gacgagaagg agatgccgaa gatcaacggc aagggcgtgt acgccatcct gcacggcaac 2760
gagtggaggc tgaggcacga gctgaacgag gagctgggcg ccaagctgga cggcatctcc 2820
gtgaagaggg tggtgtccga gccgaacaag gtgaagacct ccctggtgaa gggctccgtg 2880
agggcctga 2889
<210> 26
<211> 2724
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.8编码核酸序列
<400> 26
atgaagaagc agaccatcgt gaagaaggac tccaaggccg agaccaagga gaacaagatg 60
tacccggaca aggacaccga cttcccggtg aactcccagt tctccaggtc catctccatc 120
agggccaacg tggacccgaa ggacctgctg gtgctgaaga ggaccttcga ggagaccacc 180
aagatctccg acgagctgct gtccaccctg ctgatgctga ggggcaagga ctactgcctg 240
gacaacgtgg tgtgcaaggg cgaggaggtg ctggagaacc tgtacaagaa gctgtccaag 300
aacgccaccg tgaacaggga caagttcatc tccaccgcca aggccttcta cgagtacttc 360
cacggctgct cctaccacaa gggcttcaag tccttcttct tctcctccaa ggagatcgac 420
tccatccagt ccgagaagtt cggctacctg agggagatcg gcctgttccc gatcaagatc 480
gacgcccaga tctccaacga cctgcagtac tccatcgtgg cctccaacca cgccaagatc 540
aagggcttcg agaagatcga caaggagtac caggccaaca aggagaagtg gaacaagacc 600
atcggcgagt ccaccctgaa gcacctgaac aggtacggcg agatgctgaa gggcctgtcc 660
gacctgggca ccatgggcaa cttcaacggc aagaagtacg acaggttcat gggccactgg 720
aggaacgagc agaagatccc ggaccacatc tccatgctgg acttcttcag gaagatctac 780
caggagaagg gcaagtccca caggttcacc gccatcgaca acttcaccta cggctacgag 840
tccgagttca tgaaccacat ctacctgaac ttctccgacc tgtggctgaa ggaggacgtg 900
atcggcgacg aggagtacgt gtccctgatc aggggcgcct accactggca gaaggacgtg 960
gtgggcatcg cctccttctc cggctacaac aagtacgaga agctgttcat gggcgacaac 1020
aagatcaact acgccctgga cttctccaac aaggaccagt ggctgatgaa gttcaacaac 1080
gtgatctcca aggagccgga gaccatcacc ctgaggctgt gcaagaacgg ctacttcaac 1140
aacctgtccg tgctggagaa gaacgacgag aacggcaggt acaagatcag gttctccacc 1200
gagaagcagg gcaagtactt ctacgaggcc ttcatcaggg agccgttcct gaggtacaac 1260
aaggacaacg acaagatcta cgtgcacttc tgcctgtccg aggagatcaa ggagaactgc 1320
ccgaaccacc tggacaccag gtccgacaag tacctgttca agtccgccct gctgaccaac 1380
tccaggcaga agctgggcaa gctgcactac agggacttcc acatcgtggg cgtggacctg 1440
ggcatcaacc cggtggccaa gatcaccgtg tgcaaggtgc acgtggacaa gaacgagaac 1500
ctgaagatca ccaagatcat caccgaggag accaggaaga acatcgacac caactacctg 1560
gaccagctga acctgctgta caagaagatc gtgtccctga agaggctgat cagggccacc 1620
gtggccttca agaaggacgg cgaggagatc ccgaagatgt tcaagatggg caagaagtcc 1680
ccgtacttcc tgaactggac cgaggtgctg aacgtgaact acgacgacta catcaaggag 1740
atctccacct tctccgtgga caggctgtcc ggcctgaccc tgccgatgca gtgggccagg 1800
tcccagaaca agtgggtggt gaaggacctg accaagatgg tgaggaaggg catctccgac 1860
ctgatctacg ccaggtactt caactgctcc gacaagaccc agtacgtgac cgagaacaac 1920
gccgtggaca tcaccacctt caagaagcac gacatcatct ccgagatcat cggcctgcag 1980
aagatgttct ccggcggcgg caaggacgtg gccaagaagg actacctgta cctgaggggc 2040
ctgaggaagc acatcggcaa ctacaccgcc tccgccatcg tgtccatcgc ccagaagtac 2100
aacgccgtgt tcatcttcat cgaggacctg gacctgaaga tctccggcat gaacggcaag 2160
aaggagaaca aggtgaagat cctgtggggc gtgggccagc tgaagaagag gctgtccgag 2220
aaggccgaga agttcggcat cggcatcgtg ccggtgaacc cggagctgac ctcccagatg 2280
gacagggaga ccttcctgct gggctacagg aacccgacca acaagaagga gctgtacgtg 2340
aagagggacg acaagatcga gatcctggac gccgacgaga ccgcctccta caacgtggcc 2400
ctgaggggcc tgggccacca cgccaacctg atccagttca gggccgacaa gatgccgaac 2460
ggctgcttca gggtgatgcc ggacaggaag tacaagcagg gcgccctgta cggctacctg 2520
aactccaccg ccgtgctgtt caaggacaag ggcgacggcg tgctgaccat ccacaagtcc 2580
aagctgacca agaaggagag ggactccagg ccgatcaagg gcaagaagac cttcgtggtg 2640
aagaacggca agaggtggat cctgaggcac gtgctggacg aggaggtgaa gaagtacccg 2700
gagatgtaca actcccagaa ctga 2724
<210> 27
<211> 2739
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.9编码核酸序列
<400> 27
atgtccgact acaagttctc caacaacggc gtgaccaaca ccggctccgc ccacatcggc 60
ctgtccccgg agaactcctc caccgtgatg gacatgttca aggtgatcac caaggacgcc 120
gacttcctgc tgaagaacct gctgatcatg gagggcggcg agtacatgct gaacagggag 180
atccacaacg gcgacaagga gttcgacaag atcatctcca agctgggcct gtccaagaag 240
gagaaggaga acctgaagat gaagtgcaag gacttcttct tcgacttcgt gaagctgcag 300
aacggcaggt ccctggccaa catcctgttc gagaccaagg gcaccaccct gatcggctgc 360
ggcaaggaca agaagggcga gaaggtggac ggcgagtacc cgaccatcta ccacgaccac 420
gagaccctga ggtccaccgg cctgctgccg ctgaagttct ccaagaacat cgacgacgtg 480
gactacaagt acctgatctg ctacctggtg cacaacgtgc tgtcctcctt catcgagaag 540
agggacgcct acaacgacaa caagaaggag tgggagtcca agctgtccaa ctccaacctg 600
ccgcagctgg agaggatgtc cgagttcctg aacggcatca accacctggg caacatcatc 660
ggctggaacg gcaagaagta catcggcttc atcaagaagt ggaccgacga ggagtcctcc 720
atgtacgact tcttcgtgca gaagctgcag gacaacccga agtacaagtt cggcaagaag 780
gaccagttcc tgtacggcta cgagccggag ttcctgaact acctgttcca cgacttcagg 840
gacctgtggc acccggacaa cctgatcggc aaggacgagt acgtggacct gatctccggc 900
aagaacaaca ccgacgccga gaccgccaac aagggcgcct accactggct gaaggacttc 960
atcaacatct cctccttcga cgcctacggc aagatggcca ccatcggcat gggcaacaac 1020
ctgatcaact actccatgaa catcgacaag gacggcaaga tcatcgtgaa catggacaac 1080
atcttcgaca ggtccaagcc gatcgtgttc aacgtgtaca ggaactccta cttcaggaac 1140
ttcaagatca tcgagtccga cgacaagaag ggcatctaca aggtggagtt ctccacctcc 1200
aacaacggcg tgatctacga gggctacatc aagtccccgt ccctgaggtt cgccaccaag 1260
ggcggcacca tcaagatcga cttcccgatc tccgacaaga ggatcaaggg cggcagggag 1320
atgaacaccg acctgatgtg gttcctgaac agggcctccc cgtgctccac caagaacaag 1380
gaggtgaact ccttcatcgg caagaacttc gtgggcctgg ccatcgacag gggcatcaac 1440
ccgctgatgg cctggtacgt ggccgagtgg acctacgaca aggacggcaa ggccaagatc 1500
gtgaggtcca tcgccaacgg cagggtggac tccggccaca acgagtccga ggtgaagttc 1560
gtgagggaga ccaccaacag gatcgtgggc atcaagtccc tggtgtggaa caccgtgaag 1620
tacaggaccg gcggctccga gggcatcgac aggtgcagga agtcccagaa cggccaggtg 1680
gacctgttcg agatgttcga catcgactac aacaactacc tgaaggaggt gaacaacctg 1740
ccgtacgacc cgaactccga gaggtccatc atccagacct gggtgtcctc cccgtggaag 1800
gtgaaggacc tggtgaagga cgccaagaac aggatggtgc agatcaagac ccagtaccac 1860
aacgccaagg acaaggagaa gtacatcacc acccagaaca gggccggctt ctacgacttc 1920
ctgaagatcg agatggagaa gcagttcacc tccctgcaga ggatgttctc cggcggccag 1980
aaggacatct gcaagaacaa cgaggagtac aggaggggcc tgaggaggag gatcaacctg 2040
tacacctcct ccgtgatcat gtccctggcc aggaagttca acgtggactg catcttcctg 2100
gaggacctgg actcctccaa gtcctcctgg gacgacgcca agaagaactc cctgaaggac 2160
ctgtggtcca ccggcggcgc cgacgacatc ctgggcaaga tggccaacaa gtacaagtac 2220
ccgatcgtga aggtgaactc ccacctgacc tccctggtgg acaacaagac cggcaagatc 2280
ggctacaggg acccgaagaa gaagtccaac ctgtacgtgg agaggggcaa gaagatcgag 2340
atcatcgact ccgacgagaa cgccgccatc aacatcctga agaggggcat ctccaagcac 2400
atcgacatca gggagttctt cgccgagaag atcgaggtgt ccggcaagac cctgtacagg 2460
atctccaaca agctgggcaa gcagaggatg ggctccctgt actacctgga gggcaacaag 2520
gagatcctgt tcggcctggg caagaacggc gagccgatcg tgtgcaagag gggcctgtgc 2580
aagaaggaga ggctggcccc gaggatcgcc gagaagaagt ccacctacct gatcatgaac 2640
ggctccaagt ggatgttcag gcacgaggcc aagaagatcg tggagaccta caaggacagg 2700
tactgcgcca accacaaggt ggcctccaag gacggctga 2739
<210> 28
<211> 3360
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.10编码核酸序列
<400> 28
atgatgaaca tcaacgagat ggtgaagctg atgaagtccg agtacctgtt cgaggacgac 60
ggcatcgtga ccaagaacaa gatccaggag aggctgagga acggcttctc cgacatcggc 120
gtggacccgt ccctggtgtc ctacgcctcc aagttcctgg actccatgtt catctgcttc 180
tccagggtga agggcgagaa gaacttcaag gccaagaacg tgaggaagaa catgtcctcc 240
gccgagaaga aggcccagaa gaagaaggag taccaggagt actaccaggg cgtgatggcc 300
cagcaggacg cctacgccca gctgctgtcc gacccgaccc aggagaacct ggacaagctg 360
aacgagctga tctccatgtc cgtgaacggc tccctggtgg aggacttctt cccggccctg 420
aagaacatga tccagaaggc cgactactcc atcgacaaga agggcctgct ggacttctcc 480
tgctgcatga tggacaggta cgaggacagg tccctgacca gggccatctc catctccgcc 540
ttcaacatcc actccggcgg cctgaggaag gccctgtccg acatctccga gaaggtgcag 600
gacctgtcca acaccctgct gatcaggatc ctgtacatga agggcgagga gctgtccatc 660
gacggcgaga agatctccaa ggaggaggtg cagaggcagc tgaaggccga ctacgaggag 720
cacaaggagt acttcgagga cttcgaggac ttcgccaaga agtgcaggtt cttctacaac 780
aagttctcca agaagaagaa gaccaggggc ttcggcacct acttcttcgg cgacaagaag 840
aaggagatct cctccgccga gtacaaggcc cacaaggagc tgagggactc cggctacctg 900
tggttcgaca tcggctggtc cgagtcctcc gacttcaagt acgtgatcgt gggcaacgtg 960
tccggcaagc tgaagtcctt cgaggagacc tccgaggagt accagaagtc caagaactgc 1020
tgggaggccg agagggtgaa gctgtacgag caggactccg acttcgtgct gttcgtggag 1080
gacatgatcg agtccaagta cggcccgatc gagaagatga agctgaggac cttcaagacc 1140
atcgtgaaga agctggacaa ggagttcggc aagaggggcg acaagacccc gtccatccac 1200
gactacttcg agtccctgga cccgaaccac accttctccc agtccgagca gttcatgtac 1260
ggcctggacg tgaccctgat gcagttcctg ttcaacaaca agaagcagtt ctacaagctg 1320
tgcaaggacc acgacggcaa gaggaccttc gccaaggtgg tggaggagtc ctaccactgg 1380
ggcaagaact ccatcaacgt gtccaccttc cagaactcca cctccatcct gctgggcggc 1440
aactacctga actactccat gtccatcgag ggcgagggcc tggtgatcaa gttcgacaac 1500
ccgctgtccg gcaaggaggt gcacttcgtg gtgtgcaaca acaagtacct gtccgacctg 1560
gagatcctgt ccggcaaccc gaacaggaag gacaacaact acaccatctc ctactccacc 1620
ggcggcaagg ccaggttcat cgccaagtcc aaggagccga ggatcttctt caacaggaag 1680
accaagaagt gggagatcgc cttccagctg tccgacgtgt ccccgctgaa cggcaagttc 1740
ggcaagcagg gcgagttcct gtccaacctg aggaagttcg tgtacaacca cgtggccaag 1800
tccccgtcca agctgaacat ctccgacaac aactgcaggg ccgtggccta cgacctgggc 1860
atcaggaacg tgggcgcctg gtcctccttc gacttctcct acaaggacgg cgtgctgggc 1920
ggctacaagt acctgacctc cggctccctg aggtccaagt ccgagtcctc cgagatggac 1980
cagggctact acttcgtgct gaacctgaag aagatcgtga agctgatccc ggtggtgaag 2040
aagtccatca tcgacgaccc ggagctgaag aggcagttca tcggcgtgct gaacgagaac 2100
ggcaacaccg tgggcctggg caacatcggc aagctggaca tcgcctccag gaaggccgtg 2160
cagtccttcc acaactgcat ccagcagatc aactactacg tggacaccta cgccgaccac 2220
atcgacaaga tctccgccaa ggacttcgtg gacgacatcg acggcatcaa ggtgctggac 2280
gaggacgacc cgtacgtggt gaagatcctg tcccacctgc cggaggacgt ggagggcaac 2340
caggacgaca tcctgaacat ctccctgctg aagtggaaga cctccaacgc ccagttcgtg 2400
ccgccgctga tccaggaggc caaggccatc atgtccagga tcaagaggga gaacctggac 2460
aacatcaggg gcaagaagac ccaggtggtg acccagaaga ccttccacaa gatcaagttc 2520
gccaaggccc tgctgtccct gatgaagtcc tggtcctcca tcggcaccgt gagggtggtg 2580
aagaccgacc agatctacgg caagaagatc tgggactaca tcaacggcct gaggaggaac 2640
gtgctgacct acctgtcctc cgccatcgtg aacaacgccc tggacctggg cgcccacatg 2700
atcatcctgg aggacctgga ctcctccgtg tccaagtaca gggagaagga caagaacgcc 2760
atccagtccc tgtggggctc cggcgagctg aagaagagga tcgaggagaa ggccgagaag 2820
cacagggtgg tggtgcagta cgtgtccccg tacctgacct cccagctgga caacgagacc 2880
aaggacatcg gctacaggaa gggcggcagg ctgtacgtgg tgaggaacgg caagatcaag 2940
tccatcgacg ccgacatcaa cgcctccaag aacatcggcg agaggttctt cgacagggac 3000
ctgatccaga ccctgtccgg cgtggtggtg gaggaccagt ccaccgtgta catcctgcag 3060
aagaggaacg tgtcctccga caacaggaag aggttctaca agaagttcct ggaggacgtg 3120
ggcggcaagt ccaagaagga cgccgtgctg aagatgggcg accacggcga gctggaggtg 3180
gagaggctga tcgacggcaa gaagctggac atcgacggca agaagatcct ggtggacggc 3240
gagaaggtgc cgttcaggaa cacctccgtg tactactccc cgaagaagaa gaagtgggtg 3300
tccaaggagc tgaggtgcaa ccacatcaag ctgaccgtgg aggagcagga catcaagtga 3360
<210> 29
<211> 3408
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.11编码核酸序列
<400> 29
atgaacaact acgacaacta cctgtccgac tacctggcca tgctgccgca caccaagagg 60
accgagatca agaagaccgc ctccaagatc tccaggaagc tgaaccagaa ggaggtgaag 120
aagcagatcg agaggtccga gtacatcagg tccaactgcg gctacatcaa catcgagagg 180
ccgcagaagt ccctgtcctt cctgtcctac tccaccatca agtccgcctg catgtccgtg 240
aacttcaggg ccttccagaa cccgatcaac gactacgaga ccgccatctg caacggcatc 300
aacgagtgcg agaggttctt ctaccagcag atcgactcca tctacatgtc ccagatcatc 360
gagcagctgt tcgacttcta catcgcctcc aggcagcacg acatgttcat caacaacacc 420
gtggtgccgt acgacgtgaa caagctgaag tcctactaca ccgccaacga gaagtactcc 480
ttcgagcagt tctgcgacga catcaaggag ttcaccaaca agggcttcac ctccggcggc 540
gtgtcctgca tcctgaacct gttctacaag ggctccgtga aggactccaa gaacaagaag 600
gactacatca agtccgtgaa gaggctggag accaacggcc tgttcaagaa gctgaacatc 660
ttcgagaaga acggcatctc caagtacttc gccgcctcca ccctgtccac cttcttcgcc 720
accatctcct cctggaagaa gcagaacgac gactggaccg gcgtggccaa ggacggcacc 780
tccctgctgt ccaagctgga gaacaagacc atcaccctgc agtccatcat caagcaccac 840
agggtgatca acgagctggc cgtgctgatc gtgaaggcct acaaggaccc ggtgaagacc 900
ctgaacaacc tgttcgagga gaggtccgac aacaacaacg acttcaagta cacctgctcc 960
gacgacgagg acaagtaccc gatgtacatc aagagggaga tcgccgagtt cgtgaagaag 1020
cacaagaccg tgtgggagga gatcaggtac ttcgacgagt ccgacaccaa gaagaagaag 1080
agggacaaga aggagtcctc ctccgacgac aagtcctacc tgtgctgcgg cgactcctgg 1140
gactacctga agacctgggt gaggctgtac ggcgagtact acttcttcga caacgccctg 1200
aaccagttcc tgaggaagcc gtccgcctcc atgcacctgt acacctccct ggactggatc 1260
aacaagaaga ccatctgcat cgtgggcgcc aactactaca agatcggcaa ggtggaggtg 1320
gtggagagga acaaccagag gttcctgctg gtgtacgtgt ccgtgccgga gatggagaac 1380
tacatcatca tcccgctgca gctgaacaag tacttcggca acttccagtg caagatcttc 1440
gagggcaggc tgcaggccat cttcaagagg tacgccaact tcaacgccct gaagaacaac 1500
aagccgcagc cgtccccgaa catctccgtg aggatcaacg agttccactt cgccctgagg 1560
tcctacagga agcagcagat ctccgccgag gacttctcca agggcaggtt ctccctgatc 1620
tccaagatcg gcttccagat gaccaacgac gaggtgttcg gcaggacccc gagggagatc 1680
gccctggtga aggaccacct gtccaagggc tacgtgcact tcggctccca gatcatcgag 1740
gactccagga aggaggtgga gcaggtgctg aagaagccga tgatcctgat gggcgtggac 1800
ttcggctact ccccgctggc ctcctacaac atcaagccgc tgcagaccgg caagccggcc 1860
accgactggg tgaagaacct gcacggcaac ttcctgtgcc agaacgtgtc cctgggcgag 1920
accatcaccg agggcgagat cggcgacgtg ccgaccgaca cctacacctc ctccaacgag 1980
atctactcca tcgccaccct gaccttcagg aacgccgacg gcaagctgga gaacaggtcc 2040
ttctccaggt tctaccacga gctgaacaac accctgaaca tcatcgagca gatcaagggc 2100
accttcaact tcatccactc catcaacacc cagttcaagg agatcaaggc cctgaagacc 2160
accgaggagt tctcctccta cgtgtccacc ctgacctggg accagttcat cgaggactcc 2220
aggaagaccg ccaggtactc caagtactgg atccacatca tcaacgagaa cccgaagagg 2280
aggaccatcg ccaccctgaa cgagaccctg aagctggtgg acgagaagca caggttcacc 2340
gtgaccatcc aggagatctt cgacctggtg aagtactgcc agcagcacgg ctactacccg 2400
aagtccaacg tgatgtccaa gctgaggaac ctggccatca agctgatcaa cgacctgatc 2460
aggtaccaga agatcggcat ccactcctgc tacctggact tctgcgtgct gatcaagaac 2520
cacatcgccc tgctgaactc ctccaccgcc ttcatcatca acttctccag gaacaaggag 2580
aacatcatca ggaacaacac ctccaagatc cactccctgt gggtgtacag ggacaacttc 2640
aggaggcaga tgatcaagaa cctgtgctcc cagatcctga agatcgccgc caagaacaag 2700
gtgcacatcg tggtggtgga gaagctgaac aacatgagga ccaacaacag gaacaacgag 2760
gacaagaaca acatgatcga cctgctggcc accggccagt tcaggaagca gctgtccgac 2820
caggccaagt ggtacggcat cgccgtggtg gacaccgccg agtacaacac ctccaaggtg 2880
gacttcatga ccggcgagta cggctacagg gacgagaaca acaagaggca cttctactgc 2940
aggaagcagg acaagaccgt gctgctggac tgcgacaaga aggcctccga gaacatcctg 3000
ctggccttcg tgacccagtc cctgctgctg aaccacctga aggtgctgat caccgaggac 3060
ggcaagaccg ccgtgatcga cctgtccgag aggaccaccg agccgcagaa gatcaggtcc 3120
aagatctgga ccaactccga cgtgcagaag atcatcttct gcaagcagga gaacggctcc 3180
tacgtgctga agaagggctc caccgacatc aaggagaaga tgcacaaggc cgtgctgcac 3240
aggcacggct ccctgtggta cgactacctg aaccacaaga acatgatcga ggacatcaag 3300
aacctgcacc tgtccaactg ctccctgacc acctccacca actccgacgt gatcaactcc 3360
cactccggct cctccaggtc cctggacaag accaagacct acgcctga 3408
<210> 30
<211> 3042
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.12编码核酸序列
<400> 30
atggcctcct ccgacgccca gaagttcccg cagacccaca acaaggtgat gtccttcagg 60
ctgaccgcct ccaacatcgg ctccgtgctg tccctgcact ccaacctgca cgacgccgcc 120
gagatcggca tcaacgagtg caggtggtgg atcggcgacg gcgagatcta cgagagggac 180
ccggcctgca ggtccatcaa gaagggcaac gacatcagga ccgtgacctc cgagaagatc 240
aaggagctgt ggaccaagca caccgaccac tccgtgccgc tggtggactt catcgacatg 300
ctgaagttcg tggcccagtg cgccatctac ggcgactcca gggccctggc ctccaccctg 360
ttcggcaagt ccaaggccga gaccaggggc gtgtccaccg aggacatgac cgtgatcagg 420
gcctggatcg ccgagaccga cgccgtgctg gcctccggcc tgtccccgaa gaagaagaag 480
aagaaggaga aggaggccgg caagaaggag aggaagccgg acgtgaagat ggagatgtgc 540
aggaggatca ggtgcaccat ggtgcagtgc ggctacttca ggaggttccc gttcgaggcc 600
aagatcgaca acggcggcga gaggggcaag atggactccg agctgtccta cgtgtccgcc 660
aggaacctgc tgaggtgcct gtccacctgg agggcctcct ccgtgatgag gagggactcc 720
tacctgatcg aggaggagag gatcaaggag gccgagtcca agatgacccc ggagatcatc 780
gacggcctga ggaggctgta caggtactgc gccgtggacc acgacttcct gaagtggttc 840
ggcggcagga tcatcaggca catcgactcc tgcctggccc cggccatcgc cggcaacacc 900
ggcaggccga ccggcggcga gtccttcacc gtgatctacg acaggaggaa gaagagggac 960
gtgaagatca cctactccgt gccggaggag atctacggct acctgtcctc ccacccggag 1020
ctggtggcca tcggcaagga cggcatgacc ccgatctcca ggcacgccga ctacctggag 1080
atgatcgcct cccacgagaa gcacaggtgg tacgccacct tcccgaccgt gggcaaggag 1140
gacggctaca ggacctccgt gctgctgggc aagaactacc tgacctacga cctgtcctac 1200
gacggcgagt ccgtgccgga caagaagatc aacgtgatct ccaagggcca gccggtgtgc 1260
ctggacctgc acgacggcag gagggtgtcc tccctgtacc tgaccgtggg cgagtccgcc 1320
gcctacgaca tcgccgtgag gaagaacaag aggcaccacg gcaagccggc cgactactgc 1380
aggatgaggg tgcacctgac ccaggagagg gaggacaaga cctacaacga cccgtacttc 1440
tccaacatgg agatctggag ggccggcgac caggtgtacg ccatcgagtt cgacaggcac 1500
ggcgccaggt acaccgccat cgtgaaggag ccgtccgtgg agtacaggaa caagaagctg 1560
tacctgaggg tgaacatggt gctggactcc ccgtccaggc aggacgacaa ggacatgtac 1620
tacgcctaca tgaccgccta cccgtcctcc aacccgccgg tggagacctc cgacaacaag 1680
aagaggttcg agaggctggg cccgggcagg agggccatcg gcggcatcga catcggcatc 1740
ggcaggccgt acgtggccgt ggtggcctcc tacgaggtgg gcccggccgg caccgagcag 1800
aagttccaga tcgaggacag gctgatcgag gacgacggct cctccccgta cgactccctg 1860
tacaacgact tcctgaccga catcaggacc gtgtccagga tcatcgaggc cgccaagaag 1920
atctccgagg gcgacctgga ggacatcccg tccgacatgt ccgtggacga ggacggctcc 1980
atcgccgcca ccatgaagag gatgtccgcc aggatcgccg agaggcacca cctgtacggc 2040
gagaggaagt ccgaggccta cgccaccttc ctgaagatga accacaagca gaggctggac 2100
atcctgctga cccagaaggc ctccaacgcc accctgaagc agctggtgga ggaggacccg 2160
tccttcctgc cgaggatctg cgtgtactac gtgatctccg tggagaggga gctgaagaac 2220
aagcacagga acgcctacct ggacggcctg accgtggacg agaagtactc cggcgagacc 2280
aagaggggct acgcccagaa gaggctgaac tccatgctga gggcctactc cgccctgggc 2340
gaggaggaga ccgacgaggt gaggaccttc tccaccaggt ccgagaaggt gaggaacatg 2400
gccaagaacg ccatcaagag gaacgccagg aagctggtga acttctacgt gggcaagggc 2460
atcaggacca tcgtggccga ggacaccgac ccgaccaagt ccaggaacga cggcaagaag 2520
tccaacagga tcaaggccgc ctggtccccg aagcagttcc tggccgccgt gaagaacgcc 2580
gcccagtggc acggcctgga gatcgccgag gtggacccga ggatgacctc ccaggtgcac 2640
ccggagaccg gcctgatcgg ctacagggac ggcgacaccc tgcactgccc ggacggctcc 2700
aagatcgacg ccgacgtggc cggcgccgcc aacgtgtgca gggtgttcgc cggcaggggc 2760
ctgtggaggt tctccatcaa caccaacatc gacatctcca acaaggacga gaagaagagg 2820
ctgagggcct acatcgtgca ccacttcggc tccgagtcca actgggagaa gttcaggaag 2880
cagtacccgt ccggcaccac cctgtacctg cacggcaggg agtggctgac cgccgaggag 2940
cacaagtccg ccatcgacag gatcagggac gacgtgggca gggacgccga gaacgaccac 3000
gtggccatcg tgaccgccgc cgagaaggtg gagatcttct ga 3042
<210> 31
<211> 3159
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.13编码核酸序列
<400> 31
atgtcccacg acctgaagcc gcagaggctg atcaggtcca acatcaccaa gacccactcc 60
gaccagaacg ccaagcaggt ggccgaggag gtgaagaagg agcacctgaa ctacctgctg 120
atcaagaacg agatgctgat ctccatcgtg ccggaggcca aggacgacga cggcaacgac 180
atcgacttca agaagcagct gaagtccctg tacaaggaga ccgaccagtc cgtgtccttc 240
tccgtgttct gccagatgat gaagttcagg aacatcgccc tgctgtacgc caagggccag 300
tccaggtggg ccgtgtcctc ctacttcacc ggcaacagga ggaaggacga ctacgccaag 360
gacctgtccc tgctggacga ggccatcgag ctgctggagt gcaagaggag gaagaaggcc 420
gaggaggaga acgaggagga gaacgagacc ccgaagaaga aggaggacaa cccgtccaac 480
atctccgagg agcagatcat gaagctgttc tacgccgtga acaagaagct gaaggagatc 540
ggctacctgg acaggtactc ccacatcgag aagcaggagc agtacgccat catcggcgtg 600
acctccagga ccgtgaaggc ctgggactac gccaacttcg ccaccaggaa ccactaccag 660
tccgtgcaga acgagtacca gaagaagctg aaggccctgc cgggcaccaa gaaggacaag 720
gtgtgcctgg agaagttctt cgaccacctg aacgagaaca acatcgccgc cgactgggac 780
aagtggaggc tgaagaagca catcctgcag tgcatcatcc cggccgccaa gatcggcctg 840
aaggagctga agcagtcctt ctacgtggac aacaagggca acaagcacaa ctacttcgtg 900
aacggcctgt acgaggagat cctgaagagg ccgttcctgt actccgccga ggacccggag 960
gagtccatcc tgtacctggg cgtggaggtg gcctccctgc actccaagct gaaccacctg 1020
aggtccgagg ccaggttctc cttcgagacc ccggacgaca tctgcaagta catgaccatc 1080
tgcggcgaca actaccacaa cttcaccatg tccgccatcg gcgaggacgt ggaggacatc 1140
gaggtggagg tgtacgacta caaccactcc aagaagtacg agaccatgag gttcatcaac 1200
ggcaagagga ccaccgacct gtccctgaac ttcaagggca tcccggtgag gctgtgcctg 1260
gagggcaaga ggaacaactc ctacttcgcc gacgccatcg tgtgggagct ggacaacaag 1320
gacaagaccg gctacctgat cgagtacggc aagtccaaca acaggctgta catgctggtg 1380
aaggagccgc tgatcggctg caggaggaag ttcggcaagg acgtgctgtt cgtgtccctg 1440
tccggcaccc tggtgaacaa gtacatcgag gacgacatcg tgtccgccag gtacctgatg 1500
cagaccgccg ccccgatctt caagacctcc agggccaaga agcaggacaa gatcggcgac 1560
aagtggttcg agcactgcca gggctccacc atcaagatcg ccggcatcga catcggcatc 1620
aacccgatcg ccgccatcac cgtggccaac gtgaccttcg acagggccct gggcaacaag 1680
atcaagaacc agaagcagat cgtgatcgac tgctacgccg aggactacaa gatcgacccg 1740
gtggtggtga agaggatgga ggacatcagg cacatcaagt acaccatcaa ctcctggtac 1800
cacctggccg actgctgcag gctgaaggcc gccaacaagg agtacgtggt gaacgagagg 1860
aagcagggct tcttcaggga gaacatcgag tacctgaagg aggtggccaa gaaggccatc 1920
accgagtccg accagcagat caaggagcag aaggccgccc tgaagaggtt cgacggcgag 1980
aagaagaagg agatccaggc caccatcaac ggcttcaacc tgaagatcaa gatcctgaag 2040
aagttcgtga ggcagtccgc caagaagatc ttcgactcca ccctggagac cctggagaag 2100
tacgacaaca acatcgagca ggccaagagg gacagggagt tcggcctgaa gatcatctac 2160
gacctgatca tcaagtacta caagaggtcc aagaaggaga gggagatgaa ccagaggatc 2220
tacgtggacg actacaacca ggaggagatc gacaccgaga ggaccaagaa gatcaggaag 2280
gagaccatca ccttctgcga caacgactgg aactccctga ccaagaggat ccacgacctg 2340
gagaagaaga tgaagaagat cggcatctcc gagccgggca gggtggagca ggagatcaac 2400
gacagggact actacaacaa catccaggac aacaccaaga agaggcaggc caagatcatc 2460
gtggacgccc tgaaggagga gggcgtgtcc atcatcgtgg tggaggacct gaccggcggc 2520
ggctccgaga acaccaagga gatcaacaag tccttcgacg ccttcgcccc gatcaggttc 2580
ctgaacgccc tgaagaactg cgccgagacc aacggcatcc aggtgaccga ggtgctgtcc 2640
ccgatgtcct ccaagatggt gccgtccacc ggcgagatcg gccacaggga caagagggac 2700
aagcagctgt actacaagga cggcgaggag ctgaagtcca tcgacggcga catctccgcc 2760
tccgagatcc tgctgaggag gggcgtgtcc aggcacaccg agctgatcgg caccatgaac 2820
gtggaggacg tgctggacaa gaacaacaac aagaacaagt gcatcaaggg ctacgtgtgc 2880
aacaggtggg gcaacatcca gaacttcgag aagatcctga aggagaaggg catcggcgag 2940
agggagatca tctacctgca cggcgacaag atcctgacca tggacgagaa gaggaccctg 3000
caggcctcca tcaggaagga gctgaaggag atgagggaga gggagtccgg cgaggagaac 3060
gccggcaccg ccaggaagaa gtccaagccg aagaagaaga agaagatcaa gaggaacaac 3120
gaccaggacc tgtccaacaa caggccggcc gcctcctga 3159
<210> 32
<211> 3138
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.14编码核酸序列
<400> 32
atgaaggaga acaagatgaa ggagaacggc tccatgacca cccactccaa ggtgatcgcc 60
ctgaagatga agtccgagaa cgtggagttc gacaccttct acaaggagtc cttcgagctg 120
ttcaagcagt tcaccaacga gttcgtggcc tggggcaacg acgagatcta ccagtacggc 180
tcctccaaga ggaagaagga cgaccagaag atctccctga tcccggtgat cgaggacatc 240
tacaagtccg tggagaagaa ggccaccgcc gagggcatct ccaagaccga cttcagggcc 300
gtgctgaagt acctgtacca ccagatcatc aacgtgggca actccggcag gtcctacggc 360
acctccctgt tcggcggctg cgaggtgaag gagaagctgt ccaagcagga catctccaac 420
atcgtggagt gcgtgaagga gctggagctg tgcaagtcca agcaggagga gtccgacgcc 480
tacgacaaga tcctgctgaa ggagaagatc acccacatcg tgaagtccgg cgagaccgcc 540
ggcgacatca ccaagaagta caaccaggcc accaccggca ggaagacctc ctccaagggc 600
ttcttcgaca agtccaccaa gaccgaggtg aagtacaagg acatcaagga cgacaccctg 660
ctgcaggacg gctccaccat cttcatcaag tcctccgtgg acctgttcgt gaagaaggtg 720
tgcaacaccc tgagggagat caacttcttc gacaggctgc cgttcaagaa caaccactcc 780
aacaactacg gcctgctgtt ctccatgctg tcccagatcg agtcctggaa gaccatctcc 840
gagaccacca agaagtccca cgaggagcac ggcgagaaga tcgcctccat ggtgaagaag 900
ctggacctga cccagaccga gctgatgaag gacttcgccg ccttctgcat cgagaacaac 960
atcaccaaga agttcgacca caagttcaag aggcacatgg aggactgcgt gatcccgtcc 1020
ttcaagaacg gcaagatccc ggacaagctg ttctacttca acatcatcct ggccaagaag 1080
accgacgagc agatcgacta ctccctgtcc tccgagttct acaccaagct gttctccatg 1140
ccgaacctgt ggcaggagga ggaggccttc atcgtgaaga acatcaacct gatcgaggag 1200
atcaccatct tcaacaagag gaggaactac gcctgctgcc cgctgatcaa ggagaaggag 1260
tacgacaggt tccagatcca gctgaacgag accaacttcc tgaagttcca gttcgacccg 1320
aagaacgtgg tgaacatcga cgagaacacc accgaggcca ccgtgggctt cgacgagaag 1380
ctgaagctgg tggtgtgcgc cgacaagaag tacgccttct ccatcttcac ccagtgcaag 1440
taccacggca acaagcacaa gccgaacacc tacttcaaca acctgaagat catcaaggtg 1500
atcgagtcca agtccaactc cgtgaagtcc atgaagtaca ccttcgagtt caccaagagg 1560
aacgagctga agagggccga gatcaagcag ccgtccatcg tgtacaagaa caacaactac 1620
tacatcagga tcaacatgaa cgtgatcctg gacgccgacc agacctccta caagatcatc 1680
aacaacaacc agaccgcctc cctgccgtcc tacttccagt cctccctgcc gttcgagaac 1740
aacaggggca agatccacga caagggcatc gtgcactggg agaagatcaa gaacaggaag 1800
atcatcgcca tgggcgtgga cctgggcgtg aggaggccgt tctcctacgc catcggcaac 1860
ttcaccctga acaaggacat cctggacaag aacgacgtga acatcgtggc ctccggcttc 1920
aacctgtgct ccgactccga cgtgtacttc caggtgttca accagatcaa gaccctggcc 1980
aagttcatcg gcaagctgaa gtcccacaac aagggcctga aggtggactt cgagaaggac 2040
aagaagtaca tcttcgacct ggtgaacgac gccaaggcct acttcaagga catgtccgcc 2100
aagaggatca acgacaccaa ggacaacatc tccaacaccg tgaccaacaa ggagaggatc 2160
tacggctcct tcgtgtccga gtccgccgag tccgccatcc agtgcgccat cgacaggtcc 2220
gagaaggagt ccggcctgac cctgaagaag gacatctcct ggctggtgaa cgtgctgtcc 2280
aagtacctgg agaggaagtt caaggaggtg aagaacaaca ggaagtacac caacgtgaac 2340
aagtgcgaca actgcttcaa ctggctgagg gtgatcgaga acatcaagag gctgaagagg 2400
tccatctcct acctgggcga ggacctgcag aagaacccgg agctgaagat cgagctgaag 2460
aacctgaacg agtacggcaa caacgtgaag tccgacttcc tgaagcagat cgcctccaac 2520
atcatcaagg tggccatcga gcacaagtgc gacatcgtgt tcatcgagaa gctgggcaag 2580
gccgactcca ggtccaggaa gctgaacgag atgttctcct tctggtcccc gaaggccatc 2640
aagaaggcca tcgagaacgc cgcctcctgg cacggcatcc cggtggtgga ggtggacccg 2700
tcctgcacct ccaaggtgca ctacgagacc aacctgttcg gccacaggat cggcaacgac 2760
ctgtactacg tggaggacca gtgcctgaag aaggtggacg ccgacatcaa cgccgccaag 2820
cagatcctgg tgaggggcgc caccaggcac ggcaacatct cctccatcaa catcaagtac 2880
ctgcaggcca agatcgccga gctgaactcc gaggccaact ccgaggagga caaggaggag 2940
atcaagcagg gcggcaagag gatccagggc ttcctgtgga agaagtacgg caacatcacc 3000
aacatcacca accagctgac cgccgcccac aaggagaggg agtccaagtt cgactacatc 3060
tacctgcaca acgacaagtg gatcgcctac gaggacagga acgagatcaa gaaggacatc 3120
gagaagaggc tggagtga 3138
<210> 33
<211> 2688
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.15编码核酸序列
<400> 33
atgaccgcca agaagaccgc caagaagtac ttcccgccga agtgcctgag gtcctcccac 60
ttcaagatct acggcatccc gaccgccatc agggccctgg aggagaccaa caccttcgtg 120
aacaaggccg ccgccgacct gatggagatg ttcttcctga tgaggggcca gccgtacagg 180
aggaggatcg gctccgagga gaagcaggtg acccaggagc acatcgacgc caggctgagg 240
gtgctggtgg gcgactactc cctgaacgag gtgaagccgc tgctgaggca gctgtacgac 300
ggcatcaagg ccaagcagaa ctacgccccg acccacttcg tgaggttctt catccagccg 360
accaagggcg ccatcgacaa gaagtccccg gtgtcccaga gggccaagaa ggccggccag 420
aagctgcaga agatgggcgt gctgccgatc ctgccgctgt ccccgggctt caagttctgg 480
accgccgcca tgatgatggc ctgctccagg atgaactcct gggaggcctg caacgagaag 540
accatcgaga accacaaggc cttcctggag ggcatcgaga actacaagaa ggagatcagg 600
ttcgaggacc tgtgcgagga gtggtccctg ttctccgact ggctgaccga ggccgagtcc 660
gacaacgagg gcggctgcaa gttcaagctg accccgaggt tcctgcagag gtgggagagg 720
atctacctga agcagatgag gaagggcaag atcccggcca ggcacaacct gggcccggtg 780
atggaggccc tggccggcga caagtacagg cagctgtggg acaacggcga ggagagggac 840
tacatcaccg agctgggcga cctggtgacc tcccagagga aggccgtgag gctgtccagg 900
gactccgccg tgaccttccc ggacgaggag ctgtccccgg tgggcaccga gttcggccac 960
aactacatgt ccttctccat cgaccaggag aactcccacc tggtgaccct ggaggtgatc 1020
ggcggcaagt accagttcga gatctccaag tccgactact tcagggacct gatcgtggag 1080
gaggccggca agcagtccaa gttctacaac gtgtcctaca ggaagggcaa cgtgagggag 1140
gagaacctgg ccggcgactt caaggaggcc accgtgagga acaggaggtc cctgaagacc 1200
ggcaagagga ggctgtactt ctacatgtcc cactccatcc cgaccaggtt cgacgacgac 1260
ctgtacgccc agttcaccga gaagggccag ccggacttct ccaagctgta caaggccgtg 1320
acctacttcc agtgctccct gggcaacaag aaggccgaca cctacagggt gtacgtgaag 1380
atgggcacca ggttcctggg cgtggacatc ggcgtgtcca ggctgttcgg cttctccctg 1440
ttcgagctga gggaggagaa gccggagaag aacccgttct tcgagctgcc ggacgacctg 1500
ggctacgccg tgtgcctgga gtcctgggtg gacggcgtgg agaagaacca caaggtggcc 1560
caggagatga aggactggag gagggagtgc ctggccgccc agaggctgat ccactacgcc 1620
aagttcctga agaagaggga caagaacgag gagatcgact acaagcacga ggagtccctg 1680
gagaccatcg ccggcctgct gggcatcgag atcgacccgg agcagatcat cgacgtgccg 1740
ctgaagctgc tggacctggt gggccaggcc atcggcgccc tgaggaagaa gtacctggtg 1800
ctgaagaaga acgaggtgag gcagggcagg atcacctccg agctgttcct gtggccggag 1860
tgcgtggaca cctacatcag gctgctgaag tcctggacct acaaggacaa gaagccgtac 1920
cagaagggcg agaccaacaa ggacgccttc aagaagctga agggctacct ggccaggctg 1980
aggaaggacc tggccccgaa gtacgccgcc gtgatcgccg acgccgccat caggcacaag 2040
gtgcacgtgg tggtggccga gaacctggag cagttcggcc tgtccatgaa gaacgagaag 2100
gacctgaaca gggtgctggc ccactggtcc caccagaaga tctggtccat ggtggaggag 2160
cagctgaggc cgtacggcat catggtggtg tacgtggacc cgaggcacac ctccaagctg 2220
gacttcgcca ccgacgagtt cggcggcagg tgcttcacct ccctgtacgt gatgagggac 2280
ggcaagaaga ccaccaccga caccgagaag aacgcctccc agaacatccc gaagaagttc 2340
ctgaccaggc acaggaacgt gtcctggctg ctggcctacg ccgtggacct gtccgactcc 2400
cagaagaaga agctgggcat cggcgacgag aaggtgtggc tgccgaacat gggcctgatg 2460
atctccggcg ccctgaaggc caagcacggc aagaactccg ccctgctggt ggaggacggc 2520
gagaactaca ggctgctgcc gatcaccgcc gcccaggcca agaagttcgt ggtgaagagg 2580
aagaaggagg agttctacag gcacggcgag atctggctga ccaaggaggc ccacaaggcc 2640
aggatcgagt acctgttccc ggagtccaag aagggcagga agtcctga 2688
<210> 34
<211> 2871
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.16编码核酸序列
<400> 34
atgaagaaga ccaactacaa gacctcccac ctgctgatcg acaacccgcc gcagtccatc 60
atcgacctgc acagggacgt gatcgagatc ggctcctacc tgaccaagtt cttcctggcc 120
tgcctgggca ggccggtgga ctccaccatc ctgtccgagc cggccctgca cttccagttc 180
gtgaacggca tcctgccggt gaagaacggc ccgggcgccg acgactcctc ctggaggcac 240
tccgagaact gctactccat gctgttcgag aagaactcca agtccggcaa gtccgacggc 300
aaggtgaggc aggtgaggga gctgaaggtg gccctgttcg gcaagaagga gaagggcaag 360
ggcatcgtgg gcaagaagac ctgggacgag ctgaaggtgg tgctggaggc cctgccggag 420
gagcaccaga tcctgtccct ggagatctgc cagaggcact acgagtccag ggacgtgaag 480
gccttcggca agctggccct gtcctccaag tccaggccgt ccgtggaggc cggcctgaag 540
ctgagggagc tgggcctgct gccgctggac tccaggggcc tggacaagaa caagctgctg 600
ggcatcctgg ccgccgtgac cggcaggctg aagtcctgga gggacaggga ctgcgcctgc 660
aaggccgaca agcaggccct gagggtgaag ttcgaggaga ggctgtccaa ggtggaccag 720
tccgcctacc agcagttcaa gcagttcgcc gacgagctgc tgacccagga gggctacagg 780
atctccggca gggtgctgag ggccgtggag aagaaggact ccgactactc cccggtgctg 840
accgtgctgg ccaagtaccc ggacctgcag gacaacttcg aggagctgtg cagggcctgc 900
ctggccgagc aggccttcaa caagaagaag gccgacgcca gggtgaccgt gtgctccgag 960
acctccccgc tgcagttccc gttcggcatg accggcaacg gctacccgtt caccctgtcc 1020
gcctgcgagg gcaggatcaa cgccaccatc cacttcccgg gcggcgacct gccgctgagg 1080
ctgaggaagt ccaagtactt ccagaacccg gagatcctgc cggtgaagga cggcttccag 1140
atcaccttca ccaggggcaa gaccccgctg gtgggcacca tcaaggagcc gtccctgctg 1200
aagaagaaca accactacta cctgtccctg agggtgaacg tgccgtccgt gaagatcccg 1260
aaggaggtga gggacaccag ggcctactac tcctccgccg tgggcggcga cgagaccacc 1320
ccggtgccgg tgaaggccgt ggccatcgac ctgggcgtga ccaccctggc cgactactcc 1380
atcatcgaca cctgcctgcc gggcgactgc aaggtgttcg gcggcgagac cgccgccttc 1440
accgcccacg gcaagatcgg ccagtgcgcc aacaagtccc tgagggacag gctgtacaag 1500
aacaccgagg aggccctgtt cctgggcaag ttcatcaggc tgtccaagaa gctgagggac 1560
ggcgagggcc tgaacaggtg ggaggtggag aagctgccgg gctacgccga gaggctgggc 1620
atcacccagc acctggacaa cgcctacacc aggaaggacg agatcgccag gaagttcaag 1680
cagatcaagg gcaacttcga caagctggtg tccgagttcg ccctgaggga ccacccgtcc 1740
aagaagggcg agtcctggga gaccatctcc gccgagacca tccaggtgct ggccgccctg 1800
aagaggatcc agtccctgct gaagtcctgg acctactact cctggaccgc cgaggactac 1860
gtgctggccc tgaccgccga cggcccggtg tgcatcgacg gcgagcacgt gaaggccgtg 1920
accgccacct ccaggaggtc cttcgccccg tgcggcaagg ccgccctgct gaggctgatc 1980
gagtccggcg agatcgtgga gaccggcggc cagtaccagc tggccaccgg cgtgaagcac 2040
aggaaccacc cggtgaactt cctgtcctcc tacatcaagc acttcaacgg cctgaggagg 2100
gacctgacca acaagctggt gagggccatc gtgaacaagg cccaggagta cagggtgcag 2160
atcgtgatcg tggaggactt cggcatcgcc gacctggagg acaggatcaa ggacgcctac 2220
gagaactaca ggtggaacct gttcgccccg gccaccatcg tgaagaagct ggaggccgcc 2280
ctgctggagg tgggcatcgc catggcccag gtggacccga ggcacacctc ccagatcgcc 2340
ccgaccggcg ccttcggctt cagggaccac gccttcctgt actaccagga cgacggcctg 2400
tgcaggatcg acgccaacac caacgcctcc atgaggatcg ccgagaggtt cttcatgagg 2460
cactccgtgc tgacccagct gagggccgcc aagatcggcg agaccgagta cctgatcccg 2520
gagtccgcct ccaagaggct gaacgccttc gtgaagctgc agaccggcaa gccgttcgcc 2580
aagctgatca tgaactgctc cggcttcgtg ctggagggcc tgaccaagaa gcagtacgcc 2640
aagctggccg agaccgccgg caagaaggag tccttctacc agtacgacga caggtggttc 2700
gacaagggcc accacttcgc ctgcagggcc accctggaga acaaggtgca ggtgtgcctg 2760
aacggcggcg gcaggatcaa ggacaccacc ccggacttca acccgaagtc cctgctgagg 2820
tccgacctgc agaccccgct ggaccagctg ttcggcaact ccggcgcctg a 2871
<210> 35
<211> 2841
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.17编码核酸序列
<400> 35
atgtccaaca ccacctacaa gacctcccac ctgctgatcg acctgccgca gcaggagctg 60
atcgacctgc acagggactc caacgagatg ggctcctacc tgaccaagtt cttcctggcc 120
gccctgggca ggccggtgga caactccatc gtgctgccgc cggagctggc cgacctgtac 180
ttccagttcg ccaacggcat cctgccggtg gacaagggcc cgggctccga cgacccgtcc 240
tggctgcact ccgagaactg ctactccatg ttcttcgaga aggactccat gtccggcaac 300
tgcaccaaca agatcaagca gtaccaggag ctgaagaccg ccctgtgcgg ccagaaggtg 360
aagggccaga agggcctggt gggcaagaag acctgggccc agctgaagaa ggtgctgacc 420
gccctgccgc agaagtacca gatcctgtcc ccgaagatct gccagaagta cttcaagtcc 480
ggcaacctgg agggcttcgg caagctggcc ctggccggca agaacaggcc gtccatgtcc 540
gccggcctgc agctgaggga gctgggcctg ctgccgctgg actccagggg catcgacaag 600
aacaagctgc tgggcatcct ggtgggcatc accggcaggc tgaagtcctg gagggacagg 660
gactgggcct gcaagaccgt gaaggaggag ctgagggtga ccttcgagaa gggcctgggc 720
gaggtggacc cgaccgccta cccgcagttc aagcagttcg ccgaccagct gttcaagcag 780
gagggctaca agatctccgg cagggtgctg agggccgtgg agggcaagga cgccgactac 840
cagccggtgc tgtccctgct gacccagtac ccggacctgc agggcgactt cgaggagctg 900
ggcagggtgt acctggccga ggccgagtac ctgaggaaga aggtggacgc cagggtgacc 960
gtgtgcgacg ccgagacctc cccgctgcag ttcccgttcg gcctgaccgg caacggctac 1020
tccatcaccc tgaccgtggt gaagggccag atcgccgcca ccctgcacct gccgggcggc 1080
gacatcaccc cgaggctgag gaggtccaag tacttccaga acccggagat cgccccggtg 1140
aaggacggca agggcaaggt gaacggcttc cagatctcct tcaagagggg caagaccccg 1200
ctggtgggca tcatcaagga gccgaagctg ctgaagaaga acggcaacta ctacctgtcc 1260
ctggccgtgg gcatcaacaa gaccgagatc ccgaaggaga tctgcgacgc cagggcctac 1320
tactcctcca cctccaggac cgacaccccg ccggccgtga aggccatgtc catcgacctg 1380
ggcgtgacca ccctggccga ctactccatc atcgacaccg gcctgccggg cgactgcggc 1440
gtgttcggcg gctccaccgc cgccttcacc gagcacggca agatcggcag gtgcggctcc 1500
aagtccctga gggacggcct gtacaagaac accgaggccg gctacttcct ggccaagtac 1560
atcaggctgt ccaagaacct gaggggcggc gtgggcctga acaagctgga gaaggagaag 1620
ctgctggagc acgtggagag gctgggcatc gagcactgcg ccgacgactt cgccaggaag 1680
gacgagatcc acaggaagtt ctccgagatc aagtccaagc tggagaagtc catctccgag 1740
ttcgccctga gggacaggcc ggacaagaag ggcgcctcct gggagggcat ctgcgccgag 1800
accgtgcagg tgctgggcgc cgtgaagagg tggcagtccc tggccaagtc ctggacctac 1860
tactcctgga ccgccgagga ctacgtgctg gccctgaccg gcgagggcag gaccagggtg 1920
tccgacgagc acgtggagtc cgtggtgaag accggcagga ggcagttcgc cccgtgcggc 1980
aaggccgccc tgctgaggct gctggagaag ggcaagatcg tggaggtgtg cccgggccag 2040
ttccagctgg ccgagggcgt ggactacaag aggcacccga ccgagttcct ggccgcccac 2100
atcaggcact tcaacggcct gaggagggac ctgaccaaca agctggtgag ggccatcgtg 2160
gagaaggccc agcagcacag ggtgcagatc gtgatcgtgg aggacttcgg catcccggac 2220
atcgagggca ggatcatgga ccactacgac aactacaggt ggaacctgtt cgccccggcc 2280
aaggtgatcg agaagctgga ggaggccctg tccgaggtgg gcatcgccat ggccgaggtg 2340
gacccgaggc acacctccca gctggccccg accggcgact tcggcttcag ggaccacgag 2400
aacctgtact tctgggagaa gggcctgtgc aggaccgacg ccaacaccaa cgcctccatg 2460
aggatcgccg agaggttctt caccaggcac tccgtgctgt cccagctgag ggccgtgaag 2520
atctccgaga ccgagttcct gatcccggtg tccaccggca agagggagaa cgccttcatc 2580
aagtcccaga ccggcaagct gttcgccaag ctggtggccg actccaacgg cttcgtgatg 2640
gtgggcctga ccgagaagca gcacggcgcc accgtgaccg tgggcaagaa ggtgtccttc 2700
tacaaccacg ccggcaggtg gctgggcaag gcccaccaca tcgcccacag ggacaggatc 2760
aagaacgagg tgaaccaggt gctgacctcc ggcaggggca ggatcaggaa catcgccccg 2820
gagctgtccc cgaagacctg a 2841
<210> 36
<211> 2793
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.18编码核酸序列
<400> 36
atgaccaacc agaagccgaa gttcaagtcc tccgacatcc agatcaagca catctccccg 60
accgacaaga agaggctgaa gaccttctac caccagctgt acgagcaggt gaacttcatc 120
ctggagagga tgatcgtgat gaggggcagg ccgaggacca tcaggaacat cgacggcacc 180
gagatcttcg tgtcccagga ggaggccgac cagcagctgc tgtccctggc cggcggctcc 240
cacgagggcg tgaagtacct gaagcagtac tacgagtcct gcgtggacgc cggcaagccg 300
gccaagtacg ccgccaacat gttcctgacc aagaccatct ccggcaccaa cccgctgcag 360
tgccacaccg ccgtgtacaa gctgtacaag aaggtgcagg ccaagcagat caccaagaag 420
gagttcatcg acaagctgta ctccaagacc aagaagaaga agtccctgaa gccggcctac 480
aaggtgttca ccgagaacga gcacatcgag ttctaccaca aggtgaggtc cggcaagctg 540
ccggcctccg aggtgaggct ggaggagtcc aggagggccc cggacgtggg cctggaggtg 600
ggcctgctgc tgagggagct gggcatcttc ccgttcaact tcccgcactt caccgagaag 660
aagtacctgg acctggcctg gaccatcgcc atcaggtggc tgaagaactg gaacgagaac 720
aacaagaaca ccgccaagga gaaggccaag cagaaggcca tcgtggacaa gctgaggacc 780
tccctggacc agaaggaggt ggacctgttc gaggagttcg ccgaggagtg ctcccaggag 840
cagttcggca tcagggaggg cttcgtgaag gccaagaaga ggctgaagtc cttcccgaag 900
ggcatcgaga agtcctccta caaggagggc atgaggatcc tggtgcagaa caagcacggc 960
tccatctggg acaacttcga gaacctggcc taccaccaca tcgccctgaa cgagtacaac 1020
aggctgaggg acgaggcctc cttctccttc ccggacccga tctaccaccc gatcagggcc 1080
gagttcggcc tgacctccct gccgaagttc aacgtgggcc tgaacgacag gggcaactac 1140
gagttcacca tcaacctgcc ggacggcccg ctgatgatgc tgggcaagaa gtccaggtac 1200
tacctgaagc cgatcatcca gggcccgctg aacaacgcct tctccttcga gttcatcaag 1260
ggcaacaaga agaggccgaa gatctccgcc aagctgaagt ccatcaccgt ggtgttcgcc 1320
aagtcctcca tctacgtggg cctgccgtac aggccgatct ccatcccgat cccgcaggcc 1380
gtgaccaact ccacctacta cttcaagaag aacctgtcct ccacctccaa gttcgacaag 1440
gacgtgttca tgggcctgac cgccgtgtcc gtggacctgg gcctgaaccc ggtgttctcc 1500
atgtccgcct gcaggctgga cgagatgaag gccgacgagc actactcctg cgaggtgccg 1560
ggcttcggct gggccaacca gatctggtcc aagagggccg gcggcgtgtg gaacaggtcc 1620
ttcagggaca agatcagggg cttcgtgccg ggcaacctgt ccgacaggat cttctgctgc 1680
aagaagtcca tcatcgtgtc caagaagctg agggacgaga agccgctgac ccagtacgag 1740
gaggagaact tcgagaggtg gatgcaggtg gtgggcgtgg acccgaacga ggaccactac 1800
aagcagctga ggatcgccat cagggacatc aagaccgagt acgagaccgt gaggtccgag 1860
ttcgccctga gggaccaccc gaacaactcc aacaagacca ccgagaacat ctgcaccgag 1920
tgcttcgaca tgctgttcgt gatcaagaac ctgatctccc tgctgaagtc ctggaacagg 1980
tggcacagga ccaccggcga catcgaggag aggggcaagg acccgaacga gtgctccacc 2040
tactggaggc actacaacgg cctgaagacc gacctgctga agaagctgac caacatcctg 2100
atcgagtccg ccaagtccat cggcgcccac atcatcatcc tggaggacct gaccctgtcc 2160
cagaggtcct ccaggtccag gagggagaac tccctggtgg ccatcttcgg cgcccagacc 2220
atcatcaaga ccatctccga ggaggccgag atcaacggca tcctggtgta cctggaggac 2280
ccgaggcact cctcccagat ctccatcgtg accaacgagt tcggctacag gccgaaggag 2340
gacaaggcca agctgtactt catggacgag gagaccgtgt gcgtgaccaa ctgcgacgac 2400
tccgccgccc tgatgctgca gcagtccttc tggtccaggc acaaggacgt ggtgaaggtg 2460
aagggcacca aggtgtccga caccgagtac ctggtgtcct ccgaggacaa ggacggcacc 2520
aagatgaggc tgaggtccta cctgaagagg aacgtgggca ccgccaacgc catcctgcag 2580
aagaactgcg acggctacga cctgaagaag atctccccgc agaagaagaa gaagatcgag 2640
gagttcggca aggacgagta cttctacagg cacggcgagc agtggttcac cgccgacgcc 2700
cacttcgaca agctgaggga gttcggcaac caggtgttcc tgaccccgca gtcccagatc 2760
aagaggatca acctgcaggt ggagggcacc tga 2793
<210> 37
<211> 2727
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.19编码核酸序列
<400> 37
atgccgtcct acaagtcctc cagggtgctg gtgagggacg tgccggagga gctggtggac 60
cactacgaga ggtcccacag ggtggccgcc ttcttcatga ggctgctgct ggccatgagg 120
agggagccgt actccctgag gatgagggac ggcaccgaga gggaggtgga cctggacgag 180
accgacgact tcctgaggtc cgccggctgc gaggagccgg acgccgtgtc cgacgacctg 240
aggtccttcg ccctggccgt gctgcaccag gacaacccga agaagagggc cttcctggag 300
tccgagaact gcgtgtccat cctgtgcctg gagaagtccg cctccggcac caggtactac 360
aagaggccgg gctaccagct gctgaagaag gccatcgagg aggagtgggg ctgggacaag 420
ttcgaggcct ccctgctgga cgagaggacc ggcgaggtgg ccgagaagtt cgccgccctg 480
tccatggagg actggaggag gttcttcgcc gccagggacc cggacgacct gggcagggag 540
ctgctgaaga ccgacaccag ggagggcatg gccgccgccc tgaggctgag ggagaggggc 600
gtgttcccgg tgtccgtgcc ggagcacctg gacctggact ccctgaaggc cgccatggcc 660
tccgccgccg agaggctgaa gtcctggctg gcctgcaacc agagggccgt ggacgagaag 720
tccgagctga ggaagaggtt cgaggaggcc ctggacggcg tggacccgga gaagtacgcc 780
ctgttcgaga agttcgccgc cgagctgcag caggccgact acaacgtgac caagaagctg 840
gtgctggccg tgtccgccaa gttcccggcc accgagccgt ccgagttcaa gaggggcgtg 900
gagatcctga aggaggacgg ctacaagccg ctgtgggagg acttcaggga gctgggcttc 960
gtgtacctgg ccgagaggaa gtgggagagg aggaggggcg gcgccgccgt gaccctgtgc 1020
gacgccgacg actccccgat caaggtgagg ttcggcctga ccggcagggg caggaagttc 1080
gtgctgtccg ccgccggctc caggttcctg atcaccgtga agctgccgtg cggcgacgtg 1140
ggcctgaccg ccgtgccgtc caggtacttc tggaacccgt ccgtgggcag gaccacctcc 1200
aactccttca ggatcgagtt caccaagagg accaccgaga acaggaggta cgtgggcgag 1260
gtgaaggaga tcggcctggt gaggcagagg ggcaggtact acttcttcat cgactacaac 1320
ttcgacccgg aggaggtgtc cgacgagacc aaggtgggca gggccttctt cagggccccg 1380
ctgaacgagt ccaggccgaa gccgaaggac aagctgaccg tgatgggcat cgacctgggc 1440
atcaacccgg ccttcgcctt cgccgtgtgc accctgggcg agtgccagga cggcatcagg 1500
tccccggtgg ccaagatgga ggacgtgtcc ttcgactcca ccggcctgag gggcggcatc 1560
ggctcccaga agctgcacag ggagatgcac aacctgtccg acaggtgctt ctacggcgcc 1620
aggtacatca ggctgtccaa gaagctgagg gacaggggcg ccctgaacga catcgaggcc 1680
aggctgctgg aggagaagta catcccgggc ttcaggatcg tgcacatcga ggacgccgac 1740
gagaggagga ggaccgtggg caggaccgtg aaggagatca agcaggagta caagaggatc 1800
aggcaccagt tctacctgag gtaccacacc tccaagaggg acaggaccga gctgatctcc 1860
gccgagtact tcaggatgct gttcctggtg aagaacctga ggaacctgct gaagtcctgg 1920
aacaggtacc actggaccac cggcgacagg gagaggaggg gcggcaaccc ggacgagctg 1980
aagtcctacg tgaggtacta caacaacctg aggatggaca ccctgaagaa gctgacctgc 2040
gccatcgtga ggaccgccaa ggagcacggc gccaccctgg tggccatgga gaacatccag 2100
agggtggaca gggacgacga ggtgaagagg aggaaggaga actccctgct gtccctgtgg 2160
gccccgggca tggtgctgga gagggtggag caggagctga agaacgaggg catcctggcc 2220
tgggaggtgg acccgaggca cacctcccag acctcctgca tcaccgacga gttcggctac 2280
aggtccctgg tggccaagga caccttctac ttcgagcagg acaggaagat ccacaggatc 2340
gacgccgacg tgaacgccgc catcaacatc gccaggaggt tcctgaccag gtacaggtcc 2400
ctgacccagc tgtgggcctc cctgctggac gacggcaggt acctggtgaa cgtgaccagg 2460
cagcacgaga gggcctacct ggagctgcag accggcgccc cggccgccac cctgaacccg 2520
accgccgagg cctcctacga gctggtgggc ctgtccccgg aggaggagga gctggcccag 2580
accaggatca agaggaagaa gagggagccg ttctacaggc acgagggcgt gtggctgacc 2640
agggagaagc acagggagca ggtgcacgag ctgaggaacc aggtgctggc cctgggcaac 2700
gccaagatcc cggagatcag gacctga 2727
<210> 38
<211> 2466
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.20编码核酸序列
<400> 38
atggccttcc agtccaagag gaggatcgtg ggcaacttcg tgaaggagca gtgcctgaag 60
gccgtggacg gcaaggtgat cctgaccgac caggagaaga gggagctgat caagaggtac 120
gagctgcacc tggagccgca caagtggctg ctgaggctgt tcctgtccgg ctacgagggc 180
agggacgacg gcttctacga ggagctgggc aacaccaacc tggacaagga gaagttcttc 240
gaggtgaccg ccggcctgag ggacgccctg ctgaggcagt ccggctcctc cagggccctg 300
aagtcctcca tgctgggcaa gtgcccgccg tccgccgccg tgggcaaggc cgccaagcac 360
atccagaccc tgagggacgc cggcatcctg ccgttcaaga ccggcctgac ctccggcgag 420
gactacaacg tgctgcagca ggccgtgcag cagctgaggt cctgggtggc ctgcgaccac 480
aggaccaggg aggcctacgc cgagcagcag gagaagacct cccaggccga ggaggccgcc 540
aagaaggccg ccaacgaggt gaagccggag gacgccaagt ccctggagag gcacgagagg 600
gtgctgacca agctgaggaa gcaggagagg aggctggaga ggatgaagtc ccacgcccag 660
ttctccctgg acgagatgga ctgcaccggc tactccctgt gcatgggcgc caactacctg 720
aaggactact gcctggagaa ggagggcagg ggcctgaggc tgaccctgaa gaactccacc 780
atggccggct cctactacgt gtccgtgggc gacggccagc acgccggcat gaagaacccg 840
ggcaccccgg ccggcggctc cccggagaag ggcaggagga ggaacatcct gttcgacttc 900
accgtggaga agtgcggcga caactacctg ttcaggtacg acgagaacgg caagaggccg 960
agggccggcg tggtgaagga gccgaggttc tgctggagga ggaagggcaa ctccgtggag 1020
ctgtacctgg ccatgccgat caacatcgag aactccatga ggaacatctt cgtgggcaag 1080
cagaagtccg gcaagcactc cgccttcacc aggcagtggc cgaaggaggt ggagggcctg 1140
gacgagctga gggacgccgt ggtgctgggc gtggacatcg gcatcaacag ggccgccttc 1200
tgcgccgccc tgaagacctc caggttcgag aacggcctgc cggccgacgt gcaggtgatg 1260
gacaccacct gcgacgccct gaccgagaag ggccaggagt acaggcagct gaggaaggac 1320
gccacctgcc tggcctggct gatcaggacc accaggaggt tcaaggccga cccgggcaac 1380
aagcacaacc agatcaagga gaaggacgtg gagaggttcg actccgccga cggcgcctac 1440
aggaggtaca tggacgccat cgccgagatg ccgtccgacc cgctgcaggt gtgggaggcc 1500
gccaggatca ccggctacgg cgagtgggcc aaggagatct tcgccaggtt caaccactac 1560
aagcacgagc acgcctgctg cgccgtgtcc ctgtccctgt ccgacaggct ggtgtggtgc 1620
aggctgatcg acaggatcct gtccctgaag aagtgcctgc acttcggcgg ctacgagtcc 1680
aagcacagga agggcttctg caagtccctg tacaggctga ggcacaacgc caggaacgac 1740
gtgaggaaga agctggccag gttcatcgtg gacgccgccg tggacgccgg cgcctccgtg 1800
atcgccatgg agaagctgcc gtcctccggc ggcaagcagt ccaaggacga caacaggatc 1860
tgggacctga tggccccgaa caccctggcc accaccgtgt gcctgatggc caaggtggag 1920
ggcatcggct tcgtgcaggt ggacccggag ttcacctccc agtgggtgtt cgagcagagg 1980
gtgatcggcg acagggaggg caggatcgtg tcctgcctgg acgccgaggg cgtgaggagg 2040
gactacgacg ccgacgagaa cgccgccaag aacatcgcct ggctggccct gaccagggag 2100
gccgagccgt tctgcatggc cttcgagaag aggaacggcg tggtggagcc gaagggcctg 2160
aggttcgaca tcccggagga gccgaccagg gagcaggacg agtccgacca ggacttcaag 2220
aagaggctgg aggagaggga caagctgatc gagaggctgc aggccaaggc cgacaggatg 2280
caggccatcg tgcagaggct gttcggcgac aggaggccgt gggacgcctt cgccgacagg 2340
atcccggagg gcaagtccaa gaggctgttc aggcacaggg acggcctggt gctgaacaag 2400
ccgttcaagg gcctgtgcgg ctccgagaac tccgagcaga aggcctccgc caggaactcc 2460
aggtga 2466
<210> 39
<211> 2514
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.21编码核酸序列
<400> 39
atgggcaggt tcggcaagaa gaagatcgcc gtgaacggct acgtggagca ggactgcatc 60
aagaccatct ccgccaagtg cctgctgacc agggcccaga tcgacgagct gagggccaag 120
tacgacgccg tgctggacac catgaggccg ctgatcaggc tgatcctggc cggctacgag 180
ggcagggacg acggcatcta cgaggagatc gccccggaga tgtccaagaa gaagttcttc 240
gaggccgcca ccgagtggag ggagtccatc gtgaagaacg cctccccgag ggccatgaag 300
gcctccgtgt tcggcgacaa ggagccgtgc aagtccaccg gcggcgccag ggccgtgatc 360
ggcaagctga ggaagtccgg cgtgttcccg atcgagaccg gcctgtccgg cggcgacgag 420
tacaacctga tcgagcaggc catcgagtac gccaagtcct ggctgaagtc cgacgaggcc 480
accagggagg cctacgccga ccagcagaag gacatcaaga ggctgatcgg cgaggccaag 540
aagctggccc tgaagatcga gaaggccgag aagaagctgg aggccaccaa cccgcagacc 600
aagtcctgga agaagaccac cgagatcatc aagaagtcca agagggagtt cggctccgtg 660
accaccaaga ccgagaaggc cgagaagagg ttcgagagga tgaagccgtt ctccaagctg 720
gagctgcaga acatggactg caccaagtac tccacctacc tgggcaccaa ctactccccg 780
ttcaagctga agaaggaggg cgacctgctg cagatcaccg tgacctcctc cgtgatgaag 840
ggcacctacc tggcctccta cggcgacggc cagtacggct ccaggaggaa caacggccag 900
tccaggaggg acgacttcgt gccgaacatg aaccagaaga ggaggaggaa cctgatgttc 960
gactgcaccg tggagccgtt cggcgacggc tccctgctga ggtacgagga gaacggcctg 1020
aggccgaggg tggccgagct gaaggagccg aggctgtgct ggaggaggag gaacggcaac 1080
tacgagctgt acctgatgat gccggtgaag atgcacgtga agtccccgga gatgttcgcc 1140
ggcgaccacc tggccttctc caggtactgg ccgaaggagg tggagggcct ggactccgac 1200
accaagatca ccgccctggg cgtggacgtg ggcatcatca ggtccgccta ctgcgtggcc 1260
gtgaccgccg agaggttcgt ggacggcctg ccgaccgaga tgaccgtggg caaggcctcc 1320
ttcgacgccc agaccgagaa gggcagggag tacttcgagc tgggcaggag ggccaccatg 1380
ctgggctggc tgatcaagac caccaggagg tacaagaagg acccgaagaa cgagcacaac 1440
cagatcaagg agtccgacgt ggccgccttc gacggctccc cgggcgcctt cgagcactac 1500
atcctggccg tggacgagat gtccgacgac ccgctggacg tgtggggcca cgccaacatc 1560
accggctacg gcaagtggac caagcagatc ttcaaggagt tcaaccagct gaagagggag 1620
agggccgagg gccaggtgga gccgaacatg accgacgacc tgacctggtg ctccctgatc 1680
gactacatca tctccctgaa gaagaccctg cacttcggcg gctacgagac caaggagagg 1740
gagtccttct gcccggccct gtacaacgag agggccaact gcagggacgt ggtgaggaag 1800
aggctggcca ggtacgtggt ggagagggcc atcgccgccg aggcccaggt gatctccgtg 1860
gagaacctgt ccaagtgcag gagggacgac aagaggaaga acagggtgtg ggacctgatg 1920
tcccagcagt cctggatcgg cgtgctgacc aacatggcca ggatggagaa catcgccgtg 1980
gtgtccgtga acccggacct gacctcccag tgggtggagc agtgcggcgc catcggcgac 2040
aggaaggcca ggaccatcgc ctgcagggac gtgaacggca agttcgtgtc cctggacgcc 2100
gacctgaacg ccgcctacaa catcgcctcc agggccctga ccaggcacgc cgagccgttc 2160
tccatcacct tcaagaagaa ggacggcatc ctggagcaga aggacgtgtg cttcgacccg 2220
ggcgtgatcc cggtgctgga gaagaacgag aacgaggaga agttcaggga gagggtggag 2280
aagtacgaga agtccctggt gatcaagcag gagagggccg tgaggtggag ggccatcctg 2340
cagcacctgt tcggcaacga gaggccgtgg gacgagttca ccgacgaggt gaaggagggc 2400
aggcacgtgt ccctgtacag gcaccacggc aagctggtga ggaccaagca gtacgccggc 2460
ctggtgaagg aggccaacaa cgagctggtg ccggtgtgcg ccgtggccag gtga 2514
<210> 40
<211> 2907
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.22编码核酸序列
<400> 40
atgtccaagg ccaccaggaa gaccaagacc accgtgccgg agtccaccga caccgagtcc 60
ccggccgccg acacccaggt gagggtgcac tggctggccg cctcccacag ggcctccccg 120
ggcctgcagc aggtgaagga gatgatccag cagcacgccg acgtggcctc cgtgctgttc 180
cagggcctgg tgaggaccgc cccgatcgtg ttcaggaacg acgacggctc cccggtgaag 240
ccgctggacc tgctgctggc ctccctgagg ccgacctaca aggtgcagag ggacaccgag 300
accgtgctgg tgaccaagga cgacgtgatc aggtgcctga ccctggccac caccgccgtg 360
aacggcggcc aggccaccaa cgtggccgtg ttcgcctccg ccgacccggc cctgtccgcc 420
ccgctggcca ccctgctggc ccagctgagg gccctggagt ccgtggactc ctcctggtcc 480
gtggtgggca agctggacat caacctgagg aagttcgtgt ggctggtgct gtccgccgcc 540
ggcgtgctgc cggccctggc cgacctggag ggctacgccg ccaagtccgt gctggccaac 600
gtgcagggca agtacaagtc cctgcaggcc tgcgccgaca cccacgccgc cctgtacaag 660
cagcaccaga ccaacaagga gcagctggag aagctgatcg ccgacccggg cttcgtggcc 720
ctgtgctccg ccctgctgca ggacccggac ctgaggtccg tggactccag gaggctggcc 780
gccctggagg agatgctggg cttcgtggcc gccgacaaga actactccga gtacacctcc 840
accaggaagt gcgacggctg ggccccgccg gccaacatgt tcgacctgct gtgcgagcac 900
aaggaggccg tgaggaggaa catcgtggtg gacaactcca agtgcctgtc caggaggatc 960
tccctggtgg ccgacggcga cgtgaacgag gtgtccgtgt tcgagctgct gaacgagatg 1020
aggtggctgt ccgtgcactc ctccggcatc aggatgccga actacccgaa gcacgcctac 1080
gccctgaagt tcggcgacaa ctacatctcc gtgaagtcct tcgagaccgt ggtggacggc 1140
ggctgctccc tgctgaggat gaccgccagg gtgggcaaga acgacctggt gtgcgacttc 1200
gtgctgggca ggggcaacga gtactggaac aacctgaaga tcaccccgat gggcaagggc 1260
atcttcgccg tggtgaagac cgtgaggagg ttcaccgcca ccggcgccaa gctggtggag 1320
ctgaggggcg tgtgcaagga gccggagatc aggtacgaga ggggcgtgct gggcctgagg 1380
ctgccgatct ccttcgacgt gtacggcaag gtggaggagg actccatcgc cttcggcaag 1440
aacagggtgt ccctgaggac caccccgttc gtggagaagg ccgacaagtt ccagggcctg 1500
ctggactaca ggaacaccac cgccagggac ggctacatct actacgccgg cttcgaccag 1560
ggcgagaacg accaggtggt gggcatctac aggaccagga cctacaagaa cgccaccatg 1620
ctggagttct tcaacgtgtc cgacaccctg gaggaggtgg cctcctgcag gttctccgac 1680
taccaggaga ggaagaggag gctgaggggc gacaccggcg tgctggacat caactccatc 1740
aacgtgctgg ccgacaaggt gcagaggctg aggaggctga tctccaccct gagggcctgc 1800
gcctcccaca ccgactggta cccgaagctg aaggagagga ggaggctgga gtgggccgtg 1860
ctggcccagg gcgtgggcgt gtccgacttc gacaccgaga tcgagagggc cgagaccgcc 1920
ctgtccgccg tggccgccgt ggacttcgtg agggacccga cctgcatcat caacgtgatg 1980
gacaagcaca tctacgccca gttcaagcag ctgaggtccg agaggaacga gaagtacagg 2040
tcccagcacc agcacgacta caagtggctg cagctggtgg actccgtgat ctccctgagg 2100
aagtccatct acaggttcgg caaggccccg gagccgaggg gcgccggcga gctgtacccg 2160
cagaacctgt acacctacag ggacaacctg atgcagcagt acaggaagga ggtggccgcc 2220
ttcatcaggg acgtgtgcct ggagcacggc gtgaggcagc tggccgtgga ggccctgaac 2280
ccgacctcct acatcggcga ggactccgac gccaacagga agagggccct gttcgccccg 2340
tccgagctgc acaacgacat cgtgctggcc tgctccctgc actccatcgc cgtggtggcc 2400
gtggacgaga ccatgacctc cagggtggcc ccgaacaaca ggctgggctt caggtcccac 2460
ggcgactacc agaagttctc cgagaccgcc cagggcaggt tcaactggaa gcacctgcac 2520
tacttcggcg acaacgacgt gtccgagcac tgcgacgccg acgagaacgc ctgcaggaac 2580
atcgtgctga gggccctgac ctgcggcgcc tccaagccga ggttctccag gcagtccctg 2640
ctgggcaaga tcaagggccc ggtgctgagg acccagctgg cctacctggc ccacaagagg 2700
ggcctgctga ccgcctccac cgagccgaag aaggccgccg agaccggctt cgagctggtg 2760
gaggccgacc tgggcggcgc cctgagggtg ggcaagggct tcatctacgt ggacgccggc 2820
atctgcatca acgccaccac caggaaggag aggtcccaca aggtgggcga ggccgtggtg 2880
tccaggtccc tggcctcccc gttctga 2907
<210> 41
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.3原型同向重复序列
<400> 41
ggugauauag uaacuggucu guuccagcac uucacc 36
<210> 42
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.4原型同向重复序列
<400> 42
gugucaaugc gaugcugaac aucgcaugag uaacac 36
<210> 43
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.5原型同向重复序列
<400> 43
gugcuggccg cucucgcuag agggagguca gagcac 36
<210> 44
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.6原型同向重复序列
<400> 44
guugcaaucu aguagagaaa cuacagguaa uugcaac 37
<210> 45
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.7原型同向重复序列
<400> 45
auuacaaccu acugaugaua caguagguga uuguaac 37
<210> 46
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.8原型同向重复序列
<400> 46
gugcaauuaa guagaaauac ugcuagugau ugcaac 36
<210> 47
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.9原型同向重复序列
<400> 47
ggugcaauca ucuggaaaua ccagagauaa uugcaac 37
<210> 48
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.10原型同向重复序列
<400> 48
gguacaggcu caagaaaaac uugagccaaa ugugac 36
<210> 49
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.11原型同向重复序列
<400> 49
guuguaauac auuauguuaa aguaauguua uacaac 36
<210> 50
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.12原型同向重复序列
<400> 50
gagguagugu ggaaguccag cagggcuucg uugacac 37
<210> 51
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.13原型同向重复序列
<400> 51
cuaucagugu aaaacccauc gaggguuuau cuacac 36
<210> 52
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.14原型同向重复序列
<400> 52
auaucagugu ggguccgcaa aacggaucaa ugacac 36
<210> 53
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.15原型同向重复序列
<400> 53
gugcagccua uugggaucgc ccauaggcau gagacac 37
<210> 54
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.16原型同向重复序列
<400> 54
gugccgucac cgccuuaguu gagcgggguc aagcac 36
<210> 55
<211> 37
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.17原型同向重复序列
<400> 55
gugccaaccu caccggagac gaguggggca ccagcac 37
<210> 56
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.18原型同向重复序列
<400> 56
gugccgcugg ccuuucgaag aggggccuuu aagcac 36
<210> 57
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.19原型同向重复序列
<400> 57
gugcugcugu cucccagacg ggaggcagaa cugcac 36
<210> 58
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.20原型同向重复序列
<400> 58
guguaggccu ccucugaaug ggguggcuaa ugacac 36
<210> 59
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.21原型同向重复序列
<400> 59
guguugaucc guucugaaug gauggauugc ugacac 36
<210> 60
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.22原型同向重复序列
<400> 60
auuucagugc uggccugugg aagcaggcuc ugucac 36
<210> 61
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.3原型同向重复序列的编码核酸序列
<400> 61
ggtgatatag taactggtct gttccagcac ttcacc 36
<210> 62
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.4原型同向重复序列的编码核酸序列
<400> 62
gtgtcaatgc gatgctgaac atcgcatgag taacac 36
<210> 63
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.5原型同向重复序列的编码核酸序列
<400> 63
gtgctggccg ctctcgctag agggaggtca gagcac 36
<210> 64
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.6原型同向重复序列的编码核酸序列
<400> 64
gttgcaatct agtagagaaa ctacaggtaa ttgcaac 37
<210> 65
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.7原型同向重复序列的编码核酸序列
<400> 65
attacaacct actgatgata cagtaggtga ttgtaac 37
<210> 66
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.8原型同向重复序列的编码核酸序列
<400> 66
gtgcaattaa gtagaaatac tgctagtgat tgcaac 36
<210> 67
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.9原型同向重复序列的编码核酸序列
<400> 67
ggtgcaatca tctggaaata ccagagataa ttgcaac 37
<210> 68
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.10原型同向重复序列的编码核酸序列
<400> 68
ggtacaggct caagaaaaac ttgagccaaa tgtgac 36
<210> 69
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.11原型同向重复序列的编码核酸序列
<400> 69
gttgtaatac attatgttaa agtaatgtta tacaac 36
<210> 70
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.12原型同向重复序列的编码核酸序列
<400> 70
gaggtagtgt ggaagtccag cagggcttcg ttgacac 37
<210> 71
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.13原型同向重复序列的编码核酸序列
<400> 71
ctatcagtgt aaaacccatc gagggtttat ctacac 36
<210> 72
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.14原型同向重复序列的编码核酸序列
<400> 72
atatcagtgt gggtccgcaa aacggatcaa tgacac 36
<210> 73
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.15原型同向重复序列的编码核酸序列
<400> 73
gtgcagccta ttgggatcgc ccataggcat gagacac 37
<210> 74
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.16原型同向重复序列的编码核酸序列
<400> 74
gtgccgtcac cgccttagtt gagcggggtc aagcac 36
<210> 75
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.17原型同向重复序列的编码核酸序列
<400> 75
gtgccaacct caccggagac gagtggggca ccagcac 37
<210> 76
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.18原型同向重复序列的编码核酸序列
<400> 76
gtgccgctgg cctttcgaag aggggccttt aagcac 36
<210> 77
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.19原型同向重复序列的编码核酸序列
<400> 77
gtgctgctgt ctcccagacg ggaggcagaa ctgcac 36
<210> 78
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.20原型同向重复序列的编码核酸序列
<400> 78
gtgtaggcct cctctgaatg gggtggctaa tgacac 36
<210> 79
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.21原型同向重复序列的编码核酸序列
<400> 79
gtgttgatcc gttctgaatg gatggattgc tgacac 36
<210> 80
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> Cas12j.22原型同向重复序列的编码核酸序列
<400> 80
atttcagtgc tggcctgtgg aagcaggctc tgtcac 36
<210> 81
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> NLS序列
<400> 81
Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
1 5 10
<210> 82
<211> 1014
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.3-NLS融合蛋白的氨基酸序列
<400> 82
Met Thr Lys Glu Lys Ile Lys Lys Thr Lys Lys Ala Lys Val Glu Lys
1 5 10 15
Asp Ser Val Thr Arg Ala Gly Ile Leu Arg Ile Leu Leu Asn Pro Asp
20 25 30
Gln His Gln Glu Leu Asp Thr Leu Ile Ser Asp His Gln Glu Ala Ala
35 40 45
Arg Glu Ile Gln Thr Ala Thr Tyr Lys Leu Ser Gly Leu Lys Leu Tyr
50 55 60
Asp Lys Thr Asn Asn Met Val Val Asp Gly Ser Lys Ala Thr Pro Glu
65 70 75 80
Glu Gln Glu Ala Tyr Tyr Lys Ile Ile Asn Trp Glu Gly Gln Pro Ile
85 90 95
Ser Ile Ser Asn Pro Met Val Arg Ala Thr Phe Lys Ser Ile Ala Lys
100 105 110
Val Lys Glu Asp Ile Arg Arg Lys Gln Glu Glu Tyr Ala Lys Leu Glu
115 120 125
Glu Ala Asp Leu Thr Lys Met Ser Thr Gly Asp Val Lys Lys His Lys
130 135 140
Asn Glu Leu Arg Lys Ala Ala Asn Arg Ile Lys His Ser Glu Glu Ile
145 150 155 160
Leu Gln Phe Ala Lys Trp Arg Leu Ala Asp Ile Phe Pro Leu Pro Leu
165 170 175
Ser His Asn Ser Gln Leu His Leu Lys Asn Asn Tyr His Gln Asn Val
180 185 190
Phe Ser Gly Phe His Ala Arg Val Lys Gly Trp Asn Ala Cys Asp Ile
195 200 205
Ala Ala Gln Ala Asn Tyr Ala Glu Ile Asp Asn Arg Leu Thr Glu Leu
210 215 220
Ser Ser Glu Leu Ser Gly Asp Tyr Gly Ser Glu Val Ile Thr Asp Leu
225 230 235 240
Met Gly Leu Leu Gln Tyr Thr Lys Glu Leu Gly Glu Gly Tyr Thr Asp
245 250 255
Thr Ser Tyr Leu Asn Tyr Lys Phe Leu Ser Phe Phe Lys Glu Cys Trp
260 265 270
Arg Pro Asn Ala Ile Ala Asn Asn Thr Gly Leu Leu Glu Gly Phe Trp
275 280 285
Leu Ala Asn Asn Lys His Thr Asn Lys Lys Asn Gln Val Ala Tyr Ser
290 295 300
Phe Asn Pro Lys Ile Ser Glu Glu Leu Phe Arg Arg Arg Ser Leu Trp
305 310 315 320
Glu Ser Asp Lys Cys Leu Leu Ser Asp Pro Arg Phe Glu Lys Tyr Val
325 330 335
Glu Leu Phe Asp Lys His Gly Arg Tyr Arg Lys Gly Ala Ser Leu Thr
340 345 350
Leu Ile Ser Lys Glu Ser Pro Ile Pro Ile Gly Phe Ser Met Asp Arg
355 360 365
Asn Ala Ala Lys Leu Val Arg Ile Asp Asn Asp Thr Ala Asn Arg Gln
370 375 380
Leu Thr Ile Thr Ile Glu Leu Pro Asn Lys Glu Glu Arg Ser Tyr Val
385 390 395 400
Ala Ala Tyr Gly Arg Lys His Glu Thr Lys Cys Tyr Tyr Asn Gly Leu
405 410 415
Thr Thr Arg Leu Pro Arg Ser Glu Lys Glu Leu Leu Ala Leu Ala Lys
420 425 430
Ala Glu Asn Arg Glu Leu Thr Asp Lys Glu Ile His Glu Ala Ser Leu
435 440 445
Glu Lys Cys Tyr Ile Phe Glu Tyr Ala Arg Ala Gly Lys Ile Pro Val
450 455 460
Phe Ala Val Val Lys Thr Leu Tyr Phe Arg Arg Asn Pro Ser Asn Gly
465 470 475 480
Glu Tyr Tyr Val Ile Leu Pro Thr Asn Ile Phe Val Glu Tyr His Ala
485 490 495
Asn Asn Glu Phe Asn Ser Lys Glu Leu Phe Lys Ile Arg Ser Glu Leu
500 505 510
Gln Lys Ala Trp Asp Glu Val Arg Thr Pro Lys Arg Asn Val Gln Ser
515 520 525
Cys Val Leu Asp Lys Asp Leu Ser Lys Arg Phe Ala Gly Arg Thr Leu
530 535 540
Lys Tyr Ala Gly Ile Asp Leu Gly Tyr Ser Asn Pro Tyr Thr Val Ser
545 550 555 560
Tyr Tyr Asn Val Val Gly Thr Glu Glu Gly Ile Gln Ile Lys Glu Thr
565 570 575
Gly Asn Glu Ile Val Ser Thr Val Phe Asn Glu Gln Tyr Ile Gln Leu
580 585 590
Lys Gly Asn Ile Tyr Gln Leu Ile Asn Ile Ile Arg Ala Ser Arg Arg
595 600 605
Tyr Leu Gln Glu Ser Gly Glu Leu Lys Leu Ser Lys Asp Asp Ile Lys
610 615 620
Ser Phe Asp Gln Leu Met Glu Leu Leu Pro Ser Glu Gln Arg Ile Thr
625 630 635 640
Ile Asp Gln Phe Ile Lys Asp Ile Lys Lys Ala Lys Gln Glu Gly Lys
645 650 655
Leu Ile Arg Asp Ile Lys Gly Lys Leu Pro Val Glu Gly Lys Lys Lys
660 665 670
Glu Tyr Trp Val Ile Ser Asn Leu Met Tyr Val Ile Thr Gln Thr Met
675 680 685
Asn Gly Ile Arg Gly Asn Arg Asp Ser Asn Asn His Leu Thr Glu Lys
690 695 700
Lys Asn Trp Leu Ser Ala Pro Pro Leu Ile Glu Leu Ile Asp Ala Tyr
705 710 715 720
Tyr Asn Leu Lys Lys Thr Phe Asn Asp Ser Gly Asp Gly Ile Lys Met
725 730 735
Leu Pro Lys Asp His Val Tyr Ala Glu Gly Glu Lys Gln Arg Cys Thr
740 745 750
Leu Arg Glu Glu Asn Phe Cys Lys Gly Ile Leu Glu Trp Arg Asp Asn
755 760 765
Val Lys Asp Tyr Phe Ile Lys Lys Leu Phe Ser Gln Ile Ala His Arg
770 775 780
Cys Tyr Glu Leu Gly Ile Gly Ile Val Ala Met Glu Asn Leu Asp Ile
785 790 795 800
Met Gly Ser Ser Lys Asn Thr Lys Gln Ser Asn Arg Met Phe Asn Ile
805 810 815
Trp Pro Arg Gly Gln Met Lys Lys Ser Ala Glu Asp Ala Phe Ser Tyr
820 825 830
Met Gly Ile Leu Ile Gln Tyr Val Asp Glu Asn Gly Thr Ser Arg His
835 840 845
Asp Ala Asp Ser Gly Ile Tyr Gly Cys Arg Asp Gly Ala Asn Leu Trp
850 855 860
Leu Pro Asn Lys Lys Leu His Ala Asp Val Asn Ala Ser Arg Met Ile
865 870 875 880
Ala Leu Arg Gly Leu Thr His His Thr Asn Leu Tyr Cys Arg Ser Leu
885 890 895
Thr Glu Ile Glu Asn Gly Lys Tyr Val Asn Thr Tyr Glu Leu Phe Asp
900 905 910
Thr Thr Lys Asn Asp Gln Ser Gly Ala Ala Lys Arg Leu Arg Gly Ala
915 920 925
Glu Thr Leu Leu His Gly Tyr Ser Ala Thr Val Tyr Gln Ile His Thr
930 935 940
Thr Asn Thr Gly Ala Gly Val Ala Leu Leu Pro Asp Leu Thr Ala Thr
945 950 955 960
Asp Val Ile Lys Asn Lys Lys Ile Thr Ala Thr Lys Glu Asn Thr Ala
965 970 975
Lys Tyr Tyr Lys Leu Asp Asn Thr Asn Thr Tyr Tyr Pro Trp Ser Val
980 985 990
Cys Glu Lys Leu His Lys Asn Trp Lys Leu Ser Ser Arg Ala Asp Pro
995 1000 1005
Lys Lys Lys Arg Lys Val
1010
<210> 83
<211> 885
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.4-NLS融合蛋白的氨基酸序列
<400> 83
Met Lys Lys Lys Lys Asn Phe Ser Val Ser Ala Thr Gly Val Phe Ser
1 5 10 15
Phe Pro Thr Thr Glu Ala Lys Met Asp Phe Phe His Arg Phe Ile Glu
20 25 30
Leu Asn Gly Leu Ala Ala Glu Ile Glu Thr His Phe Leu Asn Leu Lys
35 40 45
Asn Asp Lys Asn Gly Glu Ser Val Tyr Asn Lys Val Leu Ser Asn Ser
50 55 60
Asn His Ser Arg Pro Phe Ser Thr Pro Leu Leu Gly Thr Met Thr Gly
65 70 75 80
Ser Thr Lys Val Thr Asp Lys Asn Ala Leu Tyr Gly Asn Asp Leu Asp
85 90 95
His Cys Arg Lys Lys Lys Ile Val Pro Phe Ser Ser Ser Ser Pro Leu
100 105 110
Ser Ser Gln Glu Lys Phe Phe Cys Ile Glu Ala Val Phe Arg Arg Ala
115 120 125
Lys Ser His Met Glu Cys Lys Lys Leu Phe Gln Asp Glu Thr Asn Arg
130 135 140
Met Asp Ser Gln Ile Asn Gly Ile Leu Asn Glu Leu Pro Tyr Gly Val
145 150 155 160
Glu Leu Ser Asn Met Leu Ser Glu Leu Ile Ala Ile Pro Phe Ala Ile
165 170 175
Gly Trp Lys Leu Glu Gly Tyr Leu Gly Gln Val Phe Phe Pro Ser Ile
180 185 190
Ala Glu Gly Leu Thr Pro Pro Lys Ser Ala Lys Ile Lys Gly Arg Arg
195 200 205
Arg Ser Ile Asp Tyr Ser Val Thr Asp Glu Ala Tyr Asp Ile Leu Met
210 215 220
Lys Tyr Ser Asn Leu His Ser Ser Phe Glu Thr Gly Leu Lys Met Ser
225 230 235 240
Asn Leu Phe Ser Ala Phe Tyr Lys Lys Ser Asn Arg Lys Asp Glu Ile
245 250 255
Gln Phe Thr Pro Ile Ser Met Glu Ser Arg Cys Asp Leu Leu Leu Gly
260 265 270
Lys Asn Phe Leu Lys Phe Asp Leu Lys Asn Cys Asp His Arg Ser Gly
275 280 285
Ser Leu Met Leu Thr Ile Asn Asp Lys Asn Arg Leu Asn Gly Asp Tyr
290 295 300
Glu Ile Arg Val Gly Ser Asp Lys Lys Asp Ser Tyr Leu Thr Gly Val
305 310 315 320
Asn Val Thr Asn Leu Gly Asp Asn Val Phe Asn Leu Asn Tyr Lys Val
325 330 335
Asn Gly Lys Arg Glu Tyr Asn Met Leu Leu Lys Glu Pro Ser Ile His
340 345 350
Ile Lys Met His Arg Met Arg Asp Asp Gly Asn Tyr Leu Ser Ser Asp
355 360 365
Phe Asp Phe Tyr Met Ile Phe Ser Met Ser Ser Glu Lys Asp Glu Glu
370 375 380
Lys Leu Ala Arg Ser Trp Asp Met Arg Ala Ala Met Ser Thr Ala Tyr
385 390 395 400
Gly Thr Asp Ile Lys Lys Tyr His Ser Ser Phe Pro Cys Arg Ile Leu
405 410 415
Ala Cys Asp Leu Gly Val Lys His Pro Tyr Ser Ala Ala Val Met Asp
420 425 430
Ile Gly Gln Leu Asn Glu Asn Gly Met Pro Val Ser Val Asp Lys Val
435 440 445
His Cys Met His Ser Glu Gly Val Ser Glu Ile Gly Gln Gly Tyr Asn
450 455 460
His Leu Ile Gln Lys Ile Leu Ala Leu Asn Tyr Ile Leu Ala Tyr Cys
465 470 475 480
Arg Glu Phe Val Ser Gly Thr Val Asp Asp Phe Asp Lys Ile Asp Tyr
485 490 495
Lys Leu Ser Gln Leu Ser Tyr Lys Gln Glu Asp Leu Leu Ile Asn Leu
500 505 510
Gln Glu Met Lys Asp His Phe Gly Asn Asp Met Gln Ala Trp Lys Lys
515 520 525
Ser Arg Thr Trp Val Val Ser Thr Leu Phe Phe Glu Leu Arg Gln Glu
530 535 540
Phe Asn Gln Leu Arg Asn Gln Arg Pro Gly Lys Lys Thr Val Ser Leu
545 550 555 560
Ala Asp Glu Phe Gln Tyr Ile Asp Met Arg Arg Lys Phe Ile Ser Leu
565 570 575
Ser Arg Ser Tyr Thr Asn Val Gly Arg Gln Ser Ser Lys His Arg His
580 585 590
Asp Ser Tyr Gln Thr His Tyr Asp Val Ile Asn Arg Cys Lys Lys Asn
595 600 605
Leu Leu Arg Asn Ile Cys Arg Arg Met Ile Asp Met Ala Val Gln Asn
610 615 620
Lys Cys Asp Ile Ile Val Val Glu Asp Leu Ser Phe Gln Leu Ser Ser
625 630 635 640
His Asn Ser Arg Arg Asp Asn Val Phe Asn Ala Leu Trp Ser Cys Lys
645 650 655
Ser Ile Lys Asn Met Leu Gly Ile Met Ala Glu Gln His Asn Ile Ile
660 665 670
Ile Ser Glu Val Asp Pro Asn His Thr Ser Lys Ile Asp Cys Glu Thr
675 680 685
Gly Asn Phe Gly Tyr Arg Tyr Ser Ser Asp Phe Tyr Ser Val Ile Asp
690 695 700
Gly Gln Leu Val Arg Arg His Ala Asp Glu Asn Ala Ala Ile Asn Ile
705 710 715 720
Gly Asn Arg Trp Ala Ser Arg His Thr Asp Leu Lys Ser Phe Asn Cys
725 730 735
Arg Gln Ile Ser Ile Asp Gly Arg Lys Val Ala Phe Pro Tyr Ala Lys
740 745 750
Gly Lys Arg Lys Ser Ala Leu Phe Gly Tyr Leu Phe Gly Asn Cys Lys
755 760 765
Thr Val Phe Val Ser Asp Asp Gly Asp Ser Tyr Thr Pro Ile Pro Tyr
770 775 780
Ser Lys Phe Arg Lys Ser Ile Ser Lys Asp Asp His Asp Val Val Asn
785 790 795 800
Tyr Leu His Asp Leu Thr Met Asn Lys Asn Val Ile Arg Val Glu Tyr
805 810 815
Asn Lys Ser Ile Lys Ser Ala Ser Val Glu Leu Tyr Leu Asn Asp Asp
820 825 830
Arg Val Ile Ser Arg Ser Leu Arg Asp Lys Glu Val Asp Ala Ile Glu
835 840 845
Lys Leu Val Ser Arg Gly Ser Leu Ile Asn Glu Ser Gly Pro Ser Leu
850 855 860
Glu His Asp Glu Val Lys Ser Val Thr His Ser Arg Ala Asp Pro Lys
865 870 875 880
Lys Lys Arg Lys Val
885
<210> 84
<211> 881
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.5-NLS融合蛋白的氨基酸序列
<400> 84
Met Lys Val His Glu Ile Pro Arg Ser Gln Leu Leu Lys Ile Lys Gln
1 5 10 15
Tyr Glu Gly Ser Phe Val Glu Trp Tyr Arg Asp Leu Gln Glu Asp Arg
20 25 30
Lys Lys Phe Ala Ser Leu Leu Phe Arg Trp Ala Ala Phe Gly Tyr Ala
35 40 45
Ala Arg Glu Asp Asp Gly Ala Thr Tyr Ile Ser Pro Ser Gln Ala Leu
50 55 60
Leu Glu Arg Arg Leu Leu Leu Gly Asp Ala Glu Asp Val Ala Ile Lys
65 70 75 80
Phe Leu Asp Val Leu Phe Lys Gly Gly Ala Pro Ser Ser Ser Cys Tyr
85 90 95
Ser Leu Phe Tyr Glu Asp Phe Ala Leu Arg Asp Lys Ala Lys Tyr Ser
100 105 110
Gly Ala Lys Arg Glu Phe Ile Glu Gly Leu Ala Thr Met Pro Leu Asp
115 120 125
Lys Ile Ile Glu Arg Ile Arg Gln Asp Glu Gln Leu Ser Lys Ile Pro
130 135 140
Ala Glu Glu Trp Leu Ile Leu Gly Ala Glu Tyr Ser Pro Glu Glu Ile
145 150 155 160
Trp Glu Gln Val Ala Pro Arg Ile Val Asn Val Asp Arg Ser Leu Gly
165 170 175
Lys Gln Leu Arg Glu Arg Leu Gly Ile Lys Cys Arg Arg Pro His Asp
180 185 190
Ala Gly Tyr Cys Lys Ile Leu Met Glu Val Val Ala Arg Gln Leu Arg
195 200 205
Ser His Asn Glu Thr Tyr His Glu Tyr Leu Asn Gln Thr His Glu Met
210 215 220
Lys Thr Lys Val Ala Asn Asn Leu Thr Asn Glu Phe Asp Leu Val Cys
225 230 235 240
Glu Phe Ala Glu Val Leu Glu Glu Lys Asn Tyr Gly Leu Gly Trp Tyr
245 250 255
Val Leu Trp Gln Gly Val Lys Gln Ala Leu Lys Glu Gln Lys Lys Pro
260 265 270
Thr Lys Ile Gln Ile Ala Val Asp Gln Leu Arg Gln Pro Lys Phe Ala
275 280 285
Gly Leu Leu Thr Ala Lys Trp Arg Ala Leu Lys Gly Ala Tyr Asp Thr
290 295 300
Trp Lys Leu Lys Lys Arg Leu Glu Lys Arg Lys Ala Phe Pro Tyr Met
305 310 315 320
Pro Asn Trp Asp Asn Asp Tyr Gln Ile Pro Val Gly Leu Thr Gly Leu
325 330 335
Gly Val Phe Thr Leu Glu Val Lys Arg Thr Glu Val Val Val Asp Leu
340 345 350
Lys Glu His Gly Lys Leu Phe Cys Ser His Ser His Tyr Phe Gly Asp
355 360 365
Leu Thr Ala Glu Lys His Pro Ser Arg Tyr His Leu Lys Phe Arg His
370 375 380
Lys Leu Lys Leu Arg Lys Arg Asp Ser Arg Val Glu Pro Thr Ile Gly
385 390 395 400
Pro Trp Ile Glu Ala Ala Leu Arg Glu Ile Thr Ile Gln Lys Lys Pro
405 410 415
Asn Gly Val Phe Tyr Leu Gly Leu Pro Tyr Ala Leu Ser His Gly Ile
420 425 430
Asp Asn Phe Gln Ile Ala Lys Arg Phe Phe Ser Ala Ala Lys Pro Asp
435 440 445
Lys Glu Val Ile Asn Gly Leu Pro Ser Glu Met Val Val Gly Ala Ala
450 455 460
Asp Leu Asn Leu Ser Asn Ile Val Ala Pro Val Lys Ala Arg Ile Gly
465 470 475 480
Lys Gly Leu Glu Gly Pro Leu His Ala Leu Asp Tyr Gly Tyr Gly Glu
485 490 495
Leu Ile Asp Gly Pro Lys Ile Leu Thr Pro Asp Gly Pro Arg Cys Gly
500 505 510
Glu Leu Ile Ser Leu Lys Arg Asp Ile Val Glu Ile Lys Ser Ala Ile
515 520 525
Lys Glu Phe Lys Ala Cys Gln Arg Glu Gly Leu Thr Met Ser Glu Glu
530 535 540
Thr Thr Thr Trp Leu Ser Glu Val Glu Ser Pro Ser Asp Ser Pro Arg
545 550 555 560
Cys Met Ile Gln Ser Arg Ile Ala Asp Thr Ser Arg Arg Leu Asn Ser
565 570 575
Phe Lys Tyr Gln Met Asn Lys Glu Gly Tyr Gln Asp Leu Ala Glu Ala
580 585 590
Leu Arg Leu Leu Asp Ala Met Asp Ser Tyr Asn Ser Leu Leu Glu Ser
595 600 605
Tyr Gln Arg Met His Leu Ser Pro Gly Glu Gln Ser Pro Lys Glu Ala
610 615 620
Lys Phe Asp Thr Lys Arg Ala Ser Phe Arg Asp Leu Leu Arg Arg Arg
625 630 635 640
Val Ala His Thr Ile Val Glu Tyr Phe Asp Asp Cys Asp Ile Val Phe
645 650 655
Phe Glu Asp Leu Asp Gly Pro Ser Asp Ser Asp Ser Arg Asn Asn Ala
660 665 670
Leu Val Lys Leu Leu Ser Pro Arg Thr Leu Leu Leu Tyr Ile Arg Gln
675 680 685
Ala Leu Glu Lys Arg Gly Ile Gly Met Val Glu Val Ala Lys Asp Gly
690 695 700
Thr Ser Gln Asn Asn Pro Ile Ser Gly His Val Gly Trp Arg Asn Lys
705 710 715 720
Gln Asn Lys Ser Glu Ile Tyr Phe Tyr Glu Asp Lys Glu Leu Leu Val
725 730 735
Met Asp Ala Asp Glu Val Gly Ala Met Asn Ile Leu Cys Arg Gly Leu
740 745 750
Asn His Ser Val Cys Pro Tyr Ser Phe Val Thr Lys Ala Pro Glu Lys
755 760 765
Lys Asn Asp Glu Lys Lys Glu Gly Asp Tyr Gly Lys Arg Val Lys Arg
770 775 780
Phe Leu Lys Asp Arg Tyr Gly Ser Ser Asn Val Arg Phe Leu Val Ala
785 790 795 800
Ser Met Gly Phe Val Thr Val Thr Thr Lys Arg Pro Lys Asp Ala Leu
805 810 815
Val Gly Lys Arg Leu Tyr Tyr His Gly Gly Glu Leu Val Thr His Asp
820 825 830
Leu His Asn Arg Met Lys Asp Glu Ile Lys Tyr Leu Val Glu Lys Glu
835 840 845
Val Leu Ala Arg Arg Val Ser Leu Ser Asp Ser Thr Ile Lys Ser Tyr
850 855 860
Lys Ser Phe Ala His Val Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys
865 870 875 880
Val
<210> 85
<211> 975
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.6-NLS融合蛋白的氨基酸序列
<400> 85
Met Ser Ala Asn Arg Val Ser Ala Asn Ser Gln Phe Glu Leu Gly Tyr
1 5 10 15
Pro Met Ser Leu Ser Leu Arg Gly Lys Val Phe Asn Ser Arg Glu Met
20 25 30
Met Lys Glu Ile Leu Pro Val Met Asn Asn Ile Val His Tyr Gln Asn
35 40 45
Asn Leu Leu Lys Leu Met Leu Ile Leu Arg Gly Glu Lys Tyr Thr Leu
50 55 60
Asp Gly Gln Phe Phe Ser Gln Lys Asp Val Asp Arg Gln Phe Gly Asp
65 70 75 80
Leu Cys Lys Glu His Asn Ile Lys Gly Ser Ile Cys Ser Leu Lys Glu
85 90 95
Lys Ser Arg Lys Leu Tyr Glu Val Phe Ser Cys Tyr Ile Asp Lys Lys
100 105 110
Gly Asn Leu Lys Thr Asn Ser Lys Ala Arg Ser Phe Ala Gly Val Leu
115 120 125
Leu Asn Pro Lys Asp Val Lys Leu Pro Pro Gln Ile Asp Ser Ile Ser
130 135 140
Ser Phe Val Val Glu Leu Arg Ala Lys Gly Val Leu Pro Ile Lys His
145 150 155 160
Glu Gly Asn Tyr Leu Ser Gly His Pro Ser Leu Lys Tyr Ser Val Ala
165 170 175
Gln Asn Val Leu Val Lys Leu Thr Ser Met Glu Lys Leu Gln Lys Ile
180 185 190
Tyr Ser Asp Glu Lys Ala Gly Trp Glu Asn Ile Val Ser Glu Val Arg
195 200 205
Ser Asp Leu Pro Lys Ile Glu Arg Tyr Glu Arg Met Leu Leu Ser Ile
210 215 220
Lys Ala Val Lys Glu Met Glu Lys Phe Gly Ile Asn Asn Tyr Arg His
225 230 235 240
Leu Leu Asn Asn Trp Arg Asp Glu Val Asp Lys Asp Ser Gly Lys Val
245 250 255
Leu Lys Gln Gly Met Arg Thr Tyr Phe Val Asn Met Leu Glu Ser Lys
260 265 270
Lys Asp Tyr Arg Phe Glu Glu Ser Asp Arg Tyr Leu Phe Gly Tyr Ala
275 280 285
Pro Glu Val Met Asn Leu Val Tyr His Asp Phe Arg Asp Leu Trp Gln
290 295 300
Gly Glu Asp Ile Ile Gly Ser Gln Ser Pro Glu Lys Lys Asp Arg Asp
305 310 315 320
Tyr Val Asp Val Ile Phe Asn Tyr Phe Asn Trp Arg Lys Glu Ser Ile
325 330 335
Asn Ile Ser Ser Phe Asp Ser Tyr Gly Lys Thr Ala Gln Ile Lys Leu
340 345 350
Gly Asp Asn Tyr Val Pro Phe Ser Asn Phe Gln Tyr Asp Lys Ile Leu
355 360 365
Asp Ala Trp Thr Leu Glu Ile Ala Asn Val Ser Gly Glu Gly Asp Asn
370 375 380
His Lys Leu Val Ile Ala Arg Ser Pro Gln Phe Asp Ser His Ser Ser
385 390 395 400
Val Lys Asp Ile Val Met Lys Asn Leu Lys Gly Lys Glu Ala Ser Lys
405 410 415
Thr Thr Leu Glu Phe Arg Tyr Ser Gly Asp Ser Lys Lys Ser Thr Trp
420 425 430
Tyr Arg Gly Thr Leu Lys Glu Pro Thr Leu Arg Tyr Ser Ser Ser Lys
435 440 445
Asn Cys Leu Tyr Val Asp Phe Ala Leu Ser Asn His Ile Val Glu Gly
450 455 460
Leu Ile Ser Asp Asn Leu Gly Ile Ser Asp Lys Met Tyr Lys Phe Arg
465 470 475 480
Gly Glu Phe Met Lys Ala Ser Pro Ser Ser Gly Lys Gln Ser Asn Ser
485 490 495
Ile Asn Leu Pro Ile Lys Lys Leu Arg Ala Met Gly Val Asp Phe Asn
500 505 510
Leu Arg Arg Pro Phe Gln Ala Ser Ile Tyr Asp Val Glu Asn Lys Asn
515 520 525
Gly Asn Leu Glu Phe Ser Phe Val Lys His Val Gln Ser Phe Ser Asn
530 535 540
Glu Asn Asp Glu Glu Arg Ala Lys Glu Leu Leu Asn Ile Glu Arg Asn
545 550 555 560
Ile Leu Ala Leu Lys Ile Leu Ile Trp Gln Thr Val Gly Tyr Val Thr
565 570 575
Gly Lys Asn Asp Thr Ile Asp Gly Val Val Thr Arg Lys Asn Asn Ala
580 585 590
Val Asp Ile Glu Lys Thr Leu Gly Ile Asn Met Lys Glu Tyr Met Ala
595 600 605
Tyr Leu Asn Gln Phe Arg Ser Tyr Glu Asp Lys Asn Lys Ala Phe Met
610 615 620
Asp Leu Arg Lys Arg Glu Tyr Ala Trp Ile Val Pro Pro Leu Ile Phe
625 630 635 640
Gln Cys Arg Ser Arg Leu Ile Ser Phe Arg Ser Glu Tyr Phe Asn Thr
645 650 655
Pro Lys Asp Glu Lys Ser His Tyr Cys Gln His Arg Asn Phe Val Asp
660 665 670
Tyr Ser Thr Phe Leu Lys Lys Asn Val Val Lys Lys Met Met Glu Leu
675 680 685
Arg Arg Ser Tyr Ser Thr Phe Gly Met Ser Ser Glu Gln Ser Ile Trp
690 695 700
Val Thr Asn Asn Asp His Ala Lys Asp Gly Ser Lys Lys Asn Gly Asn
705 710 715 720
Met Phe Asp Asp Asp Leu His Gln Trp Tyr Asn Gly Leu Val Arg Lys
725 730 735
Cys Ser Ser Leu Ala Ser Ser Ile Ile Asn Val Ala Arg Asp Asn Gly
740 745 750
Ala Ile Leu Val Phe Ile Glu Asp Leu Asp Cys His Pro Ser Ala Phe
755 760 765
Asp Ser Glu Glu Asp Asn Ser Leu Lys Ser Ile Trp Gly Trp Gly Ser
770 775 780
Ile Lys Ala Ser Leu Ala His Gln Ala Arg Lys His Asn Ile Ala Val
785 790 795 800
Val Ala Asn Asp Pro His Leu Thr Ser Leu Val Ser Ser Thr Thr Gly
805 810 815
Glu Leu Gly Ile Ala Lys Gly Arg Asp Val Leu Phe Phe Asp Ser Lys
820 825 830
Gly Lys Leu Thr Ser Lys Val Asn Arg Asp Glu Asn Ala Ala Gln Asn
835 840 845
Ile Ala Ile Arg Gly Phe Val Arg His Ser Asp Leu Arg Glu Phe Val
850 855 860
Ala Glu Lys Ile Glu Glu Asn Arg Tyr Arg Val Val Val Asn Lys Thr
865 870 875 880
His Lys Arg Lys Ala Gly Ala Ile Tyr Arg His Ile Gly Ser Thr Glu
885 890 895
Cys Ile Met Ser Lys Gln Ala Asp Gly Ser Leu Lys Ile Asp Lys Thr
900 905 910
Glu Leu Thr Pro Leu Glu Ile Lys Met Glu Lys Lys Asn Asp Lys Lys
915 920 925
Met Tyr Val Ile Leu His Gly Lys Thr Trp Arg Leu Arg His Glu Leu
930 935 940
Asn Glu Lys Leu Glu Lys Asp Leu Asp Asn His Leu Lys Ser Lys Ser
945 950 955 960
Ser Val Ile Ser Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
965 970 975
<210> 86
<211> 973
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.7-NLS融合蛋白的氨基酸序列
<400> 86
Met Ser Ser Ala Asn Asp Gln Leu Gly Leu Gly Tyr Pro Leu Thr Leu
1 5 10 15
Thr Leu Arg Gly Lys Val Tyr Asn His Asp Thr Ala Met Glu Ala Phe
20 25 30
Ala Pro Val Met Lys Gly Met Val Pro Tyr Ala Asn Asn Leu Met Arg
35 40 45
Ile Leu Leu Thr Leu Arg Leu Glu Lys Tyr Thr Leu Asp Gly Ile His
50 55 60
His Thr Lys Glu Glu Val Glu Lys Asp Leu Arg Gly Leu Met Lys Glu
65 70 75 80
Tyr Gly Ile Asn Leu Ser Phe Ala Lys Phe Ser Glu Met Ala Gly Glu
85 90 95
Val Tyr Arg Val Phe Val Cys Tyr Val Asp Ala Lys Gly Lys Leu Lys
100 105 110
Val Asn Gly Lys Ala Arg Gly Phe Ala Asn Val Phe Phe Ser Glu Asp
115 120 125
Asp Ala Thr Ile Pro Glu Asn Cys Pro Ser Met Glu Leu Leu Arg Lys
130 135 140
Lys Gly Met Phe Pro Ile Leu Val Asp Gly Lys Pro Ile Ser Ser Ile
145 150 155 160
Ser Arg Glu Lys Thr Pro Leu Lys Tyr Ser Val Ala Gln Asp Val Leu
165 170 175
Thr Lys Leu Thr Ser Met Glu Glu Ile Ser Lys Glu Tyr Glu Lys Ala
180 185 190
Lys Thr Asp Trp Glu Asn Glu Cys Gln Lys Val Ile Ser Gln Leu Pro
195 200 205
Leu Ile Gly Arg Tyr Glu Ala Leu Leu Thr Thr Ile Pro Leu Ile Pro
210 215 220
Glu Met Arg Gly Phe Asp Gly Asp Asn Tyr Arg Lys Met Leu Asn Arg
225 230 235 240
Trp Arg Asp Tyr Val Asn Glu Asp Gly Glu Leu Val Arg Gly Gly Met
245 250 255
Lys Thr Tyr Phe Leu Asp Leu Leu Ser Lys Asp Thr Ser His Lys Phe
260 265 270
Asn Glu Glu Glu Arg Tyr Leu Phe Gly Tyr Cys Pro Glu Phe Met Asn
275 280 285
Leu Ile Tyr His Asp Phe Arg Asp Leu Trp Ser Lys Glu Asp Ile Ile
290 295 300
Gly Ser Gln Arg Lys Gly Lys Gly Leu Lys Gly Lys Asp Tyr Val Asp
305 310 315 320
Val Ile Phe Asn Cys Phe His Trp Arg Arg Glu Ser Ile Asn Ile Ser
325 330 335
Ser Phe Gly Asn Asn Asp Lys Val Met Asn Ile His Leu Gly Asp Asn
340 345 350
Phe Val Pro Phe Glu Leu Lys Ser Gln Asn Gly Ile Trp Glu Val His
355 360 365
Val Gln Asn Leu His Gly Gln Asn Asp Pro His Arg Val Ile Val Cys
370 375 380
Arg Cys Pro Gln Phe Asn Glu Asp Ser Ser Met Lys Met Val His Pro
385 390 395 400
Leu Ala Lys Asn Gly Glu Glu Ser Asp Lys Glu Asn Ile Glu Phe Arg
405 410 415
Tyr Ser Gly Asp Ser Lys Arg Glu Thr Trp Tyr Thr Gly Leu Leu Lys
420 425 430
Glu Pro Thr Leu Arg Tyr Asp Val Glu Arg Lys Ser Leu Tyr Val Asp
435 440 445
Phe Ile Leu Ser Asn His Arg Val Glu Gly Val Val Thr Asn Glu Tyr
450 455 460
Leu Lys Asp Pro Arg Asp Leu Phe Gly Val Arg Gly Tyr Phe Leu Ser
465 470 475 480
Ser Ser Val Ser Asn Pro Arg Gln Lys Asp Lys Thr Ser Leu Pro Asp
485 490 495
Gly Lys Phe Asn Val Met Gly Val Asp Leu Gly Leu Lys Cys Pro Tyr
500 505 510
Glu Cys Ala Ile Tyr Gly Ile Thr Val Lys Asn Gly Lys Met Gln His
515 520 525
Lys Trp Ser His Asn Val Ser Ala Glu Asp Asn Asn Asn Val Ser Glu
530 535 540
Arg Leu Ala Asn Leu Lys Lys Ile Asp Glu Lys Ile Leu Ala Thr Gln
545 550 555 560
Val Leu Ile Ser Leu Thr Lys Met Cys Val Val Lys Asp Glu Glu Ile
565 570 575
Pro Asp Ser Tyr Thr Leu Arg Glu His Arg Val Asp Ile Ala Lys Ser
580 585 590
Leu Asp Leu Asp Met Asp Lys Tyr Arg Arg Tyr Val Glu Lys Cys Lys
595 600 605
Lys Asn Pro Asp Lys Ile Gln Ala Leu Lys Asp Ile Arg Lys Ser Glu
610 615 620
Asn Asn Trp Ile Val Ala Glu Lys Ile Asn Glu Ile Arg Ser Leu Ile
625 630 635 640
Ser Glu Ile Arg Ser Glu Tyr Tyr Ala Ser Lys Asp Lys Arg Asn Tyr
645 650 655
Cys Arg Asn Leu Asn Gly Val Asp Leu Ser Val Phe Leu Lys Lys Lys
660 665 670
Val Val Lys Asn Trp Ile Ser Leu Leu Arg Ser Phe Ser Thr Phe Gly
675 680 685
Met Thr Pro Gln Glu Ser Ala Tyr Ile Arg Lys Asp Phe Ala Lys Asn
690 695 700
Leu Ser Lys Trp Tyr Lys Gly Leu Val Arg Lys Cys Gly Ser Ile Ala
705 710 715 720
Ala His Ile Val Asn Ile Ala Arg Asp Asn Lys Val Met Val Ile Phe
725 730 735
Ile Glu Asp Leu Asp Ala Arg Thr Ser Ala Phe Asp Ser Lys Glu Asp
740 745 750
Asn Glu Leu Lys Ile Leu Trp Gly Trp Gly Glu Ile Lys Lys Trp Ile
755 760 765
Gly His Gln Ala Arg Lys His Asn Ile Ala Val Val Ala Val Asp Pro
770 775 780
His Leu Thr Ser Leu Val Asn His Glu Ser Gly Leu Leu Gly Ile Ala
785 790 795 800
Gly Ser Gly Asn Asp Arg Asn Ile Tyr Thr Phe Gln Lys Asn Lys Lys
805 810 815
Tyr Val Val Ile Asn Arg Asp Asn Asn Ala Ala His Asn Ile Ala Leu
820 825 830
Arg Gly Leu Ser Lys His Thr Asp Ile Arg Glu Phe Tyr Val Glu Gln
835 840 845
Ile Asp Val Asp His Tyr Arg Leu Met Tyr Gly Pro Glu Ala Glu Asn
850 855 860
Gly Lys Arg Arg Ser Gly Ala Ile Tyr Lys His Ile Gly Ser Thr Glu
865 870 875 880
Cys Val Phe Ser Lys Gln Lys Asn Gly Thr Leu Lys Val Glu Lys Thr
885 890 895
Ser Leu Thr Lys Asp Glu Lys Glu Met Pro Lys Ile Asn Gly Lys Gly
900 905 910
Val Tyr Ala Ile Leu His Gly Asn Glu Trp Arg Leu Arg His Glu Leu
915 920 925
Asn Glu Glu Leu Gly Ala Lys Leu Asp Gly Ile Ser Val Lys Arg Val
930 935 940
Val Ser Glu Pro Asn Lys Val Lys Thr Ser Leu Val Lys Gly Ser Val
945 950 955 960
Arg Ala Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
965 970
<210> 87
<211> 918
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.8-NLS融合蛋白的氨基酸序列
<400> 87
Met Lys Lys Gln Thr Ile Val Lys Lys Asp Ser Lys Ala Glu Thr Lys
1 5 10 15
Glu Asn Lys Met Tyr Pro Asp Lys Asp Thr Asp Phe Pro Val Asn Ser
20 25 30
Gln Phe Ser Arg Ser Ile Ser Ile Arg Ala Asn Val Asp Pro Lys Asp
35 40 45
Leu Leu Val Leu Lys Arg Thr Phe Glu Glu Thr Thr Lys Ile Ser Asp
50 55 60
Glu Leu Leu Ser Thr Leu Leu Met Leu Arg Gly Lys Asp Tyr Cys Leu
65 70 75 80
Asp Asn Val Val Cys Lys Gly Glu Glu Val Leu Glu Asn Leu Tyr Lys
85 90 95
Lys Leu Ser Lys Asn Ala Thr Val Asn Arg Asp Lys Phe Ile Ser Thr
100 105 110
Ala Lys Ala Phe Tyr Glu Tyr Phe His Gly Cys Ser Tyr His Lys Gly
115 120 125
Phe Lys Ser Phe Phe Phe Ser Ser Lys Glu Ile Asp Ser Ile Gln Ser
130 135 140
Glu Lys Phe Gly Tyr Leu Arg Glu Ile Gly Leu Phe Pro Ile Lys Ile
145 150 155 160
Asp Ala Gln Ile Ser Asn Asp Leu Gln Tyr Ser Ile Val Ala Ser Asn
165 170 175
His Ala Lys Ile Lys Gly Phe Glu Lys Ile Asp Lys Glu Tyr Gln Ala
180 185 190
Asn Lys Glu Lys Trp Asn Lys Thr Ile Gly Glu Ser Thr Leu Lys His
195 200 205
Leu Asn Arg Tyr Gly Glu Met Leu Lys Gly Leu Ser Asp Leu Gly Thr
210 215 220
Met Gly Asn Phe Asn Gly Lys Lys Tyr Asp Arg Phe Met Gly His Trp
225 230 235 240
Arg Asn Glu Gln Lys Ile Pro Asp His Ile Ser Met Leu Asp Phe Phe
245 250 255
Arg Lys Ile Tyr Gln Glu Lys Gly Lys Ser His Arg Phe Thr Ala Ile
260 265 270
Asp Asn Phe Thr Tyr Gly Tyr Glu Ser Glu Phe Met Asn His Ile Tyr
275 280 285
Leu Asn Phe Ser Asp Leu Trp Leu Lys Glu Asp Val Ile Gly Asp Glu
290 295 300
Glu Tyr Val Ser Leu Ile Arg Gly Ala Tyr His Trp Gln Lys Asp Val
305 310 315 320
Val Gly Ile Ala Ser Phe Ser Gly Tyr Asn Lys Tyr Glu Lys Leu Phe
325 330 335
Met Gly Asp Asn Lys Ile Asn Tyr Ala Leu Asp Phe Ser Asn Lys Asp
340 345 350
Gln Trp Leu Met Lys Phe Asn Asn Val Ile Ser Lys Glu Pro Glu Thr
355 360 365
Ile Thr Leu Arg Leu Cys Lys Asn Gly Tyr Phe Asn Asn Leu Ser Val
370 375 380
Leu Glu Lys Asn Asp Glu Asn Gly Arg Tyr Lys Ile Arg Phe Ser Thr
385 390 395 400
Glu Lys Gln Gly Lys Tyr Phe Tyr Glu Ala Phe Ile Arg Glu Pro Phe
405 410 415
Leu Arg Tyr Asn Lys Asp Asn Asp Lys Ile Tyr Val His Phe Cys Leu
420 425 430
Ser Glu Glu Ile Lys Glu Asn Cys Pro Asn His Leu Asp Thr Arg Ser
435 440 445
Asp Lys Tyr Leu Phe Lys Ser Ala Leu Leu Thr Asn Ser Arg Gln Lys
450 455 460
Leu Gly Lys Leu His Tyr Arg Asp Phe His Ile Val Gly Val Asp Leu
465 470 475 480
Gly Ile Asn Pro Val Ala Lys Ile Thr Val Cys Lys Val His Val Asp
485 490 495
Lys Asn Glu Asn Leu Lys Ile Thr Lys Ile Ile Thr Glu Glu Thr Arg
500 505 510
Lys Asn Ile Asp Thr Asn Tyr Leu Asp Gln Leu Asn Leu Leu Tyr Lys
515 520 525
Lys Ile Val Ser Leu Lys Arg Leu Ile Arg Ala Thr Val Ala Phe Lys
530 535 540
Lys Asp Gly Glu Glu Ile Pro Lys Met Phe Lys Met Gly Lys Lys Ser
545 550 555 560
Pro Tyr Phe Leu Asn Trp Thr Glu Val Leu Asn Val Asn Tyr Asp Asp
565 570 575
Tyr Ile Lys Glu Ile Ser Thr Phe Ser Val Asp Arg Leu Ser Gly Leu
580 585 590
Thr Leu Pro Met Gln Trp Ala Arg Ser Gln Asn Lys Trp Val Val Lys
595 600 605
Asp Leu Thr Lys Met Val Arg Lys Gly Ile Ser Asp Leu Ile Tyr Ala
610 615 620
Arg Tyr Phe Asn Cys Ser Asp Lys Thr Gln Tyr Val Thr Glu Asn Asn
625 630 635 640
Ala Val Asp Ile Thr Thr Phe Lys Lys His Asp Ile Ile Ser Glu Ile
645 650 655
Ile Gly Leu Gln Lys Met Phe Ser Gly Gly Gly Lys Asp Val Ala Lys
660 665 670
Lys Asp Tyr Leu Tyr Leu Arg Gly Leu Arg Lys His Ile Gly Asn Tyr
675 680 685
Thr Ala Ser Ala Ile Val Ser Ile Ala Gln Lys Tyr Asn Ala Val Phe
690 695 700
Ile Phe Ile Glu Asp Leu Asp Leu Lys Ile Ser Gly Met Asn Gly Lys
705 710 715 720
Lys Glu Asn Lys Val Lys Ile Leu Trp Gly Val Gly Gln Leu Lys Lys
725 730 735
Arg Leu Ser Glu Lys Ala Glu Lys Phe Gly Ile Gly Ile Val Pro Val
740 745 750
Asn Pro Glu Leu Thr Ser Gln Met Asp Arg Glu Thr Phe Leu Leu Gly
755 760 765
Tyr Arg Asn Pro Thr Asn Lys Lys Glu Leu Tyr Val Lys Arg Asp Asp
770 775 780
Lys Ile Glu Ile Leu Asp Ala Asp Glu Thr Ala Ser Tyr Asn Val Ala
785 790 795 800
Leu Arg Gly Leu Gly His His Ala Asn Leu Ile Gln Phe Arg Ala Asp
805 810 815
Lys Met Pro Asn Gly Cys Phe Arg Val Met Pro Asp Arg Lys Tyr Lys
820 825 830
Gln Gly Ala Leu Tyr Gly Tyr Leu Asn Ser Thr Ala Val Leu Phe Lys
835 840 845
Asp Lys Gly Asp Gly Val Leu Thr Ile His Lys Ser Lys Leu Thr Lys
850 855 860
Lys Glu Arg Asp Ser Arg Pro Ile Lys Gly Lys Lys Thr Phe Val Val
865 870 875 880
Lys Asn Gly Lys Arg Trp Ile Leu Arg His Val Leu Asp Glu Glu Val
885 890 895
Lys Lys Tyr Pro Glu Met Tyr Asn Ser Gln Asn Ser Arg Ala Asp Pro
900 905 910
Lys Lys Lys Arg Lys Val
915
<210> 88
<211> 923
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.9-NLS融合蛋白的氨基酸序列
<400> 88
Met Ser Asp Tyr Lys Phe Ser Asn Asn Gly Val Thr Asn Thr Gly Ser
1 5 10 15
Ala His Ile Gly Leu Ser Pro Glu Asn Ser Ser Thr Val Met Asp Met
20 25 30
Phe Lys Val Ile Thr Lys Asp Ala Asp Phe Leu Leu Lys Asn Leu Leu
35 40 45
Ile Met Glu Gly Gly Glu Tyr Met Leu Asn Arg Glu Ile His Asn Gly
50 55 60
Asp Lys Glu Phe Asp Lys Ile Ile Ser Lys Leu Gly Leu Ser Lys Lys
65 70 75 80
Glu Lys Glu Asn Leu Lys Met Lys Cys Lys Asp Phe Phe Phe Asp Phe
85 90 95
Val Lys Leu Gln Asn Gly Arg Ser Leu Ala Asn Ile Leu Phe Glu Thr
100 105 110
Lys Gly Thr Thr Leu Ile Gly Cys Gly Lys Asp Lys Lys Gly Glu Lys
115 120 125
Val Asp Gly Glu Tyr Pro Thr Ile Tyr His Asp His Glu Thr Leu Arg
130 135 140
Ser Thr Gly Leu Leu Pro Leu Lys Phe Ser Lys Asn Ile Asp Asp Val
145 150 155 160
Asp Tyr Lys Tyr Leu Ile Cys Tyr Leu Val His Asn Val Leu Ser Ser
165 170 175
Phe Ile Glu Lys Arg Asp Ala Tyr Asn Asp Asn Lys Lys Glu Trp Glu
180 185 190
Ser Lys Leu Ser Asn Ser Asn Leu Pro Gln Leu Glu Arg Met Ser Glu
195 200 205
Phe Leu Asn Gly Ile Asn His Leu Gly Asn Ile Ile Gly Trp Asn Gly
210 215 220
Lys Lys Tyr Ile Gly Phe Ile Lys Lys Trp Thr Asp Glu Glu Ser Ser
225 230 235 240
Met Tyr Asp Phe Phe Val Gln Lys Leu Gln Asp Asn Pro Lys Tyr Lys
245 250 255
Phe Gly Lys Lys Asp Gln Phe Leu Tyr Gly Tyr Glu Pro Glu Phe Leu
260 265 270
Asn Tyr Leu Phe His Asp Phe Arg Asp Leu Trp His Pro Asp Asn Leu
275 280 285
Ile Gly Lys Asp Glu Tyr Val Asp Leu Ile Ser Gly Lys Asn Asn Thr
290 295 300
Asp Ala Glu Thr Ala Asn Lys Gly Ala Tyr His Trp Leu Lys Asp Phe
305 310 315 320
Ile Asn Ile Ser Ser Phe Asp Ala Tyr Gly Lys Met Ala Thr Ile Gly
325 330 335
Met Gly Asn Asn Leu Ile Asn Tyr Ser Met Asn Ile Asp Lys Asp Gly
340 345 350
Lys Ile Ile Val Asn Met Asp Asn Ile Phe Asp Arg Ser Lys Pro Ile
355 360 365
Val Phe Asn Val Tyr Arg Asn Ser Tyr Phe Arg Asn Phe Lys Ile Ile
370 375 380
Glu Ser Asp Asp Lys Lys Gly Ile Tyr Lys Val Glu Phe Ser Thr Ser
385 390 395 400
Asn Asn Gly Val Ile Tyr Glu Gly Tyr Ile Lys Ser Pro Ser Leu Arg
405 410 415
Phe Ala Thr Lys Gly Gly Thr Ile Lys Ile Asp Phe Pro Ile Ser Asp
420 425 430
Lys Arg Ile Lys Gly Gly Arg Glu Met Asn Thr Asp Leu Met Trp Phe
435 440 445
Leu Asn Arg Ala Ser Pro Cys Ser Thr Lys Asn Lys Glu Val Asn Ser
450 455 460
Phe Ile Gly Lys Asn Phe Val Gly Leu Ala Ile Asp Arg Gly Ile Asn
465 470 475 480
Pro Leu Met Ala Trp Tyr Val Ala Glu Trp Thr Tyr Asp Lys Asp Gly
485 490 495
Lys Ala Lys Ile Val Arg Ser Ile Ala Asn Gly Arg Val Asp Ser Gly
500 505 510
His Asn Glu Ser Glu Val Lys Phe Val Arg Glu Thr Thr Asn Arg Ile
515 520 525
Val Gly Ile Lys Ser Leu Val Trp Asn Thr Val Lys Tyr Arg Thr Gly
530 535 540
Gly Ser Glu Gly Ile Asp Arg Cys Arg Lys Ser Gln Asn Gly Gln Val
545 550 555 560
Asp Leu Phe Glu Met Phe Asp Ile Asp Tyr Asn Asn Tyr Leu Lys Glu
565 570 575
Val Asn Asn Leu Pro Tyr Asp Pro Asn Ser Glu Arg Ser Ile Ile Gln
580 585 590
Thr Trp Val Ser Ser Pro Trp Lys Val Lys Asp Leu Val Lys Asp Ala
595 600 605
Lys Asn Arg Met Val Gln Ile Lys Thr Gln Tyr His Asn Ala Lys Asp
610 615 620
Lys Glu Lys Tyr Ile Thr Thr Gln Asn Arg Ala Gly Phe Tyr Asp Phe
625 630 635 640
Leu Lys Ile Glu Met Glu Lys Gln Phe Thr Ser Leu Gln Arg Met Phe
645 650 655
Ser Gly Gly Gln Lys Asp Ile Cys Lys Asn Asn Glu Glu Tyr Arg Arg
660 665 670
Gly Leu Arg Arg Arg Ile Asn Leu Tyr Thr Ser Ser Val Ile Met Ser
675 680 685
Leu Ala Arg Lys Phe Asn Val Asp Cys Ile Phe Leu Glu Asp Leu Asp
690 695 700
Ser Ser Lys Ser Ser Trp Asp Asp Ala Lys Lys Asn Ser Leu Lys Asp
705 710 715 720
Leu Trp Ser Thr Gly Gly Ala Asp Asp Ile Leu Gly Lys Met Ala Asn
725 730 735
Lys Tyr Lys Tyr Pro Ile Val Lys Val Asn Ser His Leu Thr Ser Leu
740 745 750
Val Asp Asn Lys Thr Gly Lys Ile Gly Tyr Arg Asp Pro Lys Lys Lys
755 760 765
Ser Asn Leu Tyr Val Glu Arg Gly Lys Lys Ile Glu Ile Ile Asp Ser
770 775 780
Asp Glu Asn Ala Ala Ile Asn Ile Leu Lys Arg Gly Ile Ser Lys His
785 790 795 800
Ile Asp Ile Arg Glu Phe Phe Ala Glu Lys Ile Glu Val Ser Gly Lys
805 810 815
Thr Leu Tyr Arg Ile Ser Asn Lys Leu Gly Lys Gln Arg Met Gly Ser
820 825 830
Leu Tyr Tyr Leu Glu Gly Asn Lys Glu Ile Leu Phe Gly Leu Gly Lys
835 840 845
Asn Gly Glu Pro Ile Val Cys Lys Arg Gly Leu Cys Lys Lys Glu Arg
850 855 860
Leu Ala Pro Arg Ile Ala Glu Lys Lys Ser Thr Tyr Leu Ile Met Asn
865 870 875 880
Gly Ser Lys Trp Met Phe Arg His Glu Ala Lys Lys Ile Val Glu Thr
885 890 895
Tyr Lys Asp Arg Tyr Cys Ala Asn His Lys Val Ala Ser Lys Asp Gly
900 905 910
Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
915 920
<210> 89
<211> 1130
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.10-NLS融合蛋白的氨基酸序列
<400> 89
Met Met Asn Ile Asn Glu Met Val Lys Leu Met Lys Ser Glu Tyr Leu
1 5 10 15
Phe Glu Asp Asp Gly Ile Val Thr Lys Asn Lys Ile Gln Glu Arg Leu
20 25 30
Arg Asn Gly Phe Ser Asp Ile Gly Val Asp Pro Ser Leu Val Ser Tyr
35 40 45
Ala Ser Lys Phe Leu Asp Ser Met Phe Ile Cys Phe Ser Arg Val Lys
50 55 60
Gly Glu Lys Asn Phe Lys Ala Lys Asn Val Arg Lys Asn Met Ser Ser
65 70 75 80
Ala Glu Lys Lys Ala Gln Lys Lys Lys Glu Tyr Gln Glu Tyr Tyr Gln
85 90 95
Gly Val Met Ala Gln Gln Asp Ala Tyr Ala Gln Leu Leu Ser Asp Pro
100 105 110
Thr Gln Glu Asn Leu Asp Lys Leu Asn Glu Leu Ile Ser Met Ser Val
115 120 125
Asn Gly Ser Leu Val Glu Asp Phe Phe Pro Ala Leu Lys Asn Met Ile
130 135 140
Gln Lys Ala Asp Tyr Ser Ile Asp Lys Lys Gly Leu Leu Asp Phe Ser
145 150 155 160
Cys Cys Met Met Asp Arg Tyr Glu Asp Arg Ser Leu Thr Arg Ala Ile
165 170 175
Ser Ile Ser Ala Phe Asn Ile His Ser Gly Gly Leu Arg Lys Ala Leu
180 185 190
Ser Asp Ile Ser Glu Lys Val Gln Asp Leu Ser Asn Thr Leu Leu Ile
195 200 205
Arg Ile Leu Tyr Met Lys Gly Glu Glu Leu Ser Ile Asp Gly Glu Lys
210 215 220
Ile Ser Lys Glu Glu Val Gln Arg Gln Leu Lys Ala Asp Tyr Glu Glu
225 230 235 240
His Lys Glu Tyr Phe Glu Asp Phe Glu Asp Phe Ala Lys Lys Cys Arg
245 250 255
Phe Phe Tyr Asn Lys Phe Ser Lys Lys Lys Lys Thr Arg Gly Phe Gly
260 265 270
Thr Tyr Phe Phe Gly Asp Lys Lys Lys Glu Ile Ser Ser Ala Glu Tyr
275 280 285
Lys Ala His Lys Glu Leu Arg Asp Ser Gly Tyr Leu Trp Phe Asp Ile
290 295 300
Gly Trp Ser Glu Ser Ser Asp Phe Lys Tyr Val Ile Val Gly Asn Val
305 310 315 320
Ser Gly Lys Leu Lys Ser Phe Glu Glu Thr Ser Glu Glu Tyr Gln Lys
325 330 335
Ser Lys Asn Cys Trp Glu Ala Glu Arg Val Lys Leu Tyr Glu Gln Asp
340 345 350
Ser Asp Phe Val Leu Phe Val Glu Asp Met Ile Glu Ser Lys Tyr Gly
355 360 365
Pro Ile Glu Lys Met Lys Leu Arg Thr Phe Lys Thr Ile Val Lys Lys
370 375 380
Leu Asp Lys Glu Phe Gly Lys Arg Gly Asp Lys Thr Pro Ser Ile His
385 390 395 400
Asp Tyr Phe Glu Ser Leu Asp Pro Asn His Thr Phe Ser Gln Ser Glu
405 410 415
Gln Phe Met Tyr Gly Leu Asp Val Thr Leu Met Gln Phe Leu Phe Asn
420 425 430
Asn Lys Lys Gln Phe Tyr Lys Leu Cys Lys Asp His Asp Gly Lys Arg
435 440 445
Thr Phe Ala Lys Val Val Glu Glu Ser Tyr His Trp Gly Lys Asn Ser
450 455 460
Ile Asn Val Ser Thr Phe Gln Asn Ser Thr Ser Ile Leu Leu Gly Gly
465 470 475 480
Asn Tyr Leu Asn Tyr Ser Met Ser Ile Glu Gly Glu Gly Leu Val Ile
485 490 495
Lys Phe Asp Asn Pro Leu Ser Gly Lys Glu Val His Phe Val Val Cys
500 505 510
Asn Asn Lys Tyr Leu Ser Asp Leu Glu Ile Leu Ser Gly Asn Pro Asn
515 520 525
Arg Lys Asp Asn Asn Tyr Thr Ile Ser Tyr Ser Thr Gly Gly Lys Ala
530 535 540
Arg Phe Ile Ala Lys Ser Lys Glu Pro Arg Ile Phe Phe Asn Arg Lys
545 550 555 560
Thr Lys Lys Trp Glu Ile Ala Phe Gln Leu Ser Asp Val Ser Pro Leu
565 570 575
Asn Gly Lys Phe Gly Lys Gln Gly Glu Phe Leu Ser Asn Leu Arg Lys
580 585 590
Phe Val Tyr Asn His Val Ala Lys Ser Pro Ser Lys Leu Asn Ile Ser
595 600 605
Asp Asn Asn Cys Arg Ala Val Ala Tyr Asp Leu Gly Ile Arg Asn Val
610 615 620
Gly Ala Trp Ser Ser Phe Asp Phe Ser Tyr Lys Asp Gly Val Leu Gly
625 630 635 640
Gly Tyr Lys Tyr Leu Thr Ser Gly Ser Leu Arg Ser Lys Ser Glu Ser
645 650 655
Ser Glu Met Asp Gln Gly Tyr Tyr Phe Val Leu Asn Leu Lys Lys Ile
660 665 670
Val Lys Leu Ile Pro Val Val Lys Lys Ser Ile Ile Asp Asp Pro Glu
675 680 685
Leu Lys Arg Gln Phe Ile Gly Val Leu Asn Glu Asn Gly Asn Thr Val
690 695 700
Gly Leu Gly Asn Ile Gly Lys Leu Asp Ile Ala Ser Arg Lys Ala Val
705 710 715 720
Gln Ser Phe His Asn Cys Ile Gln Gln Ile Asn Tyr Tyr Val Asp Thr
725 730 735
Tyr Ala Asp His Ile Asp Lys Ile Ser Ala Lys Asp Phe Val Asp Asp
740 745 750
Ile Asp Gly Ile Lys Val Leu Asp Glu Asp Asp Pro Tyr Val Val Lys
755 760 765
Ile Leu Ser His Leu Pro Glu Asp Val Glu Gly Asn Gln Asp Asp Ile
770 775 780
Leu Asn Ile Ser Leu Leu Lys Trp Lys Thr Ser Asn Ala Gln Phe Val
785 790 795 800
Pro Pro Leu Ile Gln Glu Ala Lys Ala Ile Met Ser Arg Ile Lys Arg
805 810 815
Glu Asn Leu Asp Asn Ile Arg Gly Lys Lys Thr Gln Val Val Thr Gln
820 825 830
Lys Thr Phe His Lys Ile Lys Phe Ala Lys Ala Leu Leu Ser Leu Met
835 840 845
Lys Ser Trp Ser Ser Ile Gly Thr Val Arg Val Val Lys Thr Asp Gln
850 855 860
Ile Tyr Gly Lys Lys Ile Trp Asp Tyr Ile Asn Gly Leu Arg Arg Asn
865 870 875 880
Val Leu Thr Tyr Leu Ser Ser Ala Ile Val Asn Asn Ala Leu Asp Leu
885 890 895
Gly Ala His Met Ile Ile Leu Glu Asp Leu Asp Ser Ser Val Ser Lys
900 905 910
Tyr Arg Glu Lys Asp Lys Asn Ala Ile Gln Ser Leu Trp Gly Ser Gly
915 920 925
Glu Leu Lys Lys Arg Ile Glu Glu Lys Ala Glu Lys His Arg Val Val
930 935 940
Val Gln Tyr Val Ser Pro Tyr Leu Thr Ser Gln Leu Asp Asn Glu Thr
945 950 955 960
Lys Asp Ile Gly Tyr Arg Lys Gly Gly Arg Leu Tyr Val Val Arg Asn
965 970 975
Gly Lys Ile Lys Ser Ile Asp Ala Asp Ile Asn Ala Ser Lys Asn Ile
980 985 990
Gly Glu Arg Phe Phe Asp Arg Asp Leu Ile Gln Thr Leu Ser Gly Val
995 1000 1005
Val Val Glu Asp Gln Ser Thr Val Tyr Ile Leu Gln Lys Arg Asn
1010 1015 1020
Val Ser Ser Asp Asn Arg Lys Arg Phe Tyr Lys Lys Phe Leu Glu
1025 1030 1035
Asp Val Gly Gly Lys Ser Lys Lys Asp Ala Val Leu Lys Met Gly
1040 1045 1050
Asp His Gly Glu Leu Glu Val Glu Arg Leu Ile Asp Gly Lys Lys
1055 1060 1065
Leu Asp Ile Asp Gly Lys Lys Ile Leu Val Asp Gly Glu Lys Val
1070 1075 1080
Pro Phe Arg Asn Thr Ser Val Tyr Tyr Ser Pro Lys Lys Lys Lys
1085 1090 1095
Trp Val Ser Lys Glu Leu Arg Cys Asn His Ile Lys Leu Thr Val
1100 1105 1110
Glu Glu Gln Asp Ile Lys Ser Arg Ala Asp Pro Lys Lys Lys Arg
1115 1120 1125
Lys Val
1130
<210> 90
<211> 1146
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.11-NLS融合蛋白的氨基酸序列
<400> 90
Met Asn Asn Tyr Asp Asn Tyr Leu Ser Asp Tyr Leu Ala Met Leu Pro
1 5 10 15
His Thr Lys Arg Thr Glu Ile Lys Lys Thr Ala Ser Lys Ile Ser Arg
20 25 30
Lys Leu Asn Gln Lys Glu Val Lys Lys Gln Ile Glu Arg Ser Glu Tyr
35 40 45
Ile Arg Ser Asn Cys Gly Tyr Ile Asn Ile Glu Arg Pro Gln Lys Ser
50 55 60
Leu Ser Phe Leu Ser Tyr Ser Thr Ile Lys Ser Ala Cys Met Ser Val
65 70 75 80
Asn Phe Arg Ala Phe Gln Asn Pro Ile Asn Asp Tyr Glu Thr Ala Ile
85 90 95
Cys Asn Gly Ile Asn Glu Cys Glu Arg Phe Phe Tyr Gln Gln Ile Asp
100 105 110
Ser Ile Tyr Met Ser Gln Ile Ile Glu Gln Leu Phe Asp Phe Tyr Ile
115 120 125
Ala Ser Arg Gln His Asp Met Phe Ile Asn Asn Thr Val Val Pro Tyr
130 135 140
Asp Val Asn Lys Leu Lys Ser Tyr Tyr Thr Ala Asn Glu Lys Tyr Ser
145 150 155 160
Phe Glu Gln Phe Cys Asp Asp Ile Lys Glu Phe Thr Asn Lys Gly Phe
165 170 175
Thr Ser Gly Gly Val Ser Cys Ile Leu Asn Leu Phe Tyr Lys Gly Ser
180 185 190
Val Lys Asp Ser Lys Asn Lys Lys Asp Tyr Ile Lys Ser Val Lys Arg
195 200 205
Leu Glu Thr Asn Gly Leu Phe Lys Lys Leu Asn Ile Phe Glu Lys Asn
210 215 220
Gly Ile Ser Lys Tyr Phe Ala Ala Ser Thr Leu Ser Thr Phe Phe Ala
225 230 235 240
Thr Ile Ser Ser Trp Lys Lys Gln Asn Asp Asp Trp Thr Gly Val Ala
245 250 255
Lys Asp Gly Thr Ser Leu Leu Ser Lys Leu Glu Asn Lys Thr Ile Thr
260 265 270
Leu Gln Ser Ile Ile Lys His His Arg Val Ile Asn Glu Leu Ala Val
275 280 285
Leu Ile Val Lys Ala Tyr Lys Asp Pro Val Lys Thr Leu Asn Asn Leu
290 295 300
Phe Glu Glu Arg Ser Asp Asn Asn Asn Asp Phe Lys Tyr Thr Cys Ser
305 310 315 320
Asp Asp Glu Asp Lys Tyr Pro Met Tyr Ile Lys Arg Glu Ile Ala Glu
325 330 335
Phe Val Lys Lys His Lys Thr Val Trp Glu Glu Ile Arg Tyr Phe Asp
340 345 350
Glu Ser Asp Thr Lys Lys Lys Lys Arg Asp Lys Lys Glu Ser Ser Ser
355 360 365
Asp Asp Lys Ser Tyr Leu Cys Cys Gly Asp Ser Trp Asp Tyr Leu Lys
370 375 380
Thr Trp Val Arg Leu Tyr Gly Glu Tyr Tyr Phe Phe Asp Asn Ala Leu
385 390 395 400
Asn Gln Phe Leu Arg Lys Pro Ser Ala Ser Met His Leu Tyr Thr Ser
405 410 415
Leu Asp Trp Ile Asn Lys Lys Thr Ile Cys Ile Val Gly Ala Asn Tyr
420 425 430
Tyr Lys Ile Gly Lys Val Glu Val Val Glu Arg Asn Asn Gln Arg Phe
435 440 445
Leu Leu Val Tyr Val Ser Val Pro Glu Met Glu Asn Tyr Ile Ile Ile
450 455 460
Pro Leu Gln Leu Asn Lys Tyr Phe Gly Asn Phe Gln Cys Lys Ile Phe
465 470 475 480
Glu Gly Arg Leu Gln Ala Ile Phe Lys Arg Tyr Ala Asn Phe Asn Ala
485 490 495
Leu Lys Asn Asn Lys Pro Gln Pro Ser Pro Asn Ile Ser Val Arg Ile
500 505 510
Asn Glu Phe His Phe Ala Leu Arg Ser Tyr Arg Lys Gln Gln Ile Ser
515 520 525
Ala Glu Asp Phe Ser Lys Gly Arg Phe Ser Leu Ile Ser Lys Ile Gly
530 535 540
Phe Gln Met Thr Asn Asp Glu Val Phe Gly Arg Thr Pro Arg Glu Ile
545 550 555 560
Ala Leu Val Lys Asp His Leu Ser Lys Gly Tyr Val His Phe Gly Ser
565 570 575
Gln Ile Ile Glu Asp Ser Arg Lys Glu Val Glu Gln Val Leu Lys Lys
580 585 590
Pro Met Ile Leu Met Gly Val Asp Phe Gly Tyr Ser Pro Leu Ala Ser
595 600 605
Tyr Asn Ile Lys Pro Leu Gln Thr Gly Lys Pro Ala Thr Asp Trp Val
610 615 620
Lys Asn Leu His Gly Asn Phe Leu Cys Gln Asn Val Ser Leu Gly Glu
625 630 635 640
Thr Ile Thr Glu Gly Glu Ile Gly Asp Val Pro Thr Asp Thr Tyr Thr
645 650 655
Ser Ser Asn Glu Ile Tyr Ser Ile Ala Thr Leu Thr Phe Arg Asn Ala
660 665 670
Asp Gly Lys Leu Glu Asn Arg Ser Phe Ser Arg Phe Tyr His Glu Leu
675 680 685
Asn Asn Thr Leu Asn Ile Ile Glu Gln Ile Lys Gly Thr Phe Asn Phe
690 695 700
Ile His Ser Ile Asn Thr Gln Phe Lys Glu Ile Lys Ala Leu Lys Thr
705 710 715 720
Thr Glu Glu Phe Ser Ser Tyr Val Ser Thr Leu Thr Trp Asp Gln Phe
725 730 735
Ile Glu Asp Ser Arg Lys Thr Ala Arg Tyr Ser Lys Tyr Trp Ile His
740 745 750
Ile Ile Asn Glu Asn Pro Lys Arg Arg Thr Ile Ala Thr Leu Asn Glu
755 760 765
Thr Leu Lys Leu Val Asp Glu Lys His Arg Phe Thr Val Thr Ile Gln
770 775 780
Glu Ile Phe Asp Leu Val Lys Tyr Cys Gln Gln His Gly Tyr Tyr Pro
785 790 795 800
Lys Ser Asn Val Met Ser Lys Leu Arg Asn Leu Ala Ile Lys Leu Ile
805 810 815
Asn Asp Leu Ile Arg Tyr Gln Lys Ile Gly Ile His Ser Cys Tyr Leu
820 825 830
Asp Phe Cys Val Leu Ile Lys Asn His Ile Ala Leu Leu Asn Ser Ser
835 840 845
Thr Ala Phe Ile Ile Asn Phe Ser Arg Asn Lys Glu Asn Ile Ile Arg
850 855 860
Asn Asn Thr Ser Lys Ile His Ser Leu Trp Val Tyr Arg Asp Asn Phe
865 870 875 880
Arg Arg Gln Met Ile Lys Asn Leu Cys Ser Gln Ile Leu Lys Ile Ala
885 890 895
Ala Lys Asn Lys Val His Ile Val Val Val Glu Lys Leu Asn Asn Met
900 905 910
Arg Thr Asn Asn Arg Asn Asn Glu Asp Lys Asn Asn Met Ile Asp Leu
915 920 925
Leu Ala Thr Gly Gln Phe Arg Lys Gln Leu Ser Asp Gln Ala Lys Trp
930 935 940
Tyr Gly Ile Ala Val Val Asp Thr Ala Glu Tyr Asn Thr Ser Lys Val
945 950 955 960
Asp Phe Met Thr Gly Glu Tyr Gly Tyr Arg Asp Glu Asn Asn Lys Arg
965 970 975
His Phe Tyr Cys Arg Lys Gln Asp Lys Thr Val Leu Leu Asp Cys Asp
980 985 990
Lys Lys Ala Ser Glu Asn Ile Leu Leu Ala Phe Val Thr Gln Ser Leu
995 1000 1005
Leu Leu Asn His Leu Lys Val Leu Ile Thr Glu Asp Gly Lys Thr
1010 1015 1020
Ala Val Ile Asp Leu Ser Glu Arg Thr Thr Glu Pro Gln Lys Ile
1025 1030 1035
Arg Ser Lys Ile Trp Thr Asn Ser Asp Val Gln Lys Ile Ile Phe
1040 1045 1050
Cys Lys Gln Glu Asn Gly Ser Tyr Val Leu Lys Lys Gly Ser Thr
1055 1060 1065
Asp Ile Lys Glu Lys Met His Lys Ala Val Leu His Arg His Gly
1070 1075 1080
Ser Leu Trp Tyr Asp Tyr Leu Asn His Lys Asn Met Ile Glu Asp
1085 1090 1095
Ile Lys Asn Leu His Leu Ser Asn Cys Ser Leu Thr Thr Ser Thr
1100 1105 1110
Asn Ser Asp Val Ile Asn Ser His Ser Gly Ser Ser Arg Ser Leu
1115 1120 1125
Asp Lys Thr Lys Thr Tyr Ala Ser Arg Ala Asp Pro Lys Lys Lys
1130 1135 1140
Arg Lys Val
1145
<210> 91
<211> 1024
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.12-NLS融合蛋白的氨基酸序列
<400> 91
Met Ala Ser Ser Asp Ala Gln Lys Phe Pro Gln Thr His Asn Lys Val
1 5 10 15
Met Ser Phe Arg Leu Thr Ala Ser Asn Ile Gly Ser Val Leu Ser Leu
20 25 30
His Ser Asn Leu His Asp Ala Ala Glu Ile Gly Ile Asn Glu Cys Arg
35 40 45
Trp Trp Ile Gly Asp Gly Glu Ile Tyr Glu Arg Asp Pro Ala Cys Arg
50 55 60
Ser Ile Lys Lys Gly Asn Asp Ile Arg Thr Val Thr Ser Glu Lys Ile
65 70 75 80
Lys Glu Leu Trp Thr Lys His Thr Asp His Ser Val Pro Leu Val Asp
85 90 95
Phe Ile Asp Met Leu Lys Phe Val Ala Gln Cys Ala Ile Tyr Gly Asp
100 105 110
Ser Arg Ala Leu Ala Ser Thr Leu Phe Gly Lys Ser Lys Ala Glu Thr
115 120 125
Arg Gly Val Ser Thr Glu Asp Met Thr Val Ile Arg Ala Trp Ile Ala
130 135 140
Glu Thr Asp Ala Val Leu Ala Ser Gly Leu Ser Pro Lys Lys Lys Lys
145 150 155 160
Lys Lys Glu Lys Glu Ala Gly Lys Lys Glu Arg Lys Pro Asp Val Lys
165 170 175
Met Glu Met Cys Arg Arg Ile Arg Cys Thr Met Val Gln Cys Gly Tyr
180 185 190
Phe Arg Arg Phe Pro Phe Glu Ala Lys Ile Asp Asn Gly Gly Glu Arg
195 200 205
Gly Lys Met Asp Ser Glu Leu Ser Tyr Val Ser Ala Arg Asn Leu Leu
210 215 220
Arg Cys Leu Ser Thr Trp Arg Ala Ser Ser Val Met Arg Arg Asp Ser
225 230 235 240
Tyr Leu Ile Glu Glu Glu Arg Ile Lys Glu Ala Glu Ser Lys Met Thr
245 250 255
Pro Glu Ile Ile Asp Gly Leu Arg Arg Leu Tyr Arg Tyr Cys Ala Val
260 265 270
Asp His Asp Phe Leu Lys Trp Phe Gly Gly Arg Ile Ile Arg His Ile
275 280 285
Asp Ser Cys Leu Ala Pro Ala Ile Ala Gly Asn Thr Gly Arg Pro Thr
290 295 300
Gly Gly Glu Ser Phe Thr Val Ile Tyr Asp Arg Arg Lys Lys Arg Asp
305 310 315 320
Val Lys Ile Thr Tyr Ser Val Pro Glu Glu Ile Tyr Gly Tyr Leu Ser
325 330 335
Ser His Pro Glu Leu Val Ala Ile Gly Lys Asp Gly Met Thr Pro Ile
340 345 350
Ser Arg His Ala Asp Tyr Leu Glu Met Ile Ala Ser His Glu Lys His
355 360 365
Arg Trp Tyr Ala Thr Phe Pro Thr Val Gly Lys Glu Asp Gly Tyr Arg
370 375 380
Thr Ser Val Leu Leu Gly Lys Asn Tyr Leu Thr Tyr Asp Leu Ser Tyr
385 390 395 400
Asp Gly Glu Ser Val Pro Asp Lys Lys Ile Asn Val Ile Ser Lys Gly
405 410 415
Gln Pro Val Cys Leu Asp Leu His Asp Gly Arg Arg Val Ser Ser Leu
420 425 430
Tyr Leu Thr Val Gly Glu Ser Ala Ala Tyr Asp Ile Ala Val Arg Lys
435 440 445
Asn Lys Arg His His Gly Lys Pro Ala Asp Tyr Cys Arg Met Arg Val
450 455 460
His Leu Thr Gln Glu Arg Glu Asp Lys Thr Tyr Asn Asp Pro Tyr Phe
465 470 475 480
Ser Asn Met Glu Ile Trp Arg Ala Gly Asp Gln Val Tyr Ala Ile Glu
485 490 495
Phe Asp Arg His Gly Ala Arg Tyr Thr Ala Ile Val Lys Glu Pro Ser
500 505 510
Val Glu Tyr Arg Asn Lys Lys Leu Tyr Leu Arg Val Asn Met Val Leu
515 520 525
Asp Ser Pro Ser Arg Gln Asp Asp Lys Asp Met Tyr Tyr Ala Tyr Met
530 535 540
Thr Ala Tyr Pro Ser Ser Asn Pro Pro Val Glu Thr Ser Asp Asn Lys
545 550 555 560
Lys Arg Phe Glu Arg Leu Gly Pro Gly Arg Arg Ala Ile Gly Gly Ile
565 570 575
Asp Ile Gly Ile Gly Arg Pro Tyr Val Ala Val Val Ala Ser Tyr Glu
580 585 590
Val Gly Pro Ala Gly Thr Glu Gln Lys Phe Gln Ile Glu Asp Arg Leu
595 600 605
Ile Glu Asp Asp Gly Ser Ser Pro Tyr Asp Ser Leu Tyr Asn Asp Phe
610 615 620
Leu Thr Asp Ile Arg Thr Val Ser Arg Ile Ile Glu Ala Ala Lys Lys
625 630 635 640
Ile Ser Glu Gly Asp Leu Glu Asp Ile Pro Ser Asp Met Ser Val Asp
645 650 655
Glu Asp Gly Ser Ile Ala Ala Thr Met Lys Arg Met Ser Ala Arg Ile
660 665 670
Ala Glu Arg His His Leu Tyr Gly Glu Arg Lys Ser Glu Ala Tyr Ala
675 680 685
Thr Phe Leu Lys Met Asn His Lys Gln Arg Leu Asp Ile Leu Leu Thr
690 695 700
Gln Lys Ala Ser Asn Ala Thr Leu Lys Gln Leu Val Glu Glu Asp Pro
705 710 715 720
Ser Phe Leu Pro Arg Ile Cys Val Tyr Tyr Val Ile Ser Val Glu Arg
725 730 735
Glu Leu Lys Asn Lys His Arg Asn Ala Tyr Leu Asp Gly Leu Thr Val
740 745 750
Asp Glu Lys Tyr Ser Gly Glu Thr Lys Arg Gly Tyr Ala Gln Lys Arg
755 760 765
Leu Asn Ser Met Leu Arg Ala Tyr Ser Ala Leu Gly Glu Glu Glu Thr
770 775 780
Asp Glu Val Arg Thr Phe Ser Thr Arg Ser Glu Lys Val Arg Asn Met
785 790 795 800
Ala Lys Asn Ala Ile Lys Arg Asn Ala Arg Lys Leu Val Asn Phe Tyr
805 810 815
Val Gly Lys Gly Ile Arg Thr Ile Val Ala Glu Asp Thr Asp Pro Thr
820 825 830
Lys Ser Arg Asn Asp Gly Lys Lys Ser Asn Arg Ile Lys Ala Ala Trp
835 840 845
Ser Pro Lys Gln Phe Leu Ala Ala Val Lys Asn Ala Ala Gln Trp His
850 855 860
Gly Leu Glu Ile Ala Glu Val Asp Pro Arg Met Thr Ser Gln Val His
865 870 875 880
Pro Glu Thr Gly Leu Ile Gly Tyr Arg Asp Gly Asp Thr Leu His Cys
885 890 895
Pro Asp Gly Ser Lys Ile Asp Ala Asp Val Ala Gly Ala Ala Asn Val
900 905 910
Cys Arg Val Phe Ala Gly Arg Gly Leu Trp Arg Phe Ser Ile Asn Thr
915 920 925
Asn Ile Asp Ile Ser Asn Lys Asp Glu Lys Lys Arg Leu Arg Ala Tyr
930 935 940
Ile Val His His Phe Gly Ser Glu Ser Asn Trp Glu Lys Phe Arg Lys
945 950 955 960
Gln Tyr Pro Ser Gly Thr Thr Leu Tyr Leu His Gly Arg Glu Trp Leu
965 970 975
Thr Ala Glu Glu His Lys Ser Ala Ile Asp Arg Ile Arg Asp Asp Val
980 985 990
Gly Arg Asp Ala Glu Asn Asp His Val Ala Ile Val Thr Ala Ala Glu
995 1000 1005
Lys Val Glu Ile Phe Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys
1010 1015 1020
Val
<210> 92
<211> 1063
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.13-NLS融合蛋白的氨基酸序列
<400> 92
Met Ser His Asp Leu Lys Pro Gln Arg Leu Ile Arg Ser Asn Ile Thr
1 5 10 15
Lys Thr His Ser Asp Gln Asn Ala Lys Gln Val Ala Glu Glu Val Lys
20 25 30
Lys Glu His Leu Asn Tyr Leu Leu Ile Lys Asn Glu Met Leu Ile Ser
35 40 45
Ile Val Pro Glu Ala Lys Asp Asp Asp Gly Asn Asp Ile Asp Phe Lys
50 55 60
Lys Gln Leu Lys Ser Leu Tyr Lys Glu Thr Asp Gln Ser Val Ser Phe
65 70 75 80
Ser Val Phe Cys Gln Met Met Lys Phe Arg Asn Ile Ala Leu Leu Tyr
85 90 95
Ala Lys Gly Gln Ser Arg Trp Ala Val Ser Ser Tyr Phe Thr Gly Asn
100 105 110
Arg Arg Lys Asp Asp Tyr Ala Lys Asp Leu Ser Leu Leu Asp Glu Ala
115 120 125
Ile Glu Leu Leu Glu Cys Lys Arg Arg Lys Lys Ala Glu Glu Glu Asn
130 135 140
Glu Glu Glu Asn Glu Thr Pro Lys Lys Lys Glu Asp Asn Pro Ser Asn
145 150 155 160
Ile Ser Glu Glu Gln Ile Met Lys Leu Phe Tyr Ala Val Asn Lys Lys
165 170 175
Leu Lys Glu Ile Gly Tyr Leu Asp Arg Tyr Ser His Ile Glu Lys Gln
180 185 190
Glu Gln Tyr Ala Ile Ile Gly Val Thr Ser Arg Thr Val Lys Ala Trp
195 200 205
Asp Tyr Ala Asn Phe Ala Thr Arg Asn His Tyr Gln Ser Val Gln Asn
210 215 220
Glu Tyr Gln Lys Lys Leu Lys Ala Leu Pro Gly Thr Lys Lys Asp Lys
225 230 235 240
Val Cys Leu Glu Lys Phe Phe Asp His Leu Asn Glu Asn Asn Ile Ala
245 250 255
Ala Asp Trp Asp Lys Trp Arg Leu Lys Lys His Ile Leu Gln Cys Ile
260 265 270
Ile Pro Ala Ala Lys Ile Gly Leu Lys Glu Leu Lys Gln Ser Phe Tyr
275 280 285
Val Asp Asn Lys Gly Asn Lys His Asn Tyr Phe Val Asn Gly Leu Tyr
290 295 300
Glu Glu Ile Leu Lys Arg Pro Phe Leu Tyr Ser Ala Glu Asp Pro Glu
305 310 315 320
Glu Ser Ile Leu Tyr Leu Gly Val Glu Val Ala Ser Leu His Ser Lys
325 330 335
Leu Asn His Leu Arg Ser Glu Ala Arg Phe Ser Phe Glu Thr Pro Asp
340 345 350
Asp Ile Cys Lys Tyr Met Thr Ile Cys Gly Asp Asn Tyr His Asn Phe
355 360 365
Thr Met Ser Ala Ile Gly Glu Asp Val Glu Asp Ile Glu Val Glu Val
370 375 380
Tyr Asp Tyr Asn His Ser Lys Lys Tyr Glu Thr Met Arg Phe Ile Asn
385 390 395 400
Gly Lys Arg Thr Thr Asp Leu Ser Leu Asn Phe Lys Gly Ile Pro Val
405 410 415
Arg Leu Cys Leu Glu Gly Lys Arg Asn Asn Ser Tyr Phe Ala Asp Ala
420 425 430
Ile Val Trp Glu Leu Asp Asn Lys Asp Lys Thr Gly Tyr Leu Ile Glu
435 440 445
Tyr Gly Lys Ser Asn Asn Arg Leu Tyr Met Leu Val Lys Glu Pro Leu
450 455 460
Ile Gly Cys Arg Arg Lys Phe Gly Lys Asp Val Leu Phe Val Ser Leu
465 470 475 480
Ser Gly Thr Leu Val Asn Lys Tyr Ile Glu Asp Asp Ile Val Ser Ala
485 490 495
Arg Tyr Leu Met Gln Thr Ala Ala Pro Ile Phe Lys Thr Ser Arg Ala
500 505 510
Lys Lys Gln Asp Lys Ile Gly Asp Lys Trp Phe Glu His Cys Gln Gly
515 520 525
Ser Thr Ile Lys Ile Ala Gly Ile Asp Ile Gly Ile Asn Pro Ile Ala
530 535 540
Ala Ile Thr Val Ala Asn Val Thr Phe Asp Arg Ala Leu Gly Asn Lys
545 550 555 560
Ile Lys Asn Gln Lys Gln Ile Val Ile Asp Cys Tyr Ala Glu Asp Tyr
565 570 575
Lys Ile Asp Pro Val Val Val Lys Arg Met Glu Asp Ile Arg His Ile
580 585 590
Lys Tyr Thr Ile Asn Ser Trp Tyr His Leu Ala Asp Cys Cys Arg Leu
595 600 605
Lys Ala Ala Asn Lys Glu Tyr Val Val Asn Glu Arg Lys Gln Gly Phe
610 615 620
Phe Arg Glu Asn Ile Glu Tyr Leu Lys Glu Val Ala Lys Lys Ala Ile
625 630 635 640
Thr Glu Ser Asp Gln Gln Ile Lys Glu Gln Lys Ala Ala Leu Lys Arg
645 650 655
Phe Asp Gly Glu Lys Lys Lys Glu Ile Gln Ala Thr Ile Asn Gly Phe
660 665 670
Asn Leu Lys Ile Lys Ile Leu Lys Lys Phe Val Arg Gln Ser Ala Lys
675 680 685
Lys Ile Phe Asp Ser Thr Leu Glu Thr Leu Glu Lys Tyr Asp Asn Asn
690 695 700
Ile Glu Gln Ala Lys Arg Asp Arg Glu Phe Gly Leu Lys Ile Ile Tyr
705 710 715 720
Asp Leu Ile Ile Lys Tyr Tyr Lys Arg Ser Lys Lys Glu Arg Glu Met
725 730 735
Asn Gln Arg Ile Tyr Val Asp Asp Tyr Asn Gln Glu Glu Ile Asp Thr
740 745 750
Glu Arg Thr Lys Lys Ile Arg Lys Glu Thr Ile Thr Phe Cys Asp Asn
755 760 765
Asp Trp Asn Ser Leu Thr Lys Arg Ile His Asp Leu Glu Lys Lys Met
770 775 780
Lys Lys Ile Gly Ile Ser Glu Pro Gly Arg Val Glu Gln Glu Ile Asn
785 790 795 800
Asp Arg Asp Tyr Tyr Asn Asn Ile Gln Asp Asn Thr Lys Lys Arg Gln
805 810 815
Ala Lys Ile Ile Val Asp Ala Leu Lys Glu Glu Gly Val Ser Ile Ile
820 825 830
Val Val Glu Asp Leu Thr Gly Gly Gly Ser Glu Asn Thr Lys Glu Ile
835 840 845
Asn Lys Ser Phe Asp Ala Phe Ala Pro Ile Arg Phe Leu Asn Ala Leu
850 855 860
Lys Asn Cys Ala Glu Thr Asn Gly Ile Gln Val Thr Glu Val Leu Ser
865 870 875 880
Pro Met Ser Ser Lys Met Val Pro Ser Thr Gly Glu Ile Gly His Arg
885 890 895
Asp Lys Arg Asp Lys Gln Leu Tyr Tyr Lys Asp Gly Glu Glu Leu Lys
900 905 910
Ser Ile Asp Gly Asp Ile Ser Ala Ser Glu Ile Leu Leu Arg Arg Gly
915 920 925
Val Ser Arg His Thr Glu Leu Ile Gly Thr Met Asn Val Glu Asp Val
930 935 940
Leu Asp Lys Asn Asn Asn Lys Asn Lys Cys Ile Lys Gly Tyr Val Cys
945 950 955 960
Asn Arg Trp Gly Asn Ile Gln Asn Phe Glu Lys Ile Leu Lys Glu Lys
965 970 975
Gly Ile Gly Glu Arg Glu Ile Ile Tyr Leu His Gly Asp Lys Ile Leu
980 985 990
Thr Met Asp Glu Lys Arg Thr Leu Gln Ala Ser Ile Arg Lys Glu Leu
995 1000 1005
Lys Glu Met Arg Glu Arg Glu Ser Gly Glu Glu Asn Ala Gly Thr
1010 1015 1020
Ala Arg Lys Lys Ser Lys Pro Lys Lys Lys Lys Lys Ile Lys Arg
1025 1030 1035
Asn Asn Asp Gln Asp Leu Ser Asn Asn Arg Pro Ala Ala Ser Ser
1040 1045 1050
Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
1055 1060
<210> 93
<211> 1056
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.14-NLS融合蛋白的氨基酸序列
<400> 93
Met Lys Glu Asn Lys Met Lys Glu Asn Gly Ser Met Thr Thr His Ser
1 5 10 15
Lys Val Ile Ala Leu Lys Met Lys Ser Glu Asn Val Glu Phe Asp Thr
20 25 30
Phe Tyr Lys Glu Ser Phe Glu Leu Phe Lys Gln Phe Thr Asn Glu Phe
35 40 45
Val Ala Trp Gly Asn Asp Glu Ile Tyr Gln Tyr Gly Ser Ser Lys Arg
50 55 60
Lys Lys Asp Asp Gln Lys Ile Ser Leu Ile Pro Val Ile Glu Asp Ile
65 70 75 80
Tyr Lys Ser Val Glu Lys Lys Ala Thr Ala Glu Gly Ile Ser Lys Thr
85 90 95
Asp Phe Arg Ala Val Leu Lys Tyr Leu Tyr His Gln Ile Ile Asn Val
100 105 110
Gly Asn Ser Gly Arg Ser Tyr Gly Thr Ser Leu Phe Gly Gly Cys Glu
115 120 125
Val Lys Glu Lys Leu Ser Lys Gln Asp Ile Ser Asn Ile Val Glu Cys
130 135 140
Val Lys Glu Leu Glu Leu Cys Lys Ser Lys Gln Glu Glu Ser Asp Ala
145 150 155 160
Tyr Asp Lys Ile Leu Leu Lys Glu Lys Ile Thr His Ile Val Lys Ser
165 170 175
Gly Glu Thr Ala Gly Asp Ile Thr Lys Lys Tyr Asn Gln Ala Thr Thr
180 185 190
Gly Arg Lys Thr Ser Ser Lys Gly Phe Phe Asp Lys Ser Thr Lys Thr
195 200 205
Glu Val Lys Tyr Lys Asp Ile Lys Asp Asp Thr Leu Leu Gln Asp Gly
210 215 220
Ser Thr Ile Phe Ile Lys Ser Ser Val Asp Leu Phe Val Lys Lys Val
225 230 235 240
Cys Asn Thr Leu Arg Glu Ile Asn Phe Phe Asp Arg Leu Pro Phe Lys
245 250 255
Asn Asn His Ser Asn Asn Tyr Gly Leu Leu Phe Ser Met Leu Ser Gln
260 265 270
Ile Glu Ser Trp Lys Thr Ile Ser Glu Thr Thr Lys Lys Ser His Glu
275 280 285
Glu His Gly Glu Lys Ile Ala Ser Met Val Lys Lys Leu Asp Leu Thr
290 295 300
Gln Thr Glu Leu Met Lys Asp Phe Ala Ala Phe Cys Ile Glu Asn Asn
305 310 315 320
Ile Thr Lys Lys Phe Asp His Lys Phe Lys Arg His Met Glu Asp Cys
325 330 335
Val Ile Pro Ser Phe Lys Asn Gly Lys Ile Pro Asp Lys Leu Phe Tyr
340 345 350
Phe Asn Ile Ile Leu Ala Lys Lys Thr Asp Glu Gln Ile Asp Tyr Ser
355 360 365
Leu Ser Ser Glu Phe Tyr Thr Lys Leu Phe Ser Met Pro Asn Leu Trp
370 375 380
Gln Glu Glu Glu Ala Phe Ile Val Lys Asn Ile Asn Leu Ile Glu Glu
385 390 395 400
Ile Thr Ile Phe Asn Lys Arg Arg Asn Tyr Ala Cys Cys Pro Leu Ile
405 410 415
Lys Glu Lys Glu Tyr Asp Arg Phe Gln Ile Gln Leu Asn Glu Thr Asn
420 425 430
Phe Leu Lys Phe Gln Phe Asp Pro Lys Asn Val Val Asn Ile Asp Glu
435 440 445
Asn Thr Thr Glu Ala Thr Val Gly Phe Asp Glu Lys Leu Lys Leu Val
450 455 460
Val Cys Ala Asp Lys Lys Tyr Ala Phe Ser Ile Phe Thr Gln Cys Lys
465 470 475 480
Tyr His Gly Asn Lys His Lys Pro Asn Thr Tyr Phe Asn Asn Leu Lys
485 490 495
Ile Ile Lys Val Ile Glu Ser Lys Ser Asn Ser Val Lys Ser Met Lys
500 505 510
Tyr Thr Phe Glu Phe Thr Lys Arg Asn Glu Leu Lys Arg Ala Glu Ile
515 520 525
Lys Gln Pro Ser Ile Val Tyr Lys Asn Asn Asn Tyr Tyr Ile Arg Ile
530 535 540
Asn Met Asn Val Ile Leu Asp Ala Asp Gln Thr Ser Tyr Lys Ile Ile
545 550 555 560
Asn Asn Asn Gln Thr Ala Ser Leu Pro Ser Tyr Phe Gln Ser Ser Leu
565 570 575
Pro Phe Glu Asn Asn Arg Gly Lys Ile His Asp Lys Gly Ile Val His
580 585 590
Trp Glu Lys Ile Lys Asn Arg Lys Ile Ile Ala Met Gly Val Asp Leu
595 600 605
Gly Val Arg Arg Pro Phe Ser Tyr Ala Ile Gly Asn Phe Thr Leu Asn
610 615 620
Lys Asp Ile Leu Asp Lys Asn Asp Val Asn Ile Val Ala Ser Gly Phe
625 630 635 640
Asn Leu Cys Ser Asp Ser Asp Val Tyr Phe Gln Val Phe Asn Gln Ile
645 650 655
Lys Thr Leu Ala Lys Phe Ile Gly Lys Leu Lys Ser His Asn Lys Gly
660 665 670
Leu Lys Val Asp Phe Glu Lys Asp Lys Lys Tyr Ile Phe Asp Leu Val
675 680 685
Asn Asp Ala Lys Ala Tyr Phe Lys Asp Met Ser Ala Lys Arg Ile Asn
690 695 700
Asp Thr Lys Asp Asn Ile Ser Asn Thr Val Thr Asn Lys Glu Arg Ile
705 710 715 720
Tyr Gly Ser Phe Val Ser Glu Ser Ala Glu Ser Ala Ile Gln Cys Ala
725 730 735
Ile Asp Arg Ser Glu Lys Glu Ser Gly Leu Thr Leu Lys Lys Asp Ile
740 745 750
Ser Trp Leu Val Asn Val Leu Ser Lys Tyr Leu Glu Arg Lys Phe Lys
755 760 765
Glu Val Lys Asn Asn Arg Lys Tyr Thr Asn Val Asn Lys Cys Asp Asn
770 775 780
Cys Phe Asn Trp Leu Arg Val Ile Glu Asn Ile Lys Arg Leu Lys Arg
785 790 795 800
Ser Ile Ser Tyr Leu Gly Glu Asp Leu Gln Lys Asn Pro Glu Leu Lys
805 810 815
Ile Glu Leu Lys Asn Leu Asn Glu Tyr Gly Asn Asn Val Lys Ser Asp
820 825 830
Phe Leu Lys Gln Ile Ala Ser Asn Ile Ile Lys Val Ala Ile Glu His
835 840 845
Lys Cys Asp Ile Val Phe Ile Glu Lys Leu Gly Lys Ala Asp Ser Arg
850 855 860
Ser Arg Lys Leu Asn Glu Met Phe Ser Phe Trp Ser Pro Lys Ala Ile
865 870 875 880
Lys Lys Ala Ile Glu Asn Ala Ala Ser Trp His Gly Ile Pro Val Val
885 890 895
Glu Val Asp Pro Ser Cys Thr Ser Lys Val His Tyr Glu Thr Asn Leu
900 905 910
Phe Gly His Arg Ile Gly Asn Asp Leu Tyr Tyr Val Glu Asp Gln Cys
915 920 925
Leu Lys Lys Val Asp Ala Asp Ile Asn Ala Ala Lys Gln Ile Leu Val
930 935 940
Arg Gly Ala Thr Arg His Gly Asn Ile Ser Ser Ile Asn Ile Lys Tyr
945 950 955 960
Leu Gln Ala Lys Ile Ala Glu Leu Asn Ser Glu Ala Asn Ser Glu Glu
965 970 975
Asp Lys Glu Glu Ile Lys Gln Gly Gly Lys Arg Ile Gln Gly Phe Leu
980 985 990
Trp Lys Lys Tyr Gly Asn Ile Thr Asn Ile Thr Asn Gln Leu Thr Ala
995 1000 1005
Ala His Lys Glu Arg Glu Ser Lys Phe Asp Tyr Ile Tyr Leu His
1010 1015 1020
Asn Asp Lys Trp Ile Ala Tyr Glu Asp Arg Asn Glu Ile Lys Lys
1025 1030 1035
Asp Ile Glu Lys Arg Leu Glu Ser Arg Ala Asp Pro Lys Lys Lys
1040 1045 1050
Arg Lys Val
1055
<210> 94
<211> 906
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.15-NLS融合蛋白的氨基酸序列
<400> 94
Met Thr Ala Lys Lys Thr Ala Lys Lys Tyr Phe Pro Pro Lys Cys Leu
1 5 10 15
Arg Ser Ser His Phe Lys Ile Tyr Gly Ile Pro Thr Ala Ile Arg Ala
20 25 30
Leu Glu Glu Thr Asn Thr Phe Val Asn Lys Ala Ala Ala Asp Leu Met
35 40 45
Glu Met Phe Phe Leu Met Arg Gly Gln Pro Tyr Arg Arg Arg Ile Gly
50 55 60
Ser Glu Glu Lys Gln Val Thr Gln Glu His Ile Asp Ala Arg Leu Arg
65 70 75 80
Val Leu Val Gly Asp Tyr Ser Leu Asn Glu Val Lys Pro Leu Leu Arg
85 90 95
Gln Leu Tyr Asp Gly Ile Lys Ala Lys Gln Asn Tyr Ala Pro Thr His
100 105 110
Phe Val Arg Phe Phe Ile Gln Pro Thr Lys Gly Ala Ile Asp Lys Lys
115 120 125
Ser Pro Val Ser Gln Arg Ala Lys Lys Ala Gly Gln Lys Leu Gln Lys
130 135 140
Met Gly Val Leu Pro Ile Leu Pro Leu Ser Pro Gly Phe Lys Phe Trp
145 150 155 160
Thr Ala Ala Met Met Met Ala Cys Ser Arg Met Asn Ser Trp Glu Ala
165 170 175
Cys Asn Glu Lys Thr Ile Glu Asn His Lys Ala Phe Leu Glu Gly Ile
180 185 190
Glu Asn Tyr Lys Lys Glu Ile Arg Phe Glu Asp Leu Cys Glu Glu Trp
195 200 205
Ser Leu Phe Ser Asp Trp Leu Thr Glu Ala Glu Ser Asp Asn Glu Gly
210 215 220
Gly Cys Lys Phe Lys Leu Thr Pro Arg Phe Leu Gln Arg Trp Glu Arg
225 230 235 240
Ile Tyr Leu Lys Gln Met Arg Lys Gly Lys Ile Pro Ala Arg His Asn
245 250 255
Leu Gly Pro Val Met Glu Ala Leu Ala Gly Asp Lys Tyr Arg Gln Leu
260 265 270
Trp Asp Asn Gly Glu Glu Arg Asp Tyr Ile Thr Glu Leu Gly Asp Leu
275 280 285
Val Thr Ser Gln Arg Lys Ala Val Arg Leu Ser Arg Asp Ser Ala Val
290 295 300
Thr Phe Pro Asp Glu Glu Leu Ser Pro Val Gly Thr Glu Phe Gly His
305 310 315 320
Asn Tyr Met Ser Phe Ser Ile Asp Gln Glu Asn Ser His Leu Val Thr
325 330 335
Leu Glu Val Ile Gly Gly Lys Tyr Gln Phe Glu Ile Ser Lys Ser Asp
340 345 350
Tyr Phe Arg Asp Leu Ile Val Glu Glu Ala Gly Lys Gln Ser Lys Phe
355 360 365
Tyr Asn Val Ser Tyr Arg Lys Gly Asn Val Arg Glu Glu Asn Leu Ala
370 375 380
Gly Asp Phe Lys Glu Ala Thr Val Arg Asn Arg Arg Ser Leu Lys Thr
385 390 395 400
Gly Lys Arg Arg Leu Tyr Phe Tyr Met Ser His Ser Ile Pro Thr Arg
405 410 415
Phe Asp Asp Asp Leu Tyr Ala Gln Phe Thr Glu Lys Gly Gln Pro Asp
420 425 430
Phe Ser Lys Leu Tyr Lys Ala Val Thr Tyr Phe Gln Cys Ser Leu Gly
435 440 445
Asn Lys Lys Ala Asp Thr Tyr Arg Val Tyr Val Lys Met Gly Thr Arg
450 455 460
Phe Leu Gly Val Asp Ile Gly Val Ser Arg Leu Phe Gly Phe Ser Leu
465 470 475 480
Phe Glu Leu Arg Glu Glu Lys Pro Glu Lys Asn Pro Phe Phe Glu Leu
485 490 495
Pro Asp Asp Leu Gly Tyr Ala Val Cys Leu Glu Ser Trp Val Asp Gly
500 505 510
Val Glu Lys Asn His Lys Val Ala Gln Glu Met Lys Asp Trp Arg Arg
515 520 525
Glu Cys Leu Ala Ala Gln Arg Leu Ile His Tyr Ala Lys Phe Leu Lys
530 535 540
Lys Arg Asp Lys Asn Glu Glu Ile Asp Tyr Lys His Glu Glu Ser Leu
545 550 555 560
Glu Thr Ile Ala Gly Leu Leu Gly Ile Glu Ile Asp Pro Glu Gln Ile
565 570 575
Ile Asp Val Pro Leu Lys Leu Leu Asp Leu Val Gly Gln Ala Ile Gly
580 585 590
Ala Leu Arg Lys Lys Tyr Leu Val Leu Lys Lys Asn Glu Val Arg Gln
595 600 605
Gly Arg Ile Thr Ser Glu Leu Phe Leu Trp Pro Glu Cys Val Asp Thr
610 615 620
Tyr Ile Arg Leu Leu Lys Ser Trp Thr Tyr Lys Asp Lys Lys Pro Tyr
625 630 635 640
Gln Lys Gly Glu Thr Asn Lys Asp Ala Phe Lys Lys Leu Lys Gly Tyr
645 650 655
Leu Ala Arg Leu Arg Lys Asp Leu Ala Pro Lys Tyr Ala Ala Val Ile
660 665 670
Ala Asp Ala Ala Ile Arg His Lys Val His Val Val Val Ala Glu Asn
675 680 685
Leu Glu Gln Phe Gly Leu Ser Met Lys Asn Glu Lys Asp Leu Asn Arg
690 695 700
Val Leu Ala His Trp Ser His Gln Lys Ile Trp Ser Met Val Glu Glu
705 710 715 720
Gln Leu Arg Pro Tyr Gly Ile Met Val Val Tyr Val Asp Pro Arg His
725 730 735
Thr Ser Lys Leu Asp Phe Ala Thr Asp Glu Phe Gly Gly Arg Cys Phe
740 745 750
Thr Ser Leu Tyr Val Met Arg Asp Gly Lys Lys Thr Thr Thr Asp Thr
755 760 765
Glu Lys Asn Ala Ser Gln Asn Ile Pro Lys Lys Phe Leu Thr Arg His
770 775 780
Arg Asn Val Ser Trp Leu Leu Ala Tyr Ala Val Asp Leu Ser Asp Ser
785 790 795 800
Gln Lys Lys Lys Leu Gly Ile Gly Asp Glu Lys Val Trp Leu Pro Asn
805 810 815
Met Gly Leu Met Ile Ser Gly Ala Leu Lys Ala Lys His Gly Lys Asn
820 825 830
Ser Ala Leu Leu Val Glu Asp Gly Glu Asn Tyr Arg Leu Leu Pro Ile
835 840 845
Thr Ala Ala Gln Ala Lys Lys Phe Val Val Lys Arg Lys Lys Glu Glu
850 855 860
Phe Tyr Arg His Gly Glu Ile Trp Leu Thr Lys Glu Ala His Lys Ala
865 870 875 880
Arg Ile Glu Tyr Leu Phe Pro Glu Ser Lys Lys Gly Arg Lys Ser Ser
885 890 895
Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
900 905
<210> 95
<211> 967
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.16-NLS融合蛋白的氨基酸序列
<400> 95
Met Lys Lys Thr Asn Tyr Lys Thr Ser His Leu Leu Ile Asp Asn Pro
1 5 10 15
Pro Gln Ser Ile Ile Asp Leu His Arg Asp Val Ile Glu Ile Gly Ser
20 25 30
Tyr Leu Thr Lys Phe Phe Leu Ala Cys Leu Gly Arg Pro Val Asp Ser
35 40 45
Thr Ile Leu Ser Glu Pro Ala Leu His Phe Gln Phe Val Asn Gly Ile
50 55 60
Leu Pro Val Lys Asn Gly Pro Gly Ala Asp Asp Ser Ser Trp Arg His
65 70 75 80
Ser Glu Asn Cys Tyr Ser Met Leu Phe Glu Lys Asn Ser Lys Ser Gly
85 90 95
Lys Ser Asp Gly Lys Val Arg Gln Val Arg Glu Leu Lys Val Ala Leu
100 105 110
Phe Gly Lys Lys Glu Lys Gly Lys Gly Ile Val Gly Lys Lys Thr Trp
115 120 125
Asp Glu Leu Lys Val Val Leu Glu Ala Leu Pro Glu Glu His Gln Ile
130 135 140
Leu Ser Leu Glu Ile Cys Gln Arg His Tyr Glu Ser Arg Asp Val Lys
145 150 155 160
Ala Phe Gly Lys Leu Ala Leu Ser Ser Lys Ser Arg Pro Ser Val Glu
165 170 175
Ala Gly Leu Lys Leu Arg Glu Leu Gly Leu Leu Pro Leu Asp Ser Arg
180 185 190
Gly Leu Asp Lys Asn Lys Leu Leu Gly Ile Leu Ala Ala Val Thr Gly
195 200 205
Arg Leu Lys Ser Trp Arg Asp Arg Asp Cys Ala Cys Lys Ala Asp Lys
210 215 220
Gln Ala Leu Arg Val Lys Phe Glu Glu Arg Leu Ser Lys Val Asp Gln
225 230 235 240
Ser Ala Tyr Gln Gln Phe Lys Gln Phe Ala Asp Glu Leu Leu Thr Gln
245 250 255
Glu Gly Tyr Arg Ile Ser Gly Arg Val Leu Arg Ala Val Glu Lys Lys
260 265 270
Asp Ser Asp Tyr Ser Pro Val Leu Thr Val Leu Ala Lys Tyr Pro Asp
275 280 285
Leu Gln Asp Asn Phe Glu Glu Leu Cys Arg Ala Cys Leu Ala Glu Gln
290 295 300
Ala Phe Asn Lys Lys Lys Ala Asp Ala Arg Val Thr Val Cys Ser Glu
305 310 315 320
Thr Ser Pro Leu Gln Phe Pro Phe Gly Met Thr Gly Asn Gly Tyr Pro
325 330 335
Phe Thr Leu Ser Ala Cys Glu Gly Arg Ile Asn Ala Thr Ile His Phe
340 345 350
Pro Gly Gly Asp Leu Pro Leu Arg Leu Arg Lys Ser Lys Tyr Phe Gln
355 360 365
Asn Pro Glu Ile Leu Pro Val Lys Asp Gly Phe Gln Ile Thr Phe Thr
370 375 380
Arg Gly Lys Thr Pro Leu Val Gly Thr Ile Lys Glu Pro Ser Leu Leu
385 390 395 400
Lys Lys Asn Asn His Tyr Tyr Leu Ser Leu Arg Val Asn Val Pro Ser
405 410 415
Val Lys Ile Pro Lys Glu Val Arg Asp Thr Arg Ala Tyr Tyr Ser Ser
420 425 430
Ala Val Gly Gly Asp Glu Thr Thr Pro Val Pro Val Lys Ala Val Ala
435 440 445
Ile Asp Leu Gly Val Thr Thr Leu Ala Asp Tyr Ser Ile Ile Asp Thr
450 455 460
Cys Leu Pro Gly Asp Cys Lys Val Phe Gly Gly Glu Thr Ala Ala Phe
465 470 475 480
Thr Ala His Gly Lys Ile Gly Gln Cys Ala Asn Lys Ser Leu Arg Asp
485 490 495
Arg Leu Tyr Lys Asn Thr Glu Glu Ala Leu Phe Leu Gly Lys Phe Ile
500 505 510
Arg Leu Ser Lys Lys Leu Arg Asp Gly Glu Gly Leu Asn Arg Trp Glu
515 520 525
Val Glu Lys Leu Pro Gly Tyr Ala Glu Arg Leu Gly Ile Thr Gln His
530 535 540
Leu Asp Asn Ala Tyr Thr Arg Lys Asp Glu Ile Ala Arg Lys Phe Lys
545 550 555 560
Gln Ile Lys Gly Asn Phe Asp Lys Leu Val Ser Glu Phe Ala Leu Arg
565 570 575
Asp His Pro Ser Lys Lys Gly Glu Ser Trp Glu Thr Ile Ser Ala Glu
580 585 590
Thr Ile Gln Val Leu Ala Ala Leu Lys Arg Ile Gln Ser Leu Leu Lys
595 600 605
Ser Trp Thr Tyr Tyr Ser Trp Thr Ala Glu Asp Tyr Val Leu Ala Leu
610 615 620
Thr Ala Asp Gly Pro Val Cys Ile Asp Gly Glu His Val Lys Ala Val
625 630 635 640
Thr Ala Thr Ser Arg Arg Ser Phe Ala Pro Cys Gly Lys Ala Ala Leu
645 650 655
Leu Arg Leu Ile Glu Ser Gly Glu Ile Val Glu Thr Gly Gly Gln Tyr
660 665 670
Gln Leu Ala Thr Gly Val Lys His Arg Asn His Pro Val Asn Phe Leu
675 680 685
Ser Ser Tyr Ile Lys His Phe Asn Gly Leu Arg Arg Asp Leu Thr Asn
690 695 700
Lys Leu Val Arg Ala Ile Val Asn Lys Ala Gln Glu Tyr Arg Val Gln
705 710 715 720
Ile Val Ile Val Glu Asp Phe Gly Ile Ala Asp Leu Glu Asp Arg Ile
725 730 735
Lys Asp Ala Tyr Glu Asn Tyr Arg Trp Asn Leu Phe Ala Pro Ala Thr
740 745 750
Ile Val Lys Lys Leu Glu Ala Ala Leu Leu Glu Val Gly Ile Ala Met
755 760 765
Ala Gln Val Asp Pro Arg His Thr Ser Gln Ile Ala Pro Thr Gly Ala
770 775 780
Phe Gly Phe Arg Asp His Ala Phe Leu Tyr Tyr Gln Asp Asp Gly Leu
785 790 795 800
Cys Arg Ile Asp Ala Asn Thr Asn Ala Ser Met Arg Ile Ala Glu Arg
805 810 815
Phe Phe Met Arg His Ser Val Leu Thr Gln Leu Arg Ala Ala Lys Ile
820 825 830
Gly Glu Thr Glu Tyr Leu Ile Pro Glu Ser Ala Ser Lys Arg Leu Asn
835 840 845
Ala Phe Val Lys Leu Gln Thr Gly Lys Pro Phe Ala Lys Leu Ile Met
850 855 860
Asn Cys Ser Gly Phe Val Leu Glu Gly Leu Thr Lys Lys Gln Tyr Ala
865 870 875 880
Lys Leu Ala Glu Thr Ala Gly Lys Lys Glu Ser Phe Tyr Gln Tyr Asp
885 890 895
Asp Arg Trp Phe Asp Lys Gly His His Phe Ala Cys Arg Ala Thr Leu
900 905 910
Glu Asn Lys Val Gln Val Cys Leu Asn Gly Gly Gly Arg Ile Lys Asp
915 920 925
Thr Thr Pro Asp Phe Asn Pro Lys Ser Leu Leu Arg Ser Asp Leu Gln
930 935 940
Thr Pro Leu Asp Gln Leu Phe Gly Asn Ser Gly Ala Ser Arg Ala Asp
945 950 955 960
Pro Lys Lys Lys Arg Lys Val
965
<210> 96
<211> 957
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.17-NLS融合蛋白的氨基酸序列
<400> 96
Met Ser Asn Thr Thr Tyr Lys Thr Ser His Leu Leu Ile Asp Leu Pro
1 5 10 15
Gln Gln Glu Leu Ile Asp Leu His Arg Asp Ser Asn Glu Met Gly Ser
20 25 30
Tyr Leu Thr Lys Phe Phe Leu Ala Ala Leu Gly Arg Pro Val Asp Asn
35 40 45
Ser Ile Val Leu Pro Pro Glu Leu Ala Asp Leu Tyr Phe Gln Phe Ala
50 55 60
Asn Gly Ile Leu Pro Val Asp Lys Gly Pro Gly Ser Asp Asp Pro Ser
65 70 75 80
Trp Leu His Ser Glu Asn Cys Tyr Ser Met Phe Phe Glu Lys Asp Ser
85 90 95
Met Ser Gly Asn Cys Thr Asn Lys Ile Lys Gln Tyr Gln Glu Leu Lys
100 105 110
Thr Ala Leu Cys Gly Gln Lys Val Lys Gly Gln Lys Gly Leu Val Gly
115 120 125
Lys Lys Thr Trp Ala Gln Leu Lys Lys Val Leu Thr Ala Leu Pro Gln
130 135 140
Lys Tyr Gln Ile Leu Ser Pro Lys Ile Cys Gln Lys Tyr Phe Lys Ser
145 150 155 160
Gly Asn Leu Glu Gly Phe Gly Lys Leu Ala Leu Ala Gly Lys Asn Arg
165 170 175
Pro Ser Met Ser Ala Gly Leu Gln Leu Arg Glu Leu Gly Leu Leu Pro
180 185 190
Leu Asp Ser Arg Gly Ile Asp Lys Asn Lys Leu Leu Gly Ile Leu Val
195 200 205
Gly Ile Thr Gly Arg Leu Lys Ser Trp Arg Asp Arg Asp Trp Ala Cys
210 215 220
Lys Thr Val Lys Glu Glu Leu Arg Val Thr Phe Glu Lys Gly Leu Gly
225 230 235 240
Glu Val Asp Pro Thr Ala Tyr Pro Gln Phe Lys Gln Phe Ala Asp Gln
245 250 255
Leu Phe Lys Gln Glu Gly Tyr Lys Ile Ser Gly Arg Val Leu Arg Ala
260 265 270
Val Glu Gly Lys Asp Ala Asp Tyr Gln Pro Val Leu Ser Leu Leu Thr
275 280 285
Gln Tyr Pro Asp Leu Gln Gly Asp Phe Glu Glu Leu Gly Arg Val Tyr
290 295 300
Leu Ala Glu Ala Glu Tyr Leu Arg Lys Lys Val Asp Ala Arg Val Thr
305 310 315 320
Val Cys Asp Ala Glu Thr Ser Pro Leu Gln Phe Pro Phe Gly Leu Thr
325 330 335
Gly Asn Gly Tyr Ser Ile Thr Leu Thr Val Val Lys Gly Gln Ile Ala
340 345 350
Ala Thr Leu His Leu Pro Gly Gly Asp Ile Thr Pro Arg Leu Arg Arg
355 360 365
Ser Lys Tyr Phe Gln Asn Pro Glu Ile Ala Pro Val Lys Asp Gly Lys
370 375 380
Gly Lys Val Asn Gly Phe Gln Ile Ser Phe Lys Arg Gly Lys Thr Pro
385 390 395 400
Leu Val Gly Ile Ile Lys Glu Pro Lys Leu Leu Lys Lys Asn Gly Asn
405 410 415
Tyr Tyr Leu Ser Leu Ala Val Gly Ile Asn Lys Thr Glu Ile Pro Lys
420 425 430
Glu Ile Cys Asp Ala Arg Ala Tyr Tyr Ser Ser Thr Ser Arg Thr Asp
435 440 445
Thr Pro Pro Ala Val Lys Ala Met Ser Ile Asp Leu Gly Val Thr Thr
450 455 460
Leu Ala Asp Tyr Ser Ile Ile Asp Thr Gly Leu Pro Gly Asp Cys Gly
465 470 475 480
Val Phe Gly Gly Ser Thr Ala Ala Phe Thr Glu His Gly Lys Ile Gly
485 490 495
Arg Cys Gly Ser Lys Ser Leu Arg Asp Gly Leu Tyr Lys Asn Thr Glu
500 505 510
Ala Gly Tyr Phe Leu Ala Lys Tyr Ile Arg Leu Ser Lys Asn Leu Arg
515 520 525
Gly Gly Val Gly Leu Asn Lys Leu Glu Lys Glu Lys Leu Leu Glu His
530 535 540
Val Glu Arg Leu Gly Ile Glu His Cys Ala Asp Asp Phe Ala Arg Lys
545 550 555 560
Asp Glu Ile His Arg Lys Phe Ser Glu Ile Lys Ser Lys Leu Glu Lys
565 570 575
Ser Ile Ser Glu Phe Ala Leu Arg Asp Arg Pro Asp Lys Lys Gly Ala
580 585 590
Ser Trp Glu Gly Ile Cys Ala Glu Thr Val Gln Val Leu Gly Ala Val
595 600 605
Lys Arg Trp Gln Ser Leu Ala Lys Ser Trp Thr Tyr Tyr Ser Trp Thr
610 615 620
Ala Glu Asp Tyr Val Leu Ala Leu Thr Gly Glu Gly Arg Thr Arg Val
625 630 635 640
Ser Asp Glu His Val Glu Ser Val Val Lys Thr Gly Arg Arg Gln Phe
645 650 655
Ala Pro Cys Gly Lys Ala Ala Leu Leu Arg Leu Leu Glu Lys Gly Lys
660 665 670
Ile Val Glu Val Cys Pro Gly Gln Phe Gln Leu Ala Glu Gly Val Asp
675 680 685
Tyr Lys Arg His Pro Thr Glu Phe Leu Ala Ala His Ile Arg His Phe
690 695 700
Asn Gly Leu Arg Arg Asp Leu Thr Asn Lys Leu Val Arg Ala Ile Val
705 710 715 720
Glu Lys Ala Gln Gln His Arg Val Gln Ile Val Ile Val Glu Asp Phe
725 730 735
Gly Ile Pro Asp Ile Glu Gly Arg Ile Met Asp His Tyr Asp Asn Tyr
740 745 750
Arg Trp Asn Leu Phe Ala Pro Ala Lys Val Ile Glu Lys Leu Glu Glu
755 760 765
Ala Leu Ser Glu Val Gly Ile Ala Met Ala Glu Val Asp Pro Arg His
770 775 780
Thr Ser Gln Leu Ala Pro Thr Gly Asp Phe Gly Phe Arg Asp His Glu
785 790 795 800
Asn Leu Tyr Phe Trp Glu Lys Gly Leu Cys Arg Thr Asp Ala Asn Thr
805 810 815
Asn Ala Ser Met Arg Ile Ala Glu Arg Phe Phe Thr Arg His Ser Val
820 825 830
Leu Ser Gln Leu Arg Ala Val Lys Ile Ser Glu Thr Glu Phe Leu Ile
835 840 845
Pro Val Ser Thr Gly Lys Arg Glu Asn Ala Phe Ile Lys Ser Gln Thr
850 855 860
Gly Lys Leu Phe Ala Lys Leu Val Ala Asp Ser Asn Gly Phe Val Met
865 870 875 880
Val Gly Leu Thr Glu Lys Gln His Gly Ala Thr Val Thr Val Gly Lys
885 890 895
Lys Val Ser Phe Tyr Asn His Ala Gly Arg Trp Leu Gly Lys Ala His
900 905 910
His Ile Ala His Arg Asp Arg Ile Lys Asn Glu Val Asn Gln Val Leu
915 920 925
Thr Ser Gly Arg Gly Arg Ile Arg Asn Ile Ala Pro Glu Leu Ser Pro
930 935 940
Lys Thr Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
945 950 955
<210> 97
<211> 941
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.18-NLS融合蛋白的氨基酸序列
<400> 97
Met Thr Asn Gln Lys Pro Lys Phe Lys Ser Ser Asp Ile Gln Ile Lys
1 5 10 15
His Ile Ser Pro Thr Asp Lys Lys Arg Leu Lys Thr Phe Tyr His Gln
20 25 30
Leu Tyr Glu Gln Val Asn Phe Ile Leu Glu Arg Met Ile Val Met Arg
35 40 45
Gly Arg Pro Arg Thr Ile Arg Asn Ile Asp Gly Thr Glu Ile Phe Val
50 55 60
Ser Gln Glu Glu Ala Asp Gln Gln Leu Leu Ser Leu Ala Gly Gly Ser
65 70 75 80
His Glu Gly Val Lys Tyr Leu Lys Gln Tyr Tyr Glu Ser Cys Val Asp
85 90 95
Ala Gly Lys Pro Ala Lys Tyr Ala Ala Asn Met Phe Leu Thr Lys Thr
100 105 110
Ile Ser Gly Thr Asn Pro Leu Gln Cys His Thr Ala Val Tyr Lys Leu
115 120 125
Tyr Lys Lys Val Gln Ala Lys Gln Ile Thr Lys Lys Glu Phe Ile Asp
130 135 140
Lys Leu Tyr Ser Lys Thr Lys Lys Lys Lys Ser Leu Lys Pro Ala Tyr
145 150 155 160
Lys Val Phe Thr Glu Asn Glu His Ile Glu Phe Tyr His Lys Val Arg
165 170 175
Ser Gly Lys Leu Pro Ala Ser Glu Val Arg Leu Glu Glu Ser Arg Arg
180 185 190
Ala Pro Asp Val Gly Leu Glu Val Gly Leu Leu Leu Arg Glu Leu Gly
195 200 205
Ile Phe Pro Phe Asn Phe Pro His Phe Thr Glu Lys Lys Tyr Leu Asp
210 215 220
Leu Ala Trp Thr Ile Ala Ile Arg Trp Leu Lys Asn Trp Asn Glu Asn
225 230 235 240
Asn Lys Asn Thr Ala Lys Glu Lys Ala Lys Gln Lys Ala Ile Val Asp
245 250 255
Lys Leu Arg Thr Ser Leu Asp Gln Lys Glu Val Asp Leu Phe Glu Glu
260 265 270
Phe Ala Glu Glu Cys Ser Gln Glu Gln Phe Gly Ile Arg Glu Gly Phe
275 280 285
Val Lys Ala Lys Lys Arg Leu Lys Ser Phe Pro Lys Gly Ile Glu Lys
290 295 300
Ser Ser Tyr Lys Glu Gly Met Arg Ile Leu Val Gln Asn Lys His Gly
305 310 315 320
Ser Ile Trp Asp Asn Phe Glu Asn Leu Ala Tyr His His Ile Ala Leu
325 330 335
Asn Glu Tyr Asn Arg Leu Arg Asp Glu Ala Ser Phe Ser Phe Pro Asp
340 345 350
Pro Ile Tyr His Pro Ile Arg Ala Glu Phe Gly Leu Thr Ser Leu Pro
355 360 365
Lys Phe Asn Val Gly Leu Asn Asp Arg Gly Asn Tyr Glu Phe Thr Ile
370 375 380
Asn Leu Pro Asp Gly Pro Leu Met Met Leu Gly Lys Lys Ser Arg Tyr
385 390 395 400
Tyr Leu Lys Pro Ile Ile Gln Gly Pro Leu Asn Asn Ala Phe Ser Phe
405 410 415
Glu Phe Ile Lys Gly Asn Lys Lys Arg Pro Lys Ile Ser Ala Lys Leu
420 425 430
Lys Ser Ile Thr Val Val Phe Ala Lys Ser Ser Ile Tyr Val Gly Leu
435 440 445
Pro Tyr Arg Pro Ile Ser Ile Pro Ile Pro Gln Ala Val Thr Asn Ser
450 455 460
Thr Tyr Tyr Phe Lys Lys Asn Leu Ser Ser Thr Ser Lys Phe Asp Lys
465 470 475 480
Asp Val Phe Met Gly Leu Thr Ala Val Ser Val Asp Leu Gly Leu Asn
485 490 495
Pro Val Phe Ser Met Ser Ala Cys Arg Leu Asp Glu Met Lys Ala Asp
500 505 510
Glu His Tyr Ser Cys Glu Val Pro Gly Phe Gly Trp Ala Asn Gln Ile
515 520 525
Trp Ser Lys Arg Ala Gly Gly Val Trp Asn Arg Ser Phe Arg Asp Lys
530 535 540
Ile Arg Gly Phe Val Pro Gly Asn Leu Ser Asp Arg Ile Phe Cys Cys
545 550 555 560
Lys Lys Ser Ile Ile Val Ser Lys Lys Leu Arg Asp Glu Lys Pro Leu
565 570 575
Thr Gln Tyr Glu Glu Glu Asn Phe Glu Arg Trp Met Gln Val Val Gly
580 585 590
Val Asp Pro Asn Glu Asp His Tyr Lys Gln Leu Arg Ile Ala Ile Arg
595 600 605
Asp Ile Lys Thr Glu Tyr Glu Thr Val Arg Ser Glu Phe Ala Leu Arg
610 615 620
Asp His Pro Asn Asn Ser Asn Lys Thr Thr Glu Asn Ile Cys Thr Glu
625 630 635 640
Cys Phe Asp Met Leu Phe Val Ile Lys Asn Leu Ile Ser Leu Leu Lys
645 650 655
Ser Trp Asn Arg Trp His Arg Thr Thr Gly Asp Ile Glu Glu Arg Gly
660 665 670
Lys Asp Pro Asn Glu Cys Ser Thr Tyr Trp Arg His Tyr Asn Gly Leu
675 680 685
Lys Thr Asp Leu Leu Lys Lys Leu Thr Asn Ile Leu Ile Glu Ser Ala
690 695 700
Lys Ser Ile Gly Ala His Ile Ile Ile Leu Glu Asp Leu Thr Leu Ser
705 710 715 720
Gln Arg Ser Ser Arg Ser Arg Arg Glu Asn Ser Leu Val Ala Ile Phe
725 730 735
Gly Ala Gln Thr Ile Ile Lys Thr Ile Ser Glu Glu Ala Glu Ile Asn
740 745 750
Gly Ile Leu Val Tyr Leu Glu Asp Pro Arg His Ser Ser Gln Ile Ser
755 760 765
Ile Val Thr Asn Glu Phe Gly Tyr Arg Pro Lys Glu Asp Lys Ala Lys
770 775 780
Leu Tyr Phe Met Asp Glu Glu Thr Val Cys Val Thr Asn Cys Asp Asp
785 790 795 800
Ser Ala Ala Leu Met Leu Gln Gln Ser Phe Trp Ser Arg His Lys Asp
805 810 815
Val Val Lys Val Lys Gly Thr Lys Val Ser Asp Thr Glu Tyr Leu Val
820 825 830
Ser Ser Glu Asp Lys Asp Gly Thr Lys Met Arg Leu Arg Ser Tyr Leu
835 840 845
Lys Arg Asn Val Gly Thr Ala Asn Ala Ile Leu Gln Lys Asn Cys Asp
850 855 860
Gly Tyr Asp Leu Lys Lys Ile Ser Pro Gln Lys Lys Lys Lys Ile Glu
865 870 875 880
Glu Phe Gly Lys Asp Glu Tyr Phe Tyr Arg His Gly Glu Gln Trp Phe
885 890 895
Thr Ala Asp Ala His Phe Asp Lys Leu Arg Glu Phe Gly Asn Gln Val
900 905 910
Phe Leu Thr Pro Gln Ser Gln Ile Lys Arg Ile Asn Leu Gln Val Glu
915 920 925
Gly Thr Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
930 935 940
<210> 98
<211> 919
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.19-NLS融合蛋白的氨基酸序列
<400> 98
Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu
1 5 10 15
Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe
20 25 30
Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met
35 40 45
Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe
50 55 60
Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu
65 70 75 80
Arg Ser Phe Ala Leu Ala Val Leu His Gln Asp Asn Pro Lys Lys Arg
85 90 95
Ala Phe Leu Glu Ser Glu Asn Cys Val Ser Ile Leu Cys Leu Glu Lys
100 105 110
Ser Ala Ser Gly Thr Arg Tyr Tyr Lys Arg Pro Gly Tyr Gln Leu Leu
115 120 125
Lys Lys Ala Ile Glu Glu Glu Trp Gly Trp Asp Lys Phe Glu Ala Ser
130 135 140
Leu Leu Asp Glu Arg Thr Gly Glu Val Ala Glu Lys Phe Ala Ala Leu
145 150 155 160
Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp
165 170 175
Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala
180 185 190
Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu
195 200 205
His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu
210 215 220
Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys
225 230 235 240
Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro
245 250 255
Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala
260 265 270
Asp Tyr Asn Val Thr Lys Lys Leu Val Leu Ala Val Ser Ala Lys Phe
275 280 285
Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys
290 295 300
Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe
305 310 315 320
Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala
325 330 335
Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly
340 345 350
Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg
355 360 365
Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala
370 375 380
Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser
385 390 395 400
Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg
405 410 415
Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg
420 425 430
Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu Glu Val Ser Asp
435 440 445
Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser
450 455 460
Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly
465 470 475 480
Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln
485 490 495
Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp
500 505 510
Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu
515 520 525
Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg
530 535 540
Leu Ser Lys Lys Leu Arg Asp Arg Gly Ala Leu Asn Asp Ile Glu Ala
545 550 555 560
Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile
565 570 575
Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu
580 585 590
Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu Arg Tyr
595 600 605
His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe
610 615 620
Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp
625 630 635 640
Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn
645 650 655
Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met
660 665 670
Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu
675 680 685
His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg
690 695 700
Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp
705 710 715 720
Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu
725 730 735
Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser
740 745 750
Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr
755 760 765
Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val
770 775 780
Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser
785 790 795 800
Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val
805 810 815
Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly
820 825 830
Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu
835 840 845
Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys
850 855 860
Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr
865 870 875 880
Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu
885 890 895
Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr Ser Arg Ala Asp
900 905 910
Pro Lys Lys Lys Arg Lys Val
915
<210> 99
<211> 832
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.20-NLS融合蛋白的氨基酸序列
<400> 99
Met Ala Phe Gln Ser Lys Arg Arg Ile Val Gly Asn Phe Val Lys Glu
1 5 10 15
Gln Cys Leu Lys Ala Val Asp Gly Lys Val Ile Leu Thr Asp Gln Glu
20 25 30
Lys Arg Glu Leu Ile Lys Arg Tyr Glu Leu His Leu Glu Pro His Lys
35 40 45
Trp Leu Leu Arg Leu Phe Leu Ser Gly Tyr Glu Gly Arg Asp Asp Gly
50 55 60
Phe Tyr Glu Glu Leu Gly Asn Thr Asn Leu Asp Lys Glu Lys Phe Phe
65 70 75 80
Glu Val Thr Ala Gly Leu Arg Asp Ala Leu Leu Arg Gln Ser Gly Ser
85 90 95
Ser Arg Ala Leu Lys Ser Ser Met Leu Gly Lys Cys Pro Pro Ser Ala
100 105 110
Ala Val Gly Lys Ala Ala Lys His Ile Gln Thr Leu Arg Asp Ala Gly
115 120 125
Ile Leu Pro Phe Lys Thr Gly Leu Thr Ser Gly Glu Asp Tyr Asn Val
130 135 140
Leu Gln Gln Ala Val Gln Gln Leu Arg Ser Trp Val Ala Cys Asp His
145 150 155 160
Arg Thr Arg Glu Ala Tyr Ala Glu Gln Gln Glu Lys Thr Ser Gln Ala
165 170 175
Glu Glu Ala Ala Lys Lys Ala Ala Asn Glu Val Lys Pro Glu Asp Ala
180 185 190
Lys Ser Leu Glu Arg His Glu Arg Val Leu Thr Lys Leu Arg Lys Gln
195 200 205
Glu Arg Arg Leu Glu Arg Met Lys Ser His Ala Gln Phe Ser Leu Asp
210 215 220
Glu Met Asp Cys Thr Gly Tyr Ser Leu Cys Met Gly Ala Asn Tyr Leu
225 230 235 240
Lys Asp Tyr Cys Leu Glu Lys Glu Gly Arg Gly Leu Arg Leu Thr Leu
245 250 255
Lys Asn Ser Thr Met Ala Gly Ser Tyr Tyr Val Ser Val Gly Asp Gly
260 265 270
Gln His Ala Gly Met Lys Asn Pro Gly Thr Pro Ala Gly Gly Ser Pro
275 280 285
Glu Lys Gly Arg Arg Arg Asn Ile Leu Phe Asp Phe Thr Val Glu Lys
290 295 300
Cys Gly Asp Asn Tyr Leu Phe Arg Tyr Asp Glu Asn Gly Lys Arg Pro
305 310 315 320
Arg Ala Gly Val Val Lys Glu Pro Arg Phe Cys Trp Arg Arg Lys Gly
325 330 335
Asn Ser Val Glu Leu Tyr Leu Ala Met Pro Ile Asn Ile Glu Asn Ser
340 345 350
Met Arg Asn Ile Phe Val Gly Lys Gln Lys Ser Gly Lys His Ser Ala
355 360 365
Phe Thr Arg Gln Trp Pro Lys Glu Val Glu Gly Leu Asp Glu Leu Arg
370 375 380
Asp Ala Val Val Leu Gly Val Asp Ile Gly Ile Asn Arg Ala Ala Phe
385 390 395 400
Cys Ala Ala Leu Lys Thr Ser Arg Phe Glu Asn Gly Leu Pro Ala Asp
405 410 415
Val Gln Val Met Asp Thr Thr Cys Asp Ala Leu Thr Glu Lys Gly Gln
420 425 430
Glu Tyr Arg Gln Leu Arg Lys Asp Ala Thr Cys Leu Ala Trp Leu Ile
435 440 445
Arg Thr Thr Arg Arg Phe Lys Ala Asp Pro Gly Asn Lys His Asn Gln
450 455 460
Ile Lys Glu Lys Asp Val Glu Arg Phe Asp Ser Ala Asp Gly Ala Tyr
465 470 475 480
Arg Arg Tyr Met Asp Ala Ile Ala Glu Met Pro Ser Asp Pro Leu Gln
485 490 495
Val Trp Glu Ala Ala Arg Ile Thr Gly Tyr Gly Glu Trp Ala Lys Glu
500 505 510
Ile Phe Ala Arg Phe Asn His Tyr Lys His Glu His Ala Cys Cys Ala
515 520 525
Val Ser Leu Ser Leu Ser Asp Arg Leu Val Trp Cys Arg Leu Ile Asp
530 535 540
Arg Ile Leu Ser Leu Lys Lys Cys Leu His Phe Gly Gly Tyr Glu Ser
545 550 555 560
Lys His Arg Lys Gly Phe Cys Lys Ser Leu Tyr Arg Leu Arg His Asn
565 570 575
Ala Arg Asn Asp Val Arg Lys Lys Leu Ala Arg Phe Ile Val Asp Ala
580 585 590
Ala Val Asp Ala Gly Ala Ser Val Ile Ala Met Glu Lys Leu Pro Ser
595 600 605
Ser Gly Gly Lys Gln Ser Lys Asp Asp Asn Arg Ile Trp Asp Leu Met
610 615 620
Ala Pro Asn Thr Leu Ala Thr Thr Val Cys Leu Met Ala Lys Val Glu
625 630 635 640
Gly Ile Gly Phe Val Gln Val Asp Pro Glu Phe Thr Ser Gln Trp Val
645 650 655
Phe Glu Gln Arg Val Ile Gly Asp Arg Glu Gly Arg Ile Val Ser Cys
660 665 670
Leu Asp Ala Glu Gly Val Arg Arg Asp Tyr Asp Ala Asp Glu Asn Ala
675 680 685
Ala Lys Asn Ile Ala Trp Leu Ala Leu Thr Arg Glu Ala Glu Pro Phe
690 695 700
Cys Met Ala Phe Glu Lys Arg Asn Gly Val Val Glu Pro Lys Gly Leu
705 710 715 720
Arg Phe Asp Ile Pro Glu Glu Pro Thr Arg Glu Gln Asp Glu Ser Asp
725 730 735
Gln Asp Phe Lys Lys Arg Leu Glu Glu Arg Asp Lys Leu Ile Glu Arg
740 745 750
Leu Gln Ala Lys Ala Asp Arg Met Gln Ala Ile Val Gln Arg Leu Phe
755 760 765
Gly Asp Arg Arg Pro Trp Asp Ala Phe Ala Asp Arg Ile Pro Glu Gly
770 775 780
Lys Ser Lys Arg Leu Phe Arg His Arg Asp Gly Leu Val Leu Asn Lys
785 790 795 800
Pro Phe Lys Gly Leu Cys Gly Ser Glu Asn Ser Glu Gln Lys Ala Ser
805 810 815
Ala Arg Asn Ser Arg Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
820 825 830
<210> 100
<211> 848
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.21-NLS融合蛋白的氨基酸序列
<400> 100
Met Gly Arg Phe Gly Lys Lys Lys Ile Ala Val Asn Gly Tyr Val Glu
1 5 10 15
Gln Asp Cys Ile Lys Thr Ile Ser Ala Lys Cys Leu Leu Thr Arg Ala
20 25 30
Gln Ile Asp Glu Leu Arg Ala Lys Tyr Asp Ala Val Leu Asp Thr Met
35 40 45
Arg Pro Leu Ile Arg Leu Ile Leu Ala Gly Tyr Glu Gly Arg Asp Asp
50 55 60
Gly Ile Tyr Glu Glu Ile Ala Pro Glu Met Ser Lys Lys Lys Phe Phe
65 70 75 80
Glu Ala Ala Thr Glu Trp Arg Glu Ser Ile Val Lys Asn Ala Ser Pro
85 90 95
Arg Ala Met Lys Ala Ser Val Phe Gly Asp Lys Glu Pro Cys Lys Ser
100 105 110
Thr Gly Gly Ala Arg Ala Val Ile Gly Lys Leu Arg Lys Ser Gly Val
115 120 125
Phe Pro Ile Glu Thr Gly Leu Ser Gly Gly Asp Glu Tyr Asn Leu Ile
130 135 140
Glu Gln Ala Ile Glu Tyr Ala Lys Ser Trp Leu Lys Ser Asp Glu Ala
145 150 155 160
Thr Arg Glu Ala Tyr Ala Asp Gln Gln Lys Asp Ile Lys Arg Leu Ile
165 170 175
Gly Glu Ala Lys Lys Leu Ala Leu Lys Ile Glu Lys Ala Glu Lys Lys
180 185 190
Leu Glu Ala Thr Asn Pro Gln Thr Lys Ser Trp Lys Lys Thr Thr Glu
195 200 205
Ile Ile Lys Lys Ser Lys Arg Glu Phe Gly Ser Val Thr Thr Lys Thr
210 215 220
Glu Lys Ala Glu Lys Arg Phe Glu Arg Met Lys Pro Phe Ser Lys Leu
225 230 235 240
Glu Leu Gln Asn Met Asp Cys Thr Lys Tyr Ser Thr Tyr Leu Gly Thr
245 250 255
Asn Tyr Ser Pro Phe Lys Leu Lys Lys Glu Gly Asp Leu Leu Gln Ile
260 265 270
Thr Val Thr Ser Ser Val Met Lys Gly Thr Tyr Leu Ala Ser Tyr Gly
275 280 285
Asp Gly Gln Tyr Gly Ser Arg Arg Asn Asn Gly Gln Ser Arg Arg Asp
290 295 300
Asp Phe Val Pro Asn Met Asn Gln Lys Arg Arg Arg Asn Leu Met Phe
305 310 315 320
Asp Cys Thr Val Glu Pro Phe Gly Asp Gly Ser Leu Leu Arg Tyr Glu
325 330 335
Glu Asn Gly Leu Arg Pro Arg Val Ala Glu Leu Lys Glu Pro Arg Leu
340 345 350
Cys Trp Arg Arg Arg Asn Gly Asn Tyr Glu Leu Tyr Leu Met Met Pro
355 360 365
Val Lys Met His Val Lys Ser Pro Glu Met Phe Ala Gly Asp His Leu
370 375 380
Ala Phe Ser Arg Tyr Trp Pro Lys Glu Val Glu Gly Leu Asp Ser Asp
385 390 395 400
Thr Lys Ile Thr Ala Leu Gly Val Asp Val Gly Ile Ile Arg Ser Ala
405 410 415
Tyr Cys Val Ala Val Thr Ala Glu Arg Phe Val Asp Gly Leu Pro Thr
420 425 430
Glu Met Thr Val Gly Lys Ala Ser Phe Asp Ala Gln Thr Glu Lys Gly
435 440 445
Arg Glu Tyr Phe Glu Leu Gly Arg Arg Ala Thr Met Leu Gly Trp Leu
450 455 460
Ile Lys Thr Thr Arg Arg Tyr Lys Lys Asp Pro Lys Asn Glu His Asn
465 470 475 480
Gln Ile Lys Glu Ser Asp Val Ala Ala Phe Asp Gly Ser Pro Gly Ala
485 490 495
Phe Glu His Tyr Ile Leu Ala Val Asp Glu Met Ser Asp Asp Pro Leu
500 505 510
Asp Val Trp Gly His Ala Asn Ile Thr Gly Tyr Gly Lys Trp Thr Lys
515 520 525
Gln Ile Phe Lys Glu Phe Asn Gln Leu Lys Arg Glu Arg Ala Glu Gly
530 535 540
Gln Val Glu Pro Asn Met Thr Asp Asp Leu Thr Trp Cys Ser Leu Ile
545 550 555 560
Asp Tyr Ile Ile Ser Leu Lys Lys Thr Leu His Phe Gly Gly Tyr Glu
565 570 575
Thr Lys Glu Arg Glu Ser Phe Cys Pro Ala Leu Tyr Asn Glu Arg Ala
580 585 590
Asn Cys Arg Asp Val Val Arg Lys Arg Leu Ala Arg Tyr Val Val Glu
595 600 605
Arg Ala Ile Ala Ala Glu Ala Gln Val Ile Ser Val Glu Asn Leu Ser
610 615 620
Lys Cys Arg Arg Asp Asp Lys Arg Lys Asn Arg Val Trp Asp Leu Met
625 630 635 640
Ser Gln Gln Ser Trp Ile Gly Val Leu Thr Asn Met Ala Arg Met Glu
645 650 655
Asn Ile Ala Val Val Ser Val Asn Pro Asp Leu Thr Ser Gln Trp Val
660 665 670
Glu Gln Cys Gly Ala Ile Gly Asp Arg Lys Ala Arg Thr Ile Ala Cys
675 680 685
Arg Asp Val Asn Gly Lys Phe Val Ser Leu Asp Ala Asp Leu Asn Ala
690 695 700
Ala Tyr Asn Ile Ala Ser Arg Ala Leu Thr Arg His Ala Glu Pro Phe
705 710 715 720
Ser Ile Thr Phe Lys Lys Lys Asp Gly Ile Leu Glu Gln Lys Asp Val
725 730 735
Cys Phe Asp Pro Gly Val Ile Pro Val Leu Glu Lys Asn Glu Asn Glu
740 745 750
Glu Lys Phe Arg Glu Arg Val Glu Lys Tyr Glu Lys Ser Leu Val Ile
755 760 765
Lys Gln Glu Arg Ala Val Arg Trp Arg Ala Ile Leu Gln His Leu Phe
770 775 780
Gly Asn Glu Arg Pro Trp Asp Glu Phe Thr Asp Glu Val Lys Glu Gly
785 790 795 800
Arg His Val Ser Leu Tyr Arg His His Gly Lys Leu Val Arg Thr Lys
805 810 815
Gln Tyr Ala Gly Leu Val Lys Glu Ala Asn Asn Glu Leu Val Pro Val
820 825 830
Cys Ala Val Ala Arg Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val
835 840 845
<210> 101
<211> 979
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.22-NLS融合蛋白的氨基酸序列
<400> 101
Met Ser Lys Ala Thr Arg Lys Thr Lys Thr Thr Val Pro Glu Ser Thr
1 5 10 15
Asp Thr Glu Ser Pro Ala Ala Asp Thr Gln Val Arg Val His Trp Leu
20 25 30
Ala Ala Ser His Arg Ala Ser Pro Gly Leu Gln Gln Val Lys Glu Met
35 40 45
Ile Gln Gln His Ala Asp Val Ala Ser Val Leu Phe Gln Gly Leu Val
50 55 60
Arg Thr Ala Pro Ile Val Phe Arg Asn Asp Asp Gly Ser Pro Val Lys
65 70 75 80
Pro Leu Asp Leu Leu Leu Ala Ser Leu Arg Pro Thr Tyr Lys Val Gln
85 90 95
Arg Asp Thr Glu Thr Val Leu Val Thr Lys Asp Asp Val Ile Arg Cys
100 105 110
Leu Thr Leu Ala Thr Thr Ala Val Asn Gly Gly Gln Ala Thr Asn Val
115 120 125
Ala Val Phe Ala Ser Ala Asp Pro Ala Leu Ser Ala Pro Leu Ala Thr
130 135 140
Leu Leu Ala Gln Leu Arg Ala Leu Glu Ser Val Asp Ser Ser Trp Ser
145 150 155 160
Val Val Gly Lys Leu Asp Ile Asn Leu Arg Lys Phe Val Trp Leu Val
165 170 175
Leu Ser Ala Ala Gly Val Leu Pro Ala Leu Ala Asp Leu Glu Gly Tyr
180 185 190
Ala Ala Lys Ser Val Leu Ala Asn Val Gln Gly Lys Tyr Lys Ser Leu
195 200 205
Gln Ala Cys Ala Asp Thr His Ala Ala Leu Tyr Lys Gln His Gln Thr
210 215 220
Asn Lys Glu Gln Leu Glu Lys Leu Ile Ala Asp Pro Gly Phe Val Ala
225 230 235 240
Leu Cys Ser Ala Leu Leu Gln Asp Pro Asp Leu Arg Ser Val Asp Ser
245 250 255
Arg Arg Leu Ala Ala Leu Glu Glu Met Leu Gly Phe Val Ala Ala Asp
260 265 270
Lys Asn Tyr Ser Glu Tyr Thr Ser Thr Arg Lys Cys Asp Gly Trp Ala
275 280 285
Pro Pro Ala Asn Met Phe Asp Leu Leu Cys Glu His Lys Glu Ala Val
290 295 300
Arg Arg Asn Ile Val Val Asp Asn Ser Lys Cys Leu Ser Arg Arg Ile
305 310 315 320
Ser Leu Val Ala Asp Gly Asp Val Asn Glu Val Ser Val Phe Glu Leu
325 330 335
Leu Asn Glu Met Arg Trp Leu Ser Val His Ser Ser Gly Ile Arg Met
340 345 350
Pro Asn Tyr Pro Lys His Ala Tyr Ala Leu Lys Phe Gly Asp Asn Tyr
355 360 365
Ile Ser Val Lys Ser Phe Glu Thr Val Val Asp Gly Gly Cys Ser Leu
370 375 380
Leu Arg Met Thr Ala Arg Val Gly Lys Asn Asp Leu Val Cys Asp Phe
385 390 395 400
Val Leu Gly Arg Gly Asn Glu Tyr Trp Asn Asn Leu Lys Ile Thr Pro
405 410 415
Met Gly Lys Gly Ile Phe Ala Val Val Lys Thr Val Arg Arg Phe Thr
420 425 430
Ala Thr Gly Ala Lys Leu Val Glu Leu Arg Gly Val Cys Lys Glu Pro
435 440 445
Glu Ile Arg Tyr Glu Arg Gly Val Leu Gly Leu Arg Leu Pro Ile Ser
450 455 460
Phe Asp Val Tyr Gly Lys Val Glu Glu Asp Ser Ile Ala Phe Gly Lys
465 470 475 480
Asn Arg Val Ser Leu Arg Thr Thr Pro Phe Val Glu Lys Ala Asp Lys
485 490 495
Phe Gln Gly Leu Leu Asp Tyr Arg Asn Thr Thr Ala Arg Asp Gly Tyr
500 505 510
Ile Tyr Tyr Ala Gly Phe Asp Gln Gly Glu Asn Asp Gln Val Val Gly
515 520 525
Ile Tyr Arg Thr Arg Thr Tyr Lys Asn Ala Thr Met Leu Glu Phe Phe
530 535 540
Asn Val Ser Asp Thr Leu Glu Glu Val Ala Ser Cys Arg Phe Ser Asp
545 550 555 560
Tyr Gln Glu Arg Lys Arg Arg Leu Arg Gly Asp Thr Gly Val Leu Asp
565 570 575
Ile Asn Ser Ile Asn Val Leu Ala Asp Lys Val Gln Arg Leu Arg Arg
580 585 590
Leu Ile Ser Thr Leu Arg Ala Cys Ala Ser His Thr Asp Trp Tyr Pro
595 600 605
Lys Leu Lys Glu Arg Arg Arg Leu Glu Trp Ala Val Leu Ala Gln Gly
610 615 620
Val Gly Val Ser Asp Phe Asp Thr Glu Ile Glu Arg Ala Glu Thr Ala
625 630 635 640
Leu Ser Ala Val Ala Ala Val Asp Phe Val Arg Asp Pro Thr Cys Ile
645 650 655
Ile Asn Val Met Asp Lys His Ile Tyr Ala Gln Phe Lys Gln Leu Arg
660 665 670
Ser Glu Arg Asn Glu Lys Tyr Arg Ser Gln His Gln His Asp Tyr Lys
675 680 685
Trp Leu Gln Leu Val Asp Ser Val Ile Ser Leu Arg Lys Ser Ile Tyr
690 695 700
Arg Phe Gly Lys Ala Pro Glu Pro Arg Gly Ala Gly Glu Leu Tyr Pro
705 710 715 720
Gln Asn Leu Tyr Thr Tyr Arg Asp Asn Leu Met Gln Gln Tyr Arg Lys
725 730 735
Glu Val Ala Ala Phe Ile Arg Asp Val Cys Leu Glu His Gly Val Arg
740 745 750
Gln Leu Ala Val Glu Ala Leu Asn Pro Thr Ser Tyr Ile Gly Glu Asp
755 760 765
Ser Asp Ala Asn Arg Lys Arg Ala Leu Phe Ala Pro Ser Glu Leu His
770 775 780
Asn Asp Ile Val Leu Ala Cys Ser Leu His Ser Ile Ala Val Val Ala
785 790 795 800
Val Asp Glu Thr Met Thr Ser Arg Val Ala Pro Asn Asn Arg Leu Gly
805 810 815
Phe Arg Ser His Gly Asp Tyr Gln Lys Phe Ser Glu Thr Ala Gln Gly
820 825 830
Arg Phe Asn Trp Lys His Leu His Tyr Phe Gly Asp Asn Asp Val Ser
835 840 845
Glu His Cys Asp Ala Asp Glu Asn Ala Cys Arg Asn Ile Val Leu Arg
850 855 860
Ala Leu Thr Cys Gly Ala Ser Lys Pro Arg Phe Ser Arg Gln Ser Leu
865 870 875 880
Leu Gly Lys Ile Lys Gly Pro Val Leu Arg Thr Gln Leu Ala Tyr Leu
885 890 895
Ala His Lys Arg Gly Leu Leu Thr Ala Ser Thr Glu Pro Lys Lys Ala
900 905 910
Ala Glu Thr Gly Phe Glu Leu Val Glu Ala Asp Leu Gly Gly Ala Leu
915 920 925
Arg Val Gly Lys Gly Phe Ile Tyr Val Asp Ala Gly Ile Cys Ile Asn
930 935 940
Ala Thr Thr Arg Lys Glu Arg Ser His Lys Val Gly Glu Ala Val Val
945 950 955 960
Ser Arg Ser Leu Ala Ser Pro Phe Ser Arg Ala Asp Pro Lys Lys Lys
965 970 975
Arg Lys Val
<210> 102
<211> 3268
<212> DNA
<213> 人工序列
<220>
<223> 表达Cas12j.3系统的质粒
<400> 102
tttacacttt atgcttccgg ctcgtatgtt aggaggtctt tatcatgacc aaggagaaga 60
tcaagaagac caagaaggcc aaggtggaga aggactccgt gaccagggcc ggcatcctga 120
ggatcctgct gaacccggac cagcaccagg agctggacac cctgatctcc gaccaccagg 180
aggccgccag ggagatccag accgccacct acaagctgtc cggcctgaag ctgtacgaca 240
agaccaacaa catggtggtg gacggctcca aggccacccc ggaggagcag gaggcctact 300
acaagatcat caactgggag ggccagccga tctccatctc caacccgatg gtgagggcca 360
ccttcaagtc catcgccaag gtgaaggagg acatcaggag gaagcaggag gagtacgcca 420
agctggagga ggccgacctg accaagatgt ccaccggcga cgtgaagaag cacaagaacg 480
agctgaggaa ggccgccaac aggatcaagc actccgagga gatcctgcag ttcgccaagt 540
ggaggctggc cgacatcttc ccgctgccgc tgtcccacaa ctcccagctg cacctgaaga 600
acaactacca ccagaacgtg ttctccggct tccacgccag ggtgaagggc tggaacgcct 660
gcgacatcgc cgcccaggcc aactacgccg agatcgacaa caggctgacc gagctgtcct 720
ccgagctgtc cggcgactac ggctccgagg tgatcaccga cctgatgggc ctgctgcagt 780
acaccaagga gctgggcgag ggctacaccg acacctccta cctgaactac aagttcctgt 840
ccttcttcaa ggagtgctgg aggccgaacg ccatcgccaa caacaccggc ctgctggagg 900
gcttctggct ggccaacaac aagcacacca acaagaagaa ccaggtggcc tactccttca 960
acccgaagat ctccgaggag ctgttcagga ggaggtccct gtgggagtcc gacaagtgcc 1020
tgctgtccga cccgaggttc gagaagtacg tggagctgtt cgacaagcac ggcaggtaca 1080
ggaagggcgc ctccctgacc ctgatctcca aggagtcccc gatcccgatc ggcttctcca 1140
tggacaggaa cgccgccaag ctggtgagga tcgacaacga caccgccaac aggcagctga 1200
ccatcaccat cgagctgccg aacaaggagg agaggtccta cgtggccgcc tacggcagga 1260
agcacgagac caagtgctac tacaacggcc tgaccaccag gctgccgagg tccgagaagg 1320
agctgctggc cctggccaag gccgagaaca gggagctgac cgacaaggag atccacgagg 1380
cctccctgga gaagtgctac atcttcgagt acgccagggc cggcaagatc ccggtgttcg 1440
ccgtggtgaa gaccctgtac ttcaggagga acccgtccaa cggcgagtac tacgtgatcc 1500
tgccgaccaa catcttcgtg gagtaccacg ccaacaacga gttcaactcc aaggagctgt 1560
tcaagatcag gtccgagctg cagaaggcct gggacgaggt gaggaccccg aagaggaacg 1620
tgcagtcctg cgtgctggac aaggacctgt ccaagaggtt cgccggcagg accctgaagt 1680
acgccggcat cgacctgggc tactccaacc cgtacaccgt gtcctactac aacgtggtgg 1740
gcaccgagga gggcatccag atcaaggaga ccggcaacga gatcgtgtcc accgtgttca 1800
acgagcagta catccagctg aagggcaaca tctaccagct gatcaacatc atcagggcct 1860
ccaggaggta cctgcaggag tccggcgagc tgaagctgtc caaggacgac atcaagtcct 1920
tcgaccagct gatggagctg ctgccgtccg agcagaggat caccatcgac cagttcatca 1980
aggacatcaa gaaggccaag caggagggca agctgatcag ggacatcaag ggcaagctgc 2040
cggtggaggg caagaagaag gagtactggg tgatctccaa cctgatgtac gtgatcaccc 2100
agaccatgaa cggcatcagg ggcaacaggg actccaacaa ccacctgacc gagaagaaga 2160
actggctgtc cgccccgccg ctgatcgagc tgatcgacgc ctactacaac ctgaagaaga 2220
ccttcaacga ctccggcgac ggcatcaaga tgctgccgaa ggaccacgtg tacgccgagg 2280
gcgagaagca gaggtgcacc ctgagggagg agaacttctg caagggcatc ctggagtgga 2340
gggacaacgt gaaggactac ttcatcaaga agctgttctc ccagatcgcc cacaggtgct 2400
acgagctggg catcggcatc gtggccatgg agaacctgga catcatgggc tcctccaaga 2460
acaccaagca gtccaacagg atgttcaaca tctggccgag gggccagatg aagaagtccg 2520
ccgaggacgc cttctcctac atgggcatcc tgatccagta cgtggacgag aacggcacct 2580
ccaggcacga cgccgactcc ggcatctacg gctgcaggga cggcgccaac ctgtggctgc 2640
cgaacaagaa gctgcacgcc gacgtgaacg cctccaggat gatcgccctg aggggcctga 2700
cccaccacac caacctgtac tgcaggtccc tgaccgagat cgagaacggc aagtacgtga 2760
acacctacga gctgttcgac accaccaaga acgaccagtc cggcgccgcc aagaggctga 2820
ggggcgccga gaccctgctg cacggctact ccgccaccgt gtaccagatc cacaccacca 2880
acaccggcgc cggcgtggcc ctgctgccgg acctgaccgc caccgacgtg atcaagaaca 2940
agaagatcac cgccaccaag gagaacaccg ccaagtacta caagctggac aacaccaaca 3000
cctactaccc gtggtccgtg tgcgagaagc tgcacaagaa ctggaagctg tcctgacaaa 3060
taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 3120
acgctctcct gagtaggaca aatttgacag ctagctcagt cctaggtata atgctagcgg 3180
tgatatagta actggtctgt tccagcactt caccggtata acaacttcga cgagctctac 3240
aagaaggcca tcctgacgga tggccttt 3268
<210> 103
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> PAM文库序列
<220>
<221> misc_feature
<222> (1)..(8)
<223> n = a or t or c or g
<400> 103
nnnnnnnngg tataacaact tcgacgagct ctaca 35
<210> 104
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> Pre-crRNA加工及PAM消耗导向RNA
<400> 104
gguauaacaa cuucgacgag cucuaca 27
<210> 105
<211> 24
<212> RNA
<213> 人工序列
<220>
<223> Cas12j.19导向RNA
<400> 105
cuuccaucag agaaccucac ugcg 24
<210> 106
<211> 1020
<212> DNA
<213> 人工序列
<220>
<223> 靶向的双链DNA序列
<400> 106
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accccctctt 420
cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc 480
cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag ctcggtacca 540
tcgtattagg tatagcaagc cgtctcgcag tgaggttctc tgatggaagc atatcgtagc 600
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 660
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 720
ctggggatcc tctagagtcg acctgcaggc atgcaagctt ggcgtaatca tggtcatagc 780
tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 840
taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 900
cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 960
gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 1020
<210> 107
<211> 882
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.1氨基酸序列
<400> 107
Met Leu Tyr Thr Met Asn Val Lys Thr Ile Lys Leu Lys Val Asp Ala
1 5 10 15
Thr Lys Glu Val Glu Ser Arg Leu Thr Lys Met Leu Leu Val His Asn
20 25 30
Asn Ile Gly Arg Glu Ile Ile Asn Phe Leu Ile Leu Cys Ser Gly Asn
35 40 45
Asp Asn Ile Arg Lys Thr Lys Phe Asp Glu Phe Gly Asn Ser Tyr Asp
50 55 60
Glu Phe Cys Asn Leu Lys Leu Asp Gln Phe Asn Leu Tyr Asp Arg Leu
65 70 75 80
Thr Glu Ile His Asp Glu Val Thr Leu Glu Asp Phe Gln Lys Thr Leu
85 90 95
Asn Asp Ile Tyr Asp Leu Val Leu Asn Ser Lys Ser Phe Ser Asn Val
100 105 110
Ser Ser Thr Ile Phe Asn Lys Asn Lys Lys Val Asn Phe Asp Glu Thr
115 120 125
Lys Lys Gly Asp Leu Ser Arg Lys Cys Leu Met Asn Ala Arg Asp Trp
130 135 140
Gly Val Leu Pro Leu Ile Ser Val Asp Asp Asp Ile Val Thr Cys Gly
145 150 155 160
Thr Leu Lys Gly Ile Leu Ser Glu Cys Gln Ser Arg Ile Leu Ser Trp
165 170 175
Asn Glu Cys Asn Leu Ser Thr Lys Glu Thr Tyr Ser Glu Lys Lys Ser
180 185 190
Glu Tyr Gln Ser Ile Leu Asp Asp Ser Met Thr Lys Asp Ala Asp Val
195 200 205
Thr Thr Ala Met Ile Gln Phe Met Asp Asp Val Ser Asn Val Tyr Gly
210 215 220
Ser Asn Asn Glu Asn Gln Leu Lys Trp Phe Asn Asn Arg Phe Leu Thr
225 230 235 240
Tyr Val Arg Asn Lys Ile Arg Pro Phe Leu Leu Thr Asn Ser Pro Ile
245 250 255
Asp Asn Phe Glu Gln Ser Asp Thr Ser Tyr Asn Cys Ser Ile Glu Ile
260 265 270
Val Arg Ile Leu Ser Lys Tyr Glu Ile Leu Trp Lys Asp Glu Val Ser
275 280 285
Val Asn Arg Tyr Lys Lys Thr Cys Asp Asp Gly Ile Asn Ile Glu Lys
290 295 300
Tyr Arg Tyr Leu Val His Ala Lys Ser Asp Phe Leu Arg Tyr Lys Glu
305 310 315 320
Thr Ala Ser Phe Lys Glu Ile His Ala Val Lys Ser Pro Ile Ser Leu
325 330 335
Cys Phe Gly Asn Asn Tyr Gln Pro Phe Ser Leu Ser Asp Val Gly Asp
340 345 350
Arg His Asn Ile Asn Phe Gly Tyr Lys Phe Gly Lys Leu Gly Lys Gln
355 360 365
Arg Lys Glu Cys Ser Phe Asn Leu Asn Tyr Arg Arg Lys Lys Val Lys
370 375 380
Tyr Ala Asn Thr Pro Val Arg Ser Asp Glu Asn Lys Cys Tyr Leu Asp
385 390 395 400
Asn Leu Glu Ile Glu Asp Ala Lys Asn Gly Ser Tyr Lys Leu Ser Tyr
405 410 415
Met Val Asn Lys Lys Tyr Lys Arg Glu Ser Phe Ile Lys Glu Pro Lys
420 425 430
Met Lys Met Tyr Asn Gly Lys Leu Tyr Met Tyr Phe Pro Met Ser Asn
435 440 445
Glu Phe Glu Glu Asp Arg Asp Ser Phe Ala Leu Leu Thr Tyr Phe Ser
450 455 460
Arg Ser Ser Asn Ser Lys Ser Gln Ile Asp Glu Ala Ser Asn Ile Leu
465 470 475 480
Gln Asn Arg Lys Ile Arg Val Cys Gly Val Asp Leu Gly Ile Asn Pro
485 490 495
Thr Phe Ala Leu Ser Val Leu Glu Tyr Ser Asp Asn Lys Ile Thr Asp
500 505 510
Thr Asn Ile Gly Met Lys His Glu Gly Ser Tyr Asn Asn Phe Ser Glu
515 520 525
Ile Arg Lys Gln Ile Asn Asp Val Thr Asp Met Ile Ser Tyr Leu Lys
530 535 540
Ser Lys Tyr Asp Asn Cys Glu Lys Asp Tyr Ser Ser Lys Ile Asp Asp
545 550 555 560
His Ile Lys Ser Arg Leu Asn Glu Glu Ile Ser Asn Phe Cys Asp Leu
565 570 575
Val Ser Tyr Lys Arg Asn Lys Asn Thr Ile Ile Arg Lys Glu Ile Lys
580 585 590
Asn Val Glu Lys Glu Ile Asn Lys Ile Lys Asn Cys Arg Arg His Thr
595 600 605
Leu Lys Lys Asp Leu Thr Glu Asn Phe Gly Trp Val Ser Ala Leu Asn
610 615 620
Glu Phe Ile Ser Leu Lys His Ser Phe Asn Asp Met Gly Glu Ser Phe
625 630 635 640
Asp Ser Lys Thr Asn Pro Ser Tyr Ser Tyr Phe Glu Lys Trp Lys Arg
645 650 655
Tyr Ile Asp Asn Ile Lys Asp Asp Ser Leu Lys Thr Val Ser Arg Glu
660 665 670
Ile Leu Asn Phe Cys Ile Glu Asn Ser Val Asp Phe Ile Ala Leu Glu
675 680 685
Asp Leu Gln Thr Phe Ala Pro Ser Asp Asp Arg Thr Lys Ser His Asn
690 695 700
Lys Leu Thr Gln Leu Trp Cys Phe Gly Lys Leu Lys Lys Cys Leu Glu
705 710 715 720
Asp Ile Ala Ser Met Tyr Gly Ile His Val Tyr Ser Ser Thr Asp Pro
725 730 735
Arg Asn Thr Ser Asp Thr His Phe Glu Ser Lys Asn Phe Gly Tyr Arg
740 745 750
Asp Glu Ser Asn Lys His Asn Leu Trp Val Asn Val Asp Gly Glu Tyr
755 760 765
Thr Val Val Asp Ser Asp Ile Asn Ala Ser Lys Asn Ile Ala Asn Arg
770 775 780
Phe Leu Thr His His Lys Asp Leu Lys Gln Leu Pro Met Ile Gly Asp
785 790 795 800
Gly Thr Leu Phe Lys Ile Asp Ser Ser Ser Lys Arg Asn Lys Ser Phe
805 810 815
Ala Val Lys Leu Asn Ile His Lys Asn Val Tyr Glu Leu Ile Asp Gly
820 825 830
Glu Phe Val Lys Ser Asn Lys Lys Pro Asn Gly Thr Ser Arg Lys Gln
835 840 845
Thr Ala Tyr Ile His Gly Asp Met Phe Ile Asp Ser Ile Ser His Lys
850 855 860
Asn Lys Lys Met Phe Leu Arg Glu Asn Leu Ile Arg Asn Gly Phe Ile
865 870 875 880
Ser Lys
<210> 108
<211> 935
<212> PRT
<213> 人工序列
<220>
<223> Cas12j.2氨基酸序列
<400> 108
Met Asn Lys Thr Asp Thr Gln Asn Asn Glu Gln Ile Asn Lys Pro Thr
1 5 10 15
Gln Leu Leu Asn Asn Lys Asp Ile Glu Leu Thr Val Lys Thr Val Lys
20 25 30
Ser Ala Thr Val Lys Val Asp Asn Asn Ser Lys Lys Glu Leu Phe Gly
35 40 45
Leu Phe Asn Tyr Phe Thr Ser Val Ala Ser Gly Ile Lys Asp Lys Val
50 55 60
Tyr Asn Leu Gln Ser Asp Glu Lys Thr Ala Pro Ile Phe Asn Asp Tyr
65 70 75 80
Val Lys Gln Pro Gln Arg Gly Arg Ser Ala Ala Thr Thr Leu Phe Thr
85 90 95
Lys Leu Asp Ala Glu Lys Thr Tyr Thr Ser Gln His Ser Phe Pro Gly
100 105 110
Lys Trp Arg Asp Ser Gly Ile Phe Pro Leu Tyr Asn Lys Glu Ser Glu
115 120 125
Lys Tyr Asp Leu Ser Thr His Gly Tyr His Tyr Ser Ala Asn Ala Glu
130 135 140
Ile His Thr Gln Leu Asp Ser His Asp Glu Cys Asn Lys Glu Cys Glu
145 150 155 160
Lys Glu Tyr Ala Ala Leu Arg Asp Glu Val Asn Asn Tyr Lys Tyr Glu
165 170 175
Phe Thr Leu Gln Phe Lys Ala Glu Asn Ala Glu Lys Phe Tyr Asn Phe
180 185 190
Val Glu Lys Leu Thr Leu Met Gly Trp Arg Tyr Asp Ala Thr Phe Arg
195 200 205
Ser Phe Phe Glu Leu His Met His Pro Lys Leu Lys Thr Gly Glu Thr
210 215 220
Thr Tyr Arg Ala Thr Tyr Lys Leu Pro Ser Gly Lys Ser Lys Arg Tyr
225 230 235 240
Ser Phe Phe Arg Asp Asp Ile Ala Asp Glu Ile Ala Lys Asn Pro Glu
245 250 255
Phe Trp Pro Met Leu Glu Ser Ser Asn Ala Ile Ser Trp Ile Asn Ser
260 265 270
Asn Asn Leu Leu Ser Arg Lys Lys Asp Lys Ala Asn Tyr Ser Ser Thr
275 280 285
Ser Leu Ile Lys Ser Gln Ile Arg Leu Tyr Leu Gly Asn Asn Gly Val
290 295 300
Pro Phe Thr Ala Arg Glu His Asp Gly Arg Ile Tyr Phe Ser Phe Arg
305 310 315 320
Leu Pro Ala Ile Asn Gly Glu Lys Gly Arg Met Val Glu Ile Pro Cys
325 330 335
Ser Tyr Lys Lys Val Phe Asn Gly Lys Ala Arg Lys Ser Cys Tyr Leu
340 345 350
Gly Gly Leu Thr Ile Glu Lys Thr Asp Ala Gly Lys His Ile Phe Lys
355 360 365
Tyr Ser Val Asn Asn Lys Lys Pro Gln Val Ala Glu Leu Asn Glu Cys
370 375 380
Phe Leu Arg Leu Val Val Arg Asn Arg Glu Tyr Phe Asn Asn Val Val
385 390 395 400
Ala Gly Lys Ile Thr Asp Ile Asn Thr Asp His Phe Asp Phe Tyr Val
405 410 415
Asp Leu Pro Leu Asn Val Lys Glu Asp Pro Ile His Asp Leu Ser Ser
420 425 430
Thr Glu Val Phe Gly Lys Asn Gly Leu Arg Ser Tyr Tyr Ser Ser Ala
435 440 445
Tyr Pro Glu Ile Lys Asn Leu Gly Ser Gln Ile Glu Thr Gly Lys Asn
450 455 460
Leu Thr Cys Pro Ile Thr Lys Thr His Asn Ile Met Gly Ile Asp Leu
465 470 475 480
Gly Gln Arg Asn Pro Phe Ala Tyr Cys Ile Lys Asp Asn Thr Gly Lys
485 490 495
Leu Ile Ala Gln Gly His Met Asp Gly Ser Lys Asn Glu Thr Tyr Lys
500 505 510
Lys Tyr Ile Asn Phe Gly Lys Glu Ser Thr Ser Val Ser His Leu Ile
515 520 525
Lys Glu Thr Arg Ser Tyr Leu His Gly Asp Pro Glu Ala Ile Ser Lys
530 535 540
Glu Leu Tyr Asn Glu Val Ala Gly Phe Cys Asn Asn Pro Val Ser Tyr
545 550 555 560
Glu Glu Tyr Leu Lys Tyr Leu Asp Ser Lys Lys Phe Leu Ile Asn Lys
565 570 575
Glu Asp Leu Ser Lys Asn Ala Met His Leu Leu Arg Gln Lys Asp His
580 585 590
Asn Trp Ile Gly Arg Asp Trp Leu Trp Tyr Ile Ser Lys Gln Tyr Lys
595 600 605
Lys His Asn Glu Asn Arg Met Gln Asp Ala Asp Trp Arg Gln Thr Leu
610 615 620
Tyr Trp Ile Asp Ser Leu Tyr Arg Tyr Ile Asp Val Met Lys Ser Phe
625 630 635 640
His Asn Phe Gly Ser Phe Tyr Asp Lys Asn Leu Lys Lys Lys Val Asn
645 650 655
Gly Thr Val Val Gly Phe Cys Lys Thr Val His Asp Gln Ile Asn Asn
660 665 670
Asn Asn Asp Asp Met Phe Lys Lys Phe Thr Asn Glu Leu Met Ser Val
675 680 685
Ile Arg Glu His Lys Val Ser Val Val Ala Leu Glu Lys Met Asp Ser
690 695 700
Met Leu Gly Asp Lys Ser Arg His Thr Phe Glu Asn Arg Asn Tyr Asn
705 710 715 720
Leu Trp Pro Val Gly Gln Leu Lys Thr Phe Met Glu Gly Lys Leu Glu
725 730 735
Ser Phe Asn Val Ala Leu Ile Glu Ile Asp Glu Arg Asn Thr Ser Gln
740 745 750
Val Cys Lys Glu Asn Trp Ser Tyr Arg Glu Ala Asp Asp Leu Tyr Tyr
755 760 765
Val Thr Asp Gly Glu Ser His Lys Val His Ala Asp Glu Asn Ala Ala
770 775 780
Asn Asn Ile Val Asp Arg Cys Ile Ser Arg His Thr Asn Met Phe Ser
785 790 795 800
Leu His Met Val Asn Pro Lys Asp Asp Tyr Tyr Val Pro Thr Cys Ile
805 810 815
Trp Asp Thr Thr Glu Glu Ser Gly Lys Arg Val Arg Gly Phe Leu Thr
820 825 830
Lys Leu Tyr Lys Asn Ser Asp Val Val Phe Thr Lys Lys Gly Asp Lys
835 840 845
Leu Val Lys Ser Lys Thr Ser Val Lys Glu Leu Lys Lys Leu Val Gly
850 855 860
Lys Thr Lys Glu Lys Arg Gly Gln Tyr Trp Tyr Arg Phe Glu Gly Lys
865 870 875 880
Ser Trp Ile Asn Glu Ala Asp Arg Asp Thr Ile Ile Leu Asn Ala Lys
885 890 895
Lys Ile Ser Arg Glu Arg Asp Asn Gly Glu Gln Ser Thr Asp Thr Arg
900 905 910
Ser Gln Asn Val Thr Val Ser Val Leu Asp Val Cys Glu Thr Ala Glu
915 920 925
Lys Lys Lys Leu Val Leu Val
930 935

Claims (82)

1.一种CRISPR/Cas系统中的效应蛋白，其由SEQ ID NO：2所示的序列组成。
2.一种缀合物，其包含权利要求1所述的蛋白以及修饰部分；其中，所述修饰部分选自另外的蛋白或多肽、可检测的标记，及其任意组合。
3.权利要求2所述的缀合物，其中，所述修饰部分任选地通过接头连接至所述蛋白的N端或C端。
4.权利要求2所述的缀合物，其中，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域、转录抑制结构域、核酸酶结构域、核苷酸脱氨酶结构域、甲基化酶结构域、去甲基化酶结构域、转录释放因子活性结构域、组蛋白修饰活性结构域和核酸结合活性结构域。
5.权利要求2所述的缀合物，其中，所述另外的蛋白或多肽选自具有单链RNA切割活性的结构域、具有双链RNA切割活性的结构域、具有单链DNA切割活性的结构域、或具有双链DNA切割活性的结构域。
6.权利要求2所述的缀合物，其中，所述缀合物包含表位标签。
7.权利要求2所述的缀合物，其中，所述缀合物包含NLS序列。
8.权利要求7所述的缀合物，其中，所述NLS序列如SEQ ID NO:81所示。
9.权利要求7所述的缀合物，其中，所述NLS序列位于所述蛋白的N端或C端。
10.一种融合蛋白，其包含权利要求1所述的蛋白以及另外的蛋白或多肽。
11.权利要求10所述的融合蛋白，其中，所述另外的蛋白或多肽任选地通过接头连接至所述蛋白的N端或C端。
12.权利要求10所述的融合蛋白，其中，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域、转录抑制结构域、核酸酶结构域、核苷酸脱氨酶结构域、甲基化酶结构域、去甲基化酶结构域、转录释放因子活性结构域、组蛋白修饰活性结构域和核酸结合活性结构域。
13.权利要求10所述的融合蛋白，其中，所述另外的蛋白或多肽选自具有单链RNA切割活性的结构域、具有双链RNA切割活性的结构域、具有单链DNA切割活性的结构域、或具有双链DNA切割活性的结构域。
14.权利要求10所述的融合蛋白，其中，所述融合蛋白包含表位标签。
15.权利要求10所述的融合蛋白，其中，所述融合蛋白包含NLS序列。
16.权利要求15所述的融合蛋白，其中，所述NLS序列如SEQ ID NO:81所示。
17.权利要求15所述的融合蛋白，其中，所述NLS序列位于所述蛋白的N端或C端。
18.权利要求10所述的融合蛋白，其中，所述融合蛋白的氨基酸序列如SEQ ID NO:83所示。
19.一种复合物，其包含：
(i)蛋白组分，其选自：权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白，及其任意组合；和
(ii)核酸组分，其从5’至3’方向包含同向重复序列和能够与靶序列杂交的导向序列，所述同向重复序列选自SEQ ID NO：42所示的序列或其互补序列；
其中，所述蛋白组分与核酸组分相互结合形成复合物。
20.权利要求19所述的复合物，其中，所述导向序列连接于所述核酸分子的3’端。
21.权利要求19所述的复合物，其中，所述导向序列包含所述靶序列的互补序列。
22.权利要求19所述的复合物，其中，所述核酸组分是CRISPR/Cas系统中的导向RNA。
23.权利要求19所述的复合物，其中，所述复合物不包含反式作用crRNA(tracrRNA)。
24.一种分离的核酸分子，其包含：
(i)编码权利要求1所述的蛋白、或权利要求2-9任一项所述的缀合物、或权利要求10-18任一项所述的融合蛋白的核苷酸序列；或，
(ii)(i)所述的核苷酸序列以及编码同向重复序列的核苷酸序列，所述同向重复序列选自SEQ ID NO：42所示的序列或其互补序列。
25.权利要求24所述的分离的核酸分子，其中，(i)或(ii)所述的核苷酸序列经密码子优化用于在原核细胞或真核细胞中进行表达。
26.一种载体，其包含权利要求24或25所述的分离的核酸分子。
27.一种宿主细胞，其包含权利要求24或25所述的分离的核酸分子或权利要求26所述的载体。
28.一种组合物，其包含：
(i)第一组分，其选自：权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、编码所述蛋白或融合蛋白的核苷酸序列，以及其任意组合；和
(ii)第二组分，其为包含导向RNA的核苷酸序列，或者编码所述包含导向RNA的核苷酸序列的核苷酸序列；
其中，所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；所述同向重复序列选自SEQ ID NO：42所示的序列或其互补序列；
所述导向RNA能够与(i)中所述的蛋白、缀合物或融合蛋白形成复合物。
29.权利要求28所述的组合物，其中，所述导向序列连接至所述同向重复序列的3’端。
30.权利要求28所述的组合物，其中，所述导向序列包含所述靶序列的互补序列。
31.权利要求28所述的组合物，其中，所述组合物不包含反式作用crRNA(tracrRNA)。
32.权利要求28所述的组合物，其中，所述组合物中的至少一个组分是非天然存在的或经修饰的。
33.权利要求28所述的组合物，其中：当所述靶序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-TTN所示的序列。
34.一种组合物，其包含一种或多种载体，所述一种或多种载体包含：
(i)第一核酸，其为编码权利要求1所述的蛋白或权利要求10-18任一项所述的融合蛋白的核苷酸序列；任选地所述第一核酸可操作地连接至第一调节元件；以及
(ii)第二核酸，其编码包含导向RNA的核苷酸序列；任选地所述第二核酸可操作地连接至第二调节元件；
其中：
所述第一核酸与第二核酸存在于相同或不同的载体上；
所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；所述同向重复序列选自SEQ ID NO：42所示的序列或其互补序列；
所述导向RNA能够与(i)中所述的效应蛋白或融合蛋白形成复合物。
35.权利要求34所述的组合物，其中，所述导向序列连接至所述同向重复序列的3’端。
36.权利要求34所述的组合物，其中，所述导向序列包含所述靶序列的互补序列。
37.权利要求34所述的组合物，其中，所述组合物不包含反式作用crRNA(tracrRNA)。
38.权利要求34所述的组合物，其中，所述组合物中的至少一个组分是非天然存在的或经修饰的。
39.权利要求34所述的组合物，其中，所述第一调节元件是启动子。
40.权利要求34所述的组合物，其中，所述第二调节元件是启动子。
41.权利要求34所述的组合物，其中：当所述靶序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-TTN所示的序列。
42.权利要求34-41任一项所述的组合物，其中，当所述靶序列为RNA时，所述靶RNA序列不具有PAM结构域限制。
43.权利要求34-41任一项所述的组合物，其中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列；或者，所述靶序列是非天然存在的DNA或RNA序列。
44.权利要求34-41任一项所述的组合物，其中，所述靶序列存在于细胞内。
45.权利要求44所述的组合物，其中，所述靶序列存在于细胞核内、细胞质或细胞器内。
46.权利要求44所述的组合物，其中，所述细胞是真核细胞或原核细胞。
47.权利要求34-41任一项所述的组合物，其中，所述蛋白连接有一个或多个NLS序列，或者，所述缀合物或融合蛋白包含一个或多个NLS序列。
48.权利要求47所述的组合物，其中，所述NLS序列连接至所述蛋白的N端或C端。
49.一种试剂盒，其包括一种或多种选自下列的组分：权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、权利要求19-23任一项所述的复合物、权利要求24或25所述的分离的核酸分子、权利要求26所述的载体、权利要求28-48任一项所述的组合物。
50.一种递送组合物，其包含递送载体，以及选自下列的一种或多种：权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、权利要求19-23任一项所述的复合物、权利要求24或25所述的分离的核酸分子、权利要求26所述的载体、权利要求28-48任一项所述的组合物。
51.权利要求50所述的递送组合物，其中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、外泌体、微泡、基因枪或病毒载体。
52.权利要求50所述的递送组合物，其中，所述递送载体选自脂质体。
53.权利要求51所述的递送组合物，其中，所述病毒载体为复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒。
54.一种用于非治疗目的的修饰靶基因的方法，其包括：将权利要求19-23任一项所述的复合物或权利要求28-48任一项所述的组合物与所述靶基因接触，或者递送至包含所述靶基因的细胞中；所述靶序列存在于所述靶基因中。
55.权利要求54所述的方法，其中，所述靶基因存在于细胞内。
56.权利要求55所述的方法，其中，所述细胞是原核细胞或真核细胞。
57.权利要求54所述的方法，其中，所述细胞选自哺乳动物细胞或植物细胞。
58.权利要求54所述的方法，其中，所述靶基因存在于体外的核酸分子中。
59.权利要求58所述的方法，其中，所述体外的核酸分子为质粒。
60.权利要求54所述的方法，其中，所述修饰是指所述靶序列的断裂。
61.权利要求60所述的方法，其中，所述修饰包括DNA的双链断裂或RNA的单链断裂。
62.权利要求60所述的方法，其中，所述修饰还包括将外源核酸插入所述断裂中。
63.一种用于非治疗目的的改变基因产物的表达的方法，其包括：将权利要求19-23任一项所述的复合物或权利要求28-48任一项所述的组合物与编码所述基因产物的核酸分子接触，或者递送至包含所述核酸分子的细胞中，所述靶序列存在于所述核酸分子中。
64.权利要求63所述的方法，其中，所述核酸分子存在于细胞内。
65.权利要求64所述的方法，其中，所述细胞是原核细胞或真核细胞。
66.权利要求64所述的方法，其中，所述细胞选自哺乳动物细胞或植物细胞。
67.权利要求63所述的方法，其中，所述核酸分子存在于体外的核酸分子中。
68.权利要求67所述的方法，其中，所述体外的核酸分子为质粒。
69.权利要求63所述的方法，其中，所述基因产物的表达被改变增强或降低。
70.权利要求63所述的方法，其中，所述基因产物是蛋白。
71.权利要求54-70任一项所述的方法，其中所述的蛋白、缀合物、融合蛋白、分离的核酸分子、复合物、载体或组合物包含于递送载体中。
72.权利要求71所述的方法，其中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、外泌体、病毒载体。
73.权利要求71所述的方法，其中，所述递送载体选自脂质体。
74.权利要求72所述的方法，其中，所述病毒载体选自复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒。
75.权利要求54-70任一项所述的方法，其用于改变靶基因或编码靶基因产物的核酸分子中的一个或多个靶序列来修饰细胞、细胞系或非人生物体。
76.一种体外的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、权利要求19-23所述的复合物、权利要求24或25所述的分离的核酸分子、权利要求26所述的载体、权利要求28-48任一项所述的组合物。
77.权利要求76所述的细胞或细胞系或它们的子代，其中，所述细胞是真核细胞。
78.权利要求76所述的细胞或细胞系或它们的子代，其中，所述细胞是动物细胞或植物细胞。
79.权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、权利要求19-23任一项所述的复合物、权利要求24或25所述的分离的核酸分子、权利要求26所述的载体、权利要求28-48任一项所述的组合物、权利要求49所述的试剂盒或权利要求50所述的递送组合物，用于非诊断治疗目的的核酸编辑的用途。
80.权利要求79所述的用途，其中，所述核酸编辑包括基因或基因组编辑。
81.权利要求80所述的用途，其中，所述基因或基因组编辑包括修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或插入多核苷酸。
82.权利要求1所述的蛋白、权利要求2-9任一项所述的缀合物、权利要求10-18任一项所述的融合蛋白、权利要求19-23任一项所述的复合物、权利要求24或25所述的分离的核酸分子、权利要求26所述的载体、权利要求28-48任一项所述的组合物、权利要求49所述的试剂盒或权利要求50所述的递送组合物，在制备制剂中的用途，所述制剂用于：
(i)离体基因或基因组编辑；
(ii)离体单链DNA的检测；
(iii)编辑靶基因座中的靶序列来修饰非人类生物；
(iv)治疗由靶基因座中的靶序列的缺陷引起的病症。
CN202110475316.6A 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统 Active CN113462671B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110475316.6A CN113462671B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
CN201811355943 2018-11-15
CN2018113559430 2018-11-15
PCT/CN2019/118702 WO2020098772A1 (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统
CN201980014005.0A CN111770992B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统
CN202110475316.6A CN113462671B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201980014005.0A Division CN111770992B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统

Publications (2)

Publication Number Publication Date
CN113462671A CN113462671A (zh) 2021-10-01
CN113462671B true CN113462671B (zh) 2023-09-12

Family

ID=70731025

Family Applications (3)

Application Number Title Priority Date Filing Date
CN201980014005.0A Active CN111770992B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统
CN202110475336.3A Pending CN113462672A (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统
CN202110475316.6A Active CN113462671B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN201980014005.0A Active CN111770992B (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统
CN202110475336.3A Pending CN113462672A (zh) 2018-11-15 2019-11-15 CRISPR-Cas12j酶和系统

Country Status (13)

Country Link
US (1) US20220002691A1 (zh)
EP (1) EP3882345A4 (zh)
JP (1) JP7460178B2 (zh)
KR (1) KR20210142586A (zh)
CN (3) CN111770992B (zh)
AU (1) AU2019381258B2 (zh)
BR (1) BR112021009330A2 (zh)
CA (1) CA3120432A1 (zh)
IL (1) IL283169A (zh)
MX (1) MX2021005723A (zh)
PH (1) PH12021551114A1 (zh)
SG (1) SG11202105121WA (zh)
WO (1) WO2020098772A1 (zh)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113811607A (zh) 2019-03-07 2021-12-17 加利福尼亚大学董事会 CRISPR-Cas效应子多肽和其使用方法
AU2020397041A1 (en) * 2019-12-04 2022-06-09 Arbor Biotechnologies, Inc. Compositions comprising a nuclease and uses thereof
US20230159943A1 (en) * 2020-04-20 2023-05-25 The Regents Of The University Of California Crispr systems in plants
CN111996236B (zh) * 2020-05-29 2021-06-29 山东舜丰生物科技有限公司 基于crispr技术进行靶核酸检测的方法
CN111690773B (zh) * 2020-06-17 2021-08-20 山东舜丰生物科技有限公司 利用新型Cas酶进行目标核酸检测的方法和系统
CN113930413A (zh) * 2020-06-29 2022-01-14 中国农业大学 新型CRISPR-Cas12j.23酶和系统
CN113881652B (zh) * 2020-11-11 2022-11-22 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
WO2022159741A1 (en) * 2021-01-22 2022-07-28 Arbor Biotechnologies, Inc. Compositions comprising a nuclease and uses thereof
CN116004572A (zh) * 2021-02-05 2023-04-25 山东舜丰生物科技有限公司 Crispr酶和系统以及应用
CN113234795B (zh) * 2021-04-15 2023-02-24 山东舜丰生物科技有限公司 利用Cas蛋白进行核酸检测的方法
WO2022253960A2 (en) * 2021-06-02 2022-12-08 University Of Copenhagen Mutant cas12j endonucleases
CN113717962B (zh) * 2021-09-10 2024-08-06 武汉艾迪晶生物科技有限公司 用于水稻基因编辑的CasΦ-2蛋白及其表达盒子和表达载体
CN114438055B (zh) * 2021-10-26 2022-08-26 山东舜丰生物科技有限公司 新型的crispr酶和系统以及应用
CN117050973B (zh) * 2021-10-29 2024-07-19 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
CN116179510A (zh) * 2022-01-29 2023-05-30 山东舜丰生物科技有限公司 Cas酶和系统以及应用
CN114507654B (zh) * 2022-04-20 2022-07-08 山东舜丰生物科技有限公司 Cas酶和系统以及应用
CN116987693A (zh) * 2022-04-25 2023-11-03 上海科技大学 一种优化的CRISPR/SpCas12f1系统、工程化向导RNA及其应用
CN116716277A (zh) * 2022-07-07 2023-09-08 山东舜丰生物科技有限公司 Cas酶及其应用
WO2024040874A1 (zh) * 2022-08-22 2024-02-29 山东舜丰生物科技有限公司 突变的Cas12j蛋白及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406838A (zh) * 2014-11-06 2017-11-28 纳幕尔杜邦公司 Rna引导的内切核酸酶向细胞中的肽介导的递送
WO2017219027A1 (en) * 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140066379A1 (en) * 2012-08-30 2014-03-06 Body Organ Biomedical Corp. Recombinant vector, transgenic fish egg using the same and biomaterial using the same
JP5774657B2 (ja) * 2013-10-04 2015-09-09 国立大学法人京都大学 エレクトロポレーションを利用した哺乳類の遺伝子改変方法
WO2015089427A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Crispr-cas systems and methods for altering expression of gene products, structural information and inducible modular cas enzymes
US9790490B2 (en) * 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
AU2016279062A1 (en) * 2015-06-18 2019-03-28 Omar O. Abudayyeh Novel CRISPR enzymes and systems
WO2017062855A1 (en) * 2015-10-09 2017-04-13 Monsanto Technology Llc Novel rna-guided nucleases and uses thereof
CN105296518A (zh) * 2015-12-01 2016-02-03 中国农业大学 一种用于CRISPR/Cas9技术的同源臂载体构建方法
CN106845151B (zh) * 2015-12-07 2019-03-26 中国农业大学 CRISPR-Cas9系统sgRNA作用靶点的筛选方法及装置
EP4159847A1 (en) * 2015-12-29 2023-04-05 Monsanto Technology LLC Novel crispr-associated transposases and uses thereof
EP3445856A1 (en) * 2016-04-19 2019-02-27 The Broad Institute Inc. Novel crispr enzymes and systems
WO2018035387A1 (en) * 2016-08-17 2018-02-22 The Broad Institute, Inc. Novel crispr enzymes and systems
CN107784200B (zh) * 2016-08-26 2020-11-06 深圳华大生命科学研究院 一种筛选新型CRISPR-Cas系统的方法和装置
CN106978428A (zh) * 2017-03-15 2017-07-25 上海吐露港生物科技有限公司 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒
CN112004932B (zh) * 2018-04-20 2023-01-10 中国农业大学 一种CRISPR/Cas效应蛋白及系统
CN112020560B (zh) * 2018-04-25 2024-02-23 中国农业大学 一种RNA编辑的CRISPR/Cas效应蛋白及系统
WO2019214604A1 (zh) * 2018-05-07 2019-11-14 中国农业大学 CRISPR/Cas效应蛋白及系统
EP3894550A4 (en) * 2018-12-14 2023-01-04 Pioneer Hi-Bred International, Inc. NEW CRISPR-CAS SYSTEMS FOR GENOME EDITING
CN113811607A (zh) * 2019-03-07 2021-12-17 加利福尼亚大学董事会 CRISPR-Cas效应子多肽和其使用方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406838A (zh) * 2014-11-06 2017-11-28 纳幕尔杜邦公司 Rna引导的内切核酸酶向细胞中的肽介导的递送
WO2017219027A1 (en) * 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
transposase, IS605 OrfB family [halophilic archaeon DL31];Lucas,S等;《Genbank Database》;ACCESSION NUMBER AEN05808.1 *

Also Published As

Publication number Publication date
EP3882345A4 (en) 2023-02-22
CN111770992A (zh) 2020-10-13
JP7460178B2 (ja) 2024-04-02
MX2021005723A (es) 2021-09-23
CN113462671A (zh) 2021-10-01
IL283169A (en) 2021-06-30
WO2020098772A1 (zh) 2020-05-22
CN113462672A (zh) 2021-10-01
JP2022518329A (ja) 2022-03-15
CN111770992B (zh) 2021-04-09
PH12021551114A1 (en) 2021-11-22
US20220002691A1 (en) 2022-01-06
AU2019381258B2 (en) 2024-02-01
EP3882345A1 (en) 2021-09-22
CA3120432A1 (en) 2020-05-22
BR112021009330A2 (pt) 2021-08-17
AU2019381258A1 (en) 2021-07-01
SG11202105121WA (en) 2021-06-29
KR20210142586A (ko) 2021-11-25

Similar Documents

Publication Publication Date Title
CN113462671B (zh) CRISPR-Cas12j酶和系统
CN113136375B (zh) 新型CRISPR/Cas12f酶和系统
AU2022275537A1 (en) Nuclease systems for genetic engineering
CN112105728B (zh) CRISPR/Cas效应蛋白及系统
CN112020560B (zh) 一种RNA编辑的CRISPR/Cas效应蛋白及系统
KR101659101B1 (ko) 박테리아 ［2Ｆｅ-2Ｓ］ 다이하이드록시산 탈수효소의 동정 및 용도
CN113015798B (zh) CRISPR-Cas12a酶和系统
CN113337533A (zh) 用于鉴定和富集包含位点特异性基因组修饰的细胞的方法和组合物
KR20150105634A (ko) 서열 조작을 위한 개선된 시스템, 방법 및 효소 조성물의 유전자 조작 및 최적화
CN113846113B (zh) 一种有限自我复制mRNA分子系统、制备方法及应用
KR20230074525A (ko) 유전자 발현을 억제하기 위한 조성물 및 방법
EP4159853A1 (en) Genome editing system and method
CN109337904B (zh) 基于C2c1核酸酶的基因组编辑系统和方法
US20240150795A1 (en) Targeted insertion via transportation
JP2023539237A (ja) カーゴヌクレオチド配列を転位させるための系および方法
KR20220066111A (ko) Dna 염기 편집을 위한 방법 및 조성물
CN114292831B (zh) 新型Cas酶以及应用
KR102114010B1 (ko) 흑부병으로부터의 셀룰로즈 및/또는 헤미셀룰로즈 분해 효소 및 이의 용도
AU2020356441A1 (en) A nucleic acid delivery vector comprising a circular single stranded polynucleotide
Brown Studying Dna
CN116615539A (zh) 抑制基因表达的组合物和方法
Collet et al. Plasmid DNA small-scale preparation based on nuclease inactivation by diethylpyrocarbonate

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant