CN110241099B - 酿脓链球菌的CRISPR核酸酶SpCas9 的截短变异体及其应用 - Google Patents

酿脓链球菌的CRISPR核酸酶SpCas9 的截短变异体及其应用 Download PDF

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CN110241099B
CN110241099B CN201910488075.1A CN201910488075A CN110241099B CN 110241099 B CN110241099 B CN 110241099B CN 201910488075 A CN201910488075 A CN 201910488075A CN 110241099 B CN110241099 B CN 110241099B
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黄强
汤洪海
杜文豪
薛冬梅
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Abstract

本发明属于蛋白质工程技术领域，具体为来源于酿脓链球菌的CRISPR核酸酶SpCas9的截短变异体及其应用。本发明中，CRISPR‑Cas9（TSpCas9）核酸酶属于CRISPR‑Cas9系统，具有野生型CRISPR‑Cas9核酸酶相当的剪切活性，所述CRISPR‑Cas9（TSpCas9）核酸酶是将野生型CRISPR‑Cas9核酸的氨基酸序列截掉120个即第180位到299位的氨基酸后重组所得；所述野生型CRISPR‑Cas9核酸酶的氨基酸序列如SEQ ID NO.7所示；或者所述CRISPR‑Cas9核酸酶含有如SEQ ID NO.7所示90%的氨基酸序列，即SEQ ID NO.15。该截短变异体可用于基因组编辑、基因打靶、基因组工程、表观基因组工程、基因治疗与体外诊断。

Description

酿脓链球菌的CRISPR核酸酶SpCas9 的截短变异体及其应用
技术领域
本发明属于蛋白质工程技术领域，具体涉及来源于酿脓链球菌(Streptococcuspyogenes) 的CRISPR核酸酶SpCas9的蛋白质工程，及其在基因组编辑、基因组打靶、表观基因组工程和体外诊断中的应用。
背景技术
由细菌免疫系统经漫长进化而产生的CRISPR-Cas9系统是一种革命性的基因编辑技术，被称为“基因魔剪”，可方便地对基因组特定基因的DNA链进行高效切割编辑，在生物医学领域有巨大的应用潜力，如进行基因组编辑、基因组打靶、表观基因组工程和体外诊断等[1-11]。
来源于酿脓链球菌(Streptococcus pyogenes)的CRISPR-Cas9核酸酶SpCas9最目前最为广泛使用的CRISPR核酸酶[12]。截至目前，该技术也被广泛应用到多个领域，包括医学研究以及生物技术等，比如细胞或动物模型的构建快速、功能基因的筛选便捷以及部分遗传疾病的治疗彻底[13-24]。
该系统的出现，大大克服了传统编辑技术耗时长的缺点，仅需数周就可以实现过去利用传统技术耗时一年之久才能完成的编辑[8]。除此之外，该技术在体内编辑不仅可以大规模、高特异地进行，而且还可以减少花费，节约成本。
虽然，CRISPR-Cas9系统的优势促使其能够广泛应用于许多领域，并最近在医学领域的研究上也异军突起，但是，许多挑战仍然悬而未决，尤其是寻找一个适合并高效将CRISPR-Cas9 靶向特异性疾病组织的运载系统，至今成为临床应用的瓶颈问题。
以现在的科学技术和认知，欲将感兴趣的基因靶向运载到体内或细胞当中，需要借助一些病毒载体，如腺病毒和慢病毒(具有致癌性)等。不过，腺病毒存在的不足(如缺少高效的包装细胞，制备复杂，滴度低的缺陷)，尤其装载量较小(小于4.7kb)的自身特性限制了将编码区大于4kp的SpCas9+sgRNA靶向运载到体内和细胞的靶标位置[25]。因此，基于这个问题，考虑将SpCas9缩小到适合的大小，以实现腺病毒将小型的SpCas9装载，并高效靶向运载到靶位点，从而以期作为临床医学治疗上的优势工具，具有极大潜在医学价值。
发明内容
本发明的目的是提供一种来源于酿脓链球菌的CRISPR-Cas9核酸酶SpCas9的截短变异体及其用途。
为实现上述目的，本发明采用以下技术方案。
本发明提供一种体积比野生型小的CRISPR-Cas9核酸酶(TSpCas9)，其是将野生型的 SpCas9第180位到299位氨基酸截掉所得，属于CRISPR-Cas9系统，具有野生型CRISPR-Cas9 (SpCas9)相当的剪切活性。
所述野生型SpCas9核酸酶的核苷酸序列和氨基酸序列分别为SEQ ID NO.6和SEQID NO.7 所示。
所述CRISPR-Cas9核酸酶(TSpCas9)的核苷酸序列和氨基酸序列分别为SEQ IDNO.15和SEQ ID NO.16所示，与野生型SpCas9的相似度达90％以上。
本发明还提供一种多核苷酸序列，可以转录和翻译所述的CRISPR-Cas9核酸酶(TSpCas9)。
本发明还提供一种表达载体，其含有上述多核苷酸序列。
本发明还提供一种宿主细胞，可以用于转化上述表达载体。
本发明还提供一种制备所述CRISPR-Cas9核酸酶(TSpCas9)的方法，具体步骤包括:首先，构建所述CRISPR-Cas9核酸酶的多核苷酸序列表达载体；然后，将所述表达载体转化至宿主细胞，筛选并挑出单克隆；最后，将所述单克隆诱导表达，并通过亲和层析、离子交换等方法从表达产物中分离出所述的CRISPR-Cas9核酸酶。
本发明还提供上述CRISPR-Cas9核酸酶、多核苷酸序列以及表达载体均可作为基因组编辑工具用途，用于基因组DNA片段的相关编辑。
本发明中所述的编辑可以是单点编辑、也可以是编辑位点大于等于两个的多点编辑。
所述编辑手段包括删除、突变、插入、倒位、移位、重复或易位。
所述CRISPR-Cas9编辑工具包括与靶标DNA片段匹配的引导sgRNA。
所述的CRISPR-Cas9核酸酶与能够介导它的sgRNA组合，能够对基因进行编辑。
与现有技术相比，本发明的CRISPR-Cas9(TSpCas9)，属于CRISPR-Cas9免疫系统，含有如:SEQ ID NO.15和SEQ ID NO.16的核苷酸序列和氨基酸序列，其体积比野生型CRISPR-Cas9 (SpCas9)小120个氨基酸，剪切DNA的活性与野生型CRISPR-Cas9(SpCas9)相当，有望方便腺病毒将其运载到细胞和动物体的靶位点，具有潜在的生物医学应用价值。本发明截短变异体可用于基因组编辑、基因打靶、基因组工程、表观基因组工程、基因治疗与体外诊断。
附图说明
图1.含有TSpCas9质粒的构建策略。
图2.含有TSpCas9的质粒转化及单克隆筛选培养。
图3.含有TSpCas9的质粒筛选。
图4.含有TSpCas9的质粒测序。
图5.TSpCas9目标蛋白的纯化方法。
图6.TSpCas9目标蛋白纯化获取的过程。
图7.TSpCas9目标蛋白的电泳鉴定。
图8.引导sgRNA的在靶和脱靶序列。
图9.野生型SpCas9和截短型TSpCas9体外剪切活性的检测。
图10.野生型SpCas9体外脱靶效应的检测。
图11.截短型TSpCas9体外脱靶效应的检测。
具体实施方式
下述实施例中所用的实验方法，如无特定说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特定说明，均为从商业途径获得。
一、CRISPR-Cas9核酸酶
本发明的CRISPR-Cas9核酸酶(TSpCas9)，其是将野生型的SpCas9第180位到299位氨基酸截掉所得，属于CRISPR-Cas9系统，具有野生型CRISPR-Cas9(SpCas9)相当的剪切活性；
所述CRISPR-Cas9核酸酶(TSpCas9)含有如SEQ ID NO.16所示的氨基酸序列。
二、编码CRISPR-Cas9核酸酶的多核苷酸
转录和翻译所述CRISPR-Cas9(TSpCas9)的多核苷酸序列，包括DNA或RNA。DNA还可以细分为质粒DNA、基因组DNA或人工合成的DNA；
编码所述CRISPR-Cas9(TSpCas9)的多核苷酸序列，可以利用该领域科研或技术人员所熟悉的相关分子生物学技术来制备，其不局限于重组DNA技术和化学合成方法。
三、表达载体
所述表达载体含有编码所述CRISPR-Cas9核酸酶(TSpCas9)的多核苷酸序列。该表达载体可以通过科研或技术人员所熟悉的分子生物学方法来构建，包括DNA重组技术和DNA合成技术等，主要将CRISPR-Cas9核酸酶(TSpCas9)的DNA有效连接到载体上的多克隆位点中，然后通过转录翻译等过程表达目的蛋白TSpCas9。
四、宿主细胞
所述宿主细胞可以用于表达CRISPR-Cas9核酸酶的重组质粒所转化。宿主细胞主要包括原核细胞(如细菌)，低等真核细胞(如酵母)，高等真核细胞(如哺乳动物细胞)等。常用的宿主细胞如大肠杆菌DH5α、毕赤酵母、HEK293、CHO、Hela细胞等。
五、CRISPR-Cas9核酸酶(TSpCas9)及其编码该酶的核苷酸序列和所述表达载体的用途
本发明的CRISPR-Cas9核酸酶(TSpCas9)及其编码该酶的多核苷酸序列和所述的表达载体能够用于基因组DNA片段的编辑或用于制备基因编辑工具。CRISPR-Cas9核酸酶(TSpCas9) 编辑包括单位点和多位点编辑，其编辑手段包括删除、突变、插入、倒位、移位、重复或易位等。
六、基因编辑工具及其方法
本发明的基因编辑工具属于CRISPR-Cas9系统，CRISPR-Cas9(TSpCas9)在特定的sgRNA 的引导下可以在目的基因DNA片段PAM(NGG)位点上游3到4位间剪切底物DNA片段。该编辑过程可以在体内或体内进行。当sgRNA是单个的时候可以进行单点编辑，当sgRNA是两个或两个以上时可以进行多位点编辑。
如本发明的一些实施方式中所列举的，CRISPR-Cas9核酸酶(TSpCas9)在sgRNA的引导下，可以在体外对底物DNA(920bp)进行剪切，其产物为760bp和260bp。
在本发明中，Cas9可作为CRISPR-Cas9核酸酶的简称使用，其含义与CRISPR-Cas9核酸酶相同。本发明中的截短突变体蛋白为TSpCas9，即对野生型SpCas9第180位氨基酸到299 位氨基酸对应的质粒片段去掉，将剩余部分氨基酸的质粒重组连接并表达得到的蛋白。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围并不局限为下述特定的具体实施方案，还应理解为，本发明实施例中的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下例实施例中未注明具体条件的试验方法，tongchang按照常规条件操作，或者按照各生产厂商所建议的条件操作。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、重组DNA技术及相关领域的常规技术。
实施例1，构建CRISPR-Cas9核酸酶(TSpCas9)的质粒。
1.突变体的设计。
以pet21-6His-TEV-SpCas9质粒，即SEQ ID NO.5为模板，截掉180aa(5822bp)～299aa (6181bp)，相当于碱基序列SEQ ID NO.8和氨基酸序列SEQ ID NO.9，把剩下部分重组成截短的SpCas9，将其称为TSpCas9。其改造设计思路如图1所示，其详细步骤简述如下：
首先，利用引物F-F和F-R，S-F和S-R分别在质粒Pet21-6His-TEV-SpCas9(相当于SEQ ID NO.5)上扩增5267～5821bp(相当于SEQ ID NO.10的1-538bp)和6182～9391bp(相当于 SEQ ID NO.12的898-4104bp)，并通过AxyPrepTM DNA Gel Extraction Kit(购于Axygen) 纯化回收，将它们称为扩增片段F和S；
其次，使用限制性内切酶NdeI和XhoI(购于NEB)分别酶切pet21-6His-TEV-SpCas9和F扩增片段，并通过AxyPrepTM DNA Gel Extraction Kit纯化回收；
再次，使用T4ligase(购于Takara)将上一步纯化回收的两个片段连接，将此连接产物称为pet21-6His-TEV-F；
最后，用XhoI和NotI(购于NEB)分别酶切pet21-6His-TEV-F和S扩增片段，并通过AxyPrepTM DNA Gel Extraction Kit纯化回收，T4ligase连接，即可获得目的产物TSpCas9。
(1)采购扩增试剂盒
所用扩增试剂盒Fast HiFidelity PCR Kit从天根生化科技(北京)有限公司订购。
(2)采购引物
所用引物均从上海生工生物工程有限公司订购，引物的5’端均加有所需的酶切位点和该酶切位点的保护碱基。它们的序列如下：
扩增5267-5821bp的正反引物：
F：GGAATTCCATATGGAAAATCTCTACTTCCAAG(SEQ ID NO.1)
R：CCGCTCGAGCGGCCGCTGTTGTCTGGGTTCAGGT(SEQ ID NO.2)
扩增6182-9391bp的正反引物：
F：ATAAGAATGCGGCCGCATTCTGCTGAGTGATATTCTGCG(SEQ ID NO.3)
R：CCGCTCGAGTCAGTCTCCACCGAGCTGAG(SEQ ID NO.4)。
扩增体系如下：
Figure BDA0002086056490000051
PCR反应条件：
Figure BDA0002086056490000052
Figure BDA0002086056490000061
扩增体系如下：
Figure BDA0002086056490000062
PCR反应条件：
Figure BDA0002086056490000063
(3)割胶回收试剂盒采购
所用割胶回收试剂盒AxyPrepTM DNA Gel Extraction Kit从Axygen公司(公司链接)订购，割胶回收操作均按其说明书进行。
(4)限制内切酶及T4连接酶
所用限制内切酶NdeI、XhoI、NotI和T4连接酶均从NEB公司订购(公司链接)订购。
酶切反应体系
Figure BDA0002086056490000071
按该反应体系37℃孵育至少2小时，并电泳割胶纯化回收。
链接反应体系
Figure BDA0002086056490000072
该反应体系16℃孵育至少2小时，并转化大肠杆菌DH5α筛选单克隆pet21-6His-TEV-F，如图2所示，其核苷酸序列为SEQ ID NO.14，培养细菌，抽提质粒，抽提方法见Qiagen质粒小提试剂盒使用说明。
Figure BDA0002086056490000073
链接反应体系
Figure BDA0002086056490000074
Figure BDA0002086056490000081
该反应体系16℃孵育至少2小时，并转化大肠杆菌DH5α筛选单克隆TSpCas9，如图2所示，其核苷酸序列和氨基酸分别为序列为SEQ ID NO.15和SEQ ID NO.16。
其筛选结果如图3示，通过常规质粒抽提方法获取12个样品质粒(1到12号)，其大小应该在参照质粒1(SEQ ID NO.5)和2(SEQ ID NO.14)之间，利用琼脂糖凝胶电泳检测，我们发现样品质粒8和10符合要求；随后，通过一代测序，发现样品质粒10构建正确，其结果如图4所示。
实施例2，制备CRISPR-Cas9(TSpCas9)核酸酶。
2.蛋白表达与纯化。
2.1蛋白表达
(1)打开超净台，用含75％酒精的棉球擦拭桌面以及各种器具，开紫外灯照射20min，启动风机备用；
(2)移液枪吸10μl表达Pet21-6His-TEV-TSpCas9的Rosetta(DE3)(购于TIANGEN)菌液转至6ml含有双抗(Amp与Cm)的LB液体培养基中，37℃，200r/min振荡培养过夜；
(3)将过夜培养的菌液按照体积比为1：100转至500ml含双抗的LB(购于生工)液体培养基中，37℃，200r/min振荡培养。在培养过程中，随时检测菌液的OD值；
(4)当菌液的OD值接近0.4～0.8时，加入蛋白诱导剂IPTG，使其终浓度为0.1mM，然后16℃，200r/min振荡培养20h；
(5)收集菌液，5000r/min离心5min使菌体沉淀，弃上清，并称重 Pet21-6His-TEV-TSpCas9菌体。
2.2蛋白纯化
所述蛋白纯化主要通过镍柱亲和层析技术，如图5所示；其纯化过程包括菌体破碎、蛋白样品离心收集、蛋白样品与镍柱介质共孵育以及目的蛋白的洗脱等，如图6所示。其详细步骤简述如下：
(1)向菌体中加入预先冰浴且PMSF终浓度为0.1mM的裂解液(20mM HEPES，500mMKCl，pH7.5；1g菌体加入5ml)，涡旋仪重悬使菌块分散混匀，细胞超声破碎仪破碎细胞，超声3 sec停3sec，一次10min，超声两次，超声过程均在冰浴中进行；
(2)向破碎的菌液中加入终浓度为10μg/ml RNase(生工)，5μg/ml DNase I(生工)，冰浴处理30min后，4℃10000r/min离心45～60min，收集上清；
(3)将上清与预先用平衡液(20mM HEPES，500mMKCl，1％蔗糖，pH7.5)处理的Qiagen Ni-NTA介质孵育，此过程在冰浴上进行，并加以振荡(150r/min)，1.5h后静置，待Qiagen Ni-NTA 沉淀；
(4)将Qiagen Ni-NTA装载到重力柱中，BioLogic LP系统的监测下，分别以流速为2 ml/min的平衡液和洗脱液(20mM HEPES，500mMKCl，500mM咪唑，1％蔗糖，pH7.5)， 20、30、40、50、100、250、500Mm洗脱液冲洗Qiagen Ni-NTA，并收集蛋白；
(5)将不同咪唑浓度下的蛋白溶液跑SDS-PAGE(购于EpiZyme Scientific)电泳，考马斯亮蓝染色，脱色剂脱色，观察目的蛋白的表达和挂柱效果。
所述蛋白TSpCas9的纯化结果如图7所示，其显示该目的蛋白TSpCas9的表达和纯化情况，从上清液观察，目的蛋白TSpCas9表达较多，从穿流液观察，Ni-NTA吸附的能力适中，从洗脱液观察，50和100mM咪唑洗脱液下的目的蛋白TSpCas9相对较纯，可以收集浓缩做活性检测。
实施例3，检验CRISPR-Cas9(TSpCas9)核酸酶剪切活性。
3.突变体活性检测。
所用底物DNA(SEQ ID NO.20)，主要利用引物QG-F：TAGTCCTGTCGGGTTTCG(SEQ IDNO.17) 和QG-R：TTCCATTCGCCATTCAGG(SEQ ID NO.18)通过常规PCR扩增获取。其反应体系和扩增条件如下：
扩增体系如下：
Figure BDA0002086056490000091
PCR反应条件：
Figure BDA0002086056490000101
(3)割胶回收试剂盒采购
所用割胶回收试剂盒AxyPrepTM DNA Gel Extraction Kit从Axygen公司订购，割胶回收操作均按其说明书进行，可以获得较纯的底物DNA(SEQ ID NO.20)。
Cas9与sgRNA以等摩尔混合，而根据实验需要，底物DNA可调为Cas9摩尔质量的0.2～1 倍。其反应体系如下
Figure BDA0002086056490000102
将反应体系置37℃孵育，1h后70℃作用10min，最后琼脂糖凝胶电泳检测评价目的蛋白TSpCas9的体外切割活性。其结果如图9所示，从图9可以发现，与野生型SpCas9相比，截短型TSpCas9在体外能够剪切底物DNA(SEQ ID NO.20)(泳道4)，生成产物1和产物2，其剪切情况与野生型SpCas9基本一致，由此说明截短型TSpCas9的剪切活性和野生型SpCas9 的剪切活性相当。虽然TSpCas9在体外的DNA剪切活性并不比野生型SpCas9的强，但是其体积在小型化上却比野生型SpCas9有优势，即我们的TSpCas9体积比野生型SpCas9小，对方便腺病毒AAV运输而言，比野生型SpCas9有较大优势。
实施例4，CRISPR-Cas9(TSpCas9)体外脱靶检测的评价方法
4.脱靶效应检测
利用不同的sgRNA，如图8中1到8号所示，引导TSpCas9的体外切割活性来评价TSpCas9 的脱靶效应，其反应体系如下：
Figure BDA0002086056490000111
首先，评价野生型SpCas9在体外的脱靶效应，如图10所示，与0号sgRNA引导的SpCas9 剪切活性相比(泳道3)，1到8号sgRNA引导的SpCas9均能够在体外剪切底物DNA(SEQID NO.20)(泳道4到11)，生成产物1和产物2。尽管只有部分sgRNA的引导活性强，即1到4 号sgRNA引导的SpCas9体外剪切活性比较强(泳道4到7)，5到8号sgRNA引导的SpCas9 体外剪切活性却依次减弱(8号到11号)，但是，该结果依然反应野生型SpCas9在体外的脱靶效应比较严重。由此说明，野生型SpCas9有较强的脱靶效应，尤其在1到4号sgRNA的引导下更为突出。
其次，评价截短型TSpCas9在体外的脱靶效应，如图11所示，与0号sgRNA引导的TSpCas9 剪切活性相比(泳道3)，1到8号sgRNA引导的截短型TSpCas9均能够在体外剪切底物DNA(SEQ ID NO.20)(泳道4到11)，生成产物1和产物2。虽然1到8号引导sgRNA整体上引导截短型TSpCas9的体外剪切活性比0号sgRNA引导的弱，但是该结果也不能排除截短型TSpCas9的脱靶效应减弱。由此说明截短型TSpCas9依然存在脱靶效应。
虽然，在脱靶效应上，截短型TSpCas9并不比野生型SpCas9优，但是，就小型化而言，我们获得的截短型TSpCas9的体积比野生型SpCas9小，方便腺病毒AAV运输，比野生型SpCas9 有较大优势。这对将来CRISPR-Cas9系统方便应用到临床医学上提供了潜在的价值。
参考文献
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序列表
<110> 复旦大学
<120> 酿脓链球菌的CRISPR核酸酶SpCas9 的截短变异体及其应
<130> 001
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggaattccat atggaaaatc tctacttcca ag 32
<210> 2
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccgctcgagc ggccgctgtt gtctgggttc aggt 34
<210> 3
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ataagaatgc ggccgcattc tgctgagtga tattctgcg 39
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccgctcgagt cagtctccac cgagctgag 29
<210> 5
<211> 9553
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160
cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca 5220
tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggaaa atctctactt 5280
ccaaggcgac aagaagtact ccattgggct cgatatcggc acaaacagcg tcggctgggc 5340
cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga 5400
tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagacggc 5460
cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat 5520
ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca 5580
taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt 5640
tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag 5700
gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc 5760
gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag 5820
cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa 5880
cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc 5940
ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg 6000
taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc 6060
cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct 6120
ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc 6180
cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc 6240
tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt 6300
cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta 6360
cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat 6420
cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt 6480
gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact 6540
gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa 6600
gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa 6660
ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga 6720
ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga 6780
taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac 6840
agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt 6900
cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt 6960
taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga 7020
aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa 7080
aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat 7140
tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta 7200
cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg 7260
ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat 7320
cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga 7380
tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag 7440
tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca 7500
gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat 7560
cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga 7620
aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca 7680
cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg 7740
cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga 7800
tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag 7860
atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat 7920
gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa 7980
tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagcaggct tcatcaaaag 8040
gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat 8100
gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa 8160
gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa 8220
caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa 8280
aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag 8340
gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta 8400
cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa 8460
gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga 8520
tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga 8580
agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct 8640
gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt 8700
cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag 8760
cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat 8820
cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc 8880
caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga 8940
gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc 9000
cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt 9060
ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag 9120
agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga 9180
taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg 9240
cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac 9300
aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag 9360
aatcgacctc tctcagctcg gtggagactg actcgagcac caccaccacc accactgaga 9420
tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata 9480
actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg 9540
aactatatcc gga 9553
<210> 6
<211> 4104
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agac 4104
<210> 7
<211> 1368
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 8
<211> 360
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac 60
ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc 120
cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt 180
aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc 240
gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg 300
gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc 360
<210> 9
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu
1 5 10 15
Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile
20 25 30
Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala
35 40 45
Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala
50 55 60
Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala
65 70 75 80
Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu
85 90 95
Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu
100 105 110
Ala Ala Lys Asn Leu Ser Asp Ala
115 120
<210> 10
<211> 537
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagc 537
<210> 11
<211> 179
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser
<210> 12
<211> 3207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct 60
agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc 120
agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac 180
gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc 240
ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg 300
cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg 360
cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag 420
attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat 480
tccagattcg cgtggatgac tcgcaaatca gaagagacca tcactccctg gaacttcgag 540
gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat 600
aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca 660
gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc 720
ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt 780
accgtgaaac agctcaaaga agactatttc aaaaagattg aatgtttcga ctctgttgaa 840
atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa 900
atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt 960
gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac 1020
gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg 1080
gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc 1140
ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat 1200
gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt 1260
cttcacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag 1320
accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc 1380
gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa 1440
aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac 1500
ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc 1560
agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat 1620
catatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga 1680
tccgataaaa atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg 1740
aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat 1800
ctgactaagg ctgaacgagg tggcctgtct gagttggata aagcaggctt catcaaaagg 1860
cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg 1920
aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag 1980
tctaagctgg tctcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac 2040
aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa 2100
aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg 2160
aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac 2220
agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag 2280
cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat 2340
ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa 2400
gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg 2460
atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc 2520
gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc 2580
gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc 2640
gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc 2700
aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag 2760
ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc 2820
agccactatg aaaagctcaa agggtctccc gaagataatg agcagaagca gctgttcgtg 2880
gaacaacaca aacactacct tgatgagatc atcgagcaaa taagcgaatt ctccaaaaga 2940
gtgatcctcg ccgacgctaa cctcgataag gtgctttctg cttacaataa gcacagggat 3000
aagcccatca gggagcaggc agaaaacatt atccacttgt ttactctgac caacttgggc 3060
gcgcctgcag ccttcaagta cttcgacacc accatagaca gaaagcggta cacctctaca 3120
aaggaggtcc tggacgccac actgattcat cagtcaatta cggggctcta tgaaacaaga 3180
atcgacctct ctcagctcgg tggagac 3207
<210> 13
<211> 1069
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala
1 5 10 15
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
20 25 30
Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr
35 40 45
Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile
50 55 60
Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile
65 70 75 80
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg
85 90 95
Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro
100 105 110
His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu
115 120 125
Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile
130 135 140
Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
145 150 155 160
Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
165 170 175
Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe
180 185 190
Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val
195 200 205
Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu
210 215 220
Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe
225 230 235 240
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
245 250 255
Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
260 265 270
Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe
275 280 285
Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp
290 295 300
Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile
305 310 315 320
Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
325 330 335
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu
340 345 350
Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile
355 360 365
Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu
370 375 380
Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp
385 390 395 400
Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly
405 410 415
Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro
420 425 430
Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
435 440 445
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met
450 455 460
Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
465 470 475 480
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile
485 490 495
Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
500 505 510
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu
515 520 525
Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro
530 535 540
Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg
545 550 555 560
Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val
565 570 575
Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu
580 585 590
Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly
595 600 605
Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu
610 615 620
Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met
625 630 635 640
Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
645 650 655
Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln
660 665 670
Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
675 680 685
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys
690 695 700
Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
705 710 715 720
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys
725 730 735
Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
740 745 750
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly
755 760 765
Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
770 775 780
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
785 790 795 800
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn
805 810 815
Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr
820 825 830
Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala
835 840 845
Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu
850 855 860
Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile
865 870 875 880
Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
885 890 895
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys
900 905 910
Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
915 920 925
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu
930 935 940
Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
945 950 955 960
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu
965 970 975
Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
980 985 990
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu
995 1000 1005
Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1010 1015 1020
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1025 1030 1035 1040
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu
1045 1050 1055
Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1060 1065
<210> 14
<211> 5986
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160
cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca 5220
tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggaaa atctctactt 5280
ccaaggcgac aagaagtact ccattgggct cgatatcggc acaaacagcg tcggctgggc 5340
cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga 5400
tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagacggc 5460
cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat 5520
ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca 5580
taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt 5640
tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag 5700
gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc 5760
gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag 5820
ctgactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 5880
agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 5940
acgggtcttg aggggttttt tgctgaaagg aggaactata tccgga 5986
<210> 15
<211> 7824
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggcgacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcatt 540
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 600
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 660
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 720
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 780
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 840
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 900
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 960
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1020
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1080
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1140
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1200
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1260
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1320
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1380
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1440
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1500
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1560
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1620
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 1680
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 1740
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 1800
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 1860
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 1920
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 1980
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2040
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2100
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2160
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2220
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2280
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2340
actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 2400
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2460
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2520
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2580
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 2640
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 2700
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 2760
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 2820
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 2880
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 2940
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3000
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3060
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3120
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3180
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3240
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3300
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3360
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3420
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3480
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3540
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3600
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 3660
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 3720
gacctctctc agctcggtgg agacggcgac aagaagtact ccattgggct cgatatcggc 3780
acaaacagcg tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc 3840
aaagttctgg gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg 3900
ttcgactccg gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat 3960
acccgcagaa agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag 4020
gtggatgact ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag 4080
cacgagcgcc acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac 4140
ccaaccatat atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg 4200
ttgatctatc tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg 4260
gacctgaacc cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac 4320
aatcagcttt tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg 4380
agcgctaggc tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag 4440
aagaagaacg gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt 4500
aaatctaact tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat 4560
gatgatctcg acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg 4620
gcaaagaacc tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc 4680
accaaagctc cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg 4740
actttgctga aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc 4800
gatcagtcta aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt 4860
tacaaattta ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag 4920
cttaacagag aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac 4980
cagattcacc tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt 5040
ttgaaagata acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta 5100
ggccccctcg cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc 5160
atcactccct ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc 5220
gaactgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 5280
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 5340
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 5400
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 5460
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 5520
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 5580
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 5640
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 5700
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 5760
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 5820
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 5880
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 5940
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 6000
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 6060
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 6120
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 6180
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 6240
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 6300
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 6360
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 6420
actaaggctg aacgaggtgg cctgtctgag ttggataaag caggcttcat caaaaggcag 6480
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 6540
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 6600
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 6660
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 6720
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 6780
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 6840
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 6900
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 6960
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 7020
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 7080
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 7140
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 7200
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 7260
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 7320
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 7380
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 7440
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 7500
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 7560
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 7620
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 7680
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 7740
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 7800
gacctctctc agctcggtgg agac 7824
<210> 16
<211> 1248
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Gly Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
180 185 190
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
195 200 205
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
210 215 220
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
225 230 235 240
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
245 250 255
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
260 265 270
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
275 280 285
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
290 295 300
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
305 310 315 320
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
325 330 335
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
340 345 350
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
355 360 365
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
370 375 380
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
385 390 395 400
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
405 410 415
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
420 425 430
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
435 440 445
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
450 455 460
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
465 470 475 480
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
485 490 495
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
500 505 510
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
515 520 525
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
530 535 540
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
545 550 555 560
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
565 570 575
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
580 585 590
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
595 600 605
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
610 615 620
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
625 630 635 640
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
645 650 655
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
660 665 670
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
675 680 685
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
690 695 700
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His
705 710 715 720
Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
725 730 735
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser
740 745 750
Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn
755 760 765
Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu
770 775 780
Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln
785 790 795 800
Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp
805 810 815
Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
820 825 830
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys
835 840 845
Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala
850 855 860
His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
865 870 875 880
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr
885 890 895
Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
900 905 910
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
915 920 925
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
930 935 940
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe
945 950 955 960
Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
965 970 975
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro
980 985 990
Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
995 1000 1005
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1010 1015 1020
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val
1025 1030 1035 1040
Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys
1045 1050 1055
Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1060 1065 1070
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn
1075 1080 1085
Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn
1090 1095 1100
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1105 1110 1115 1120
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln
1125 1130 1135
Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1140 1145 1150
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1155 1160 1165
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1170 1175 1180
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala
1185 1190 1195 1200
Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1205 1210 1215
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile
1220 1225 1230
Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1235 1240 1245
<210> 17
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tagtcctgtc gggtttcg 18
<210> 18
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttccattcgc cattcagg 18
<210> 19
<211> 3046
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 60
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 120
agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 180
ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 240
gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 300
gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 360
tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 420
acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 480
aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 540
cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 600
gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 660
cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 720
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 780
tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 840
ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 900
tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 960
gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1020
ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1080
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1140
agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1200
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1260
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1320
agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1380
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1440
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1500
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1560
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1620
gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1680
ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1740
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 1800
acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 1860
gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 1920
cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 1980
gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 2040
gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 2100
gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 2160
agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc 2220
gctctagaac tagtggatcc cccgggctgc aggaattcga tatcaagctt atcgattacc 2280
gctccagtcg ttcatgaggt tagagctaga aatagcaagt taaaataagg ctagtccgtt 2340
atcaacttga aaaagtggca ccgagtcggt gctctcgagg gggggcccgg tacccaattc 2400
gccctatagt gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga 2460
aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 2520
taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 2580
atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 2640
tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 2700
gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 2760
tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 2820
ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 2880
agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 2940
aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 3000
accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtg 3046
<210> 20
<211> 920
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 60
ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 120
ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 180
taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 240
agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 300
gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 360
cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 420
ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 480
ccatgattac gccaagctcg aaattaaccc tcactaaagg gaacaaaagc tggagctcca 540
ccgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat tcgatatcaa 600
gcttatcgat taccgctcca gtcgttcatg aggttagagc tagaaatagc aagttaaaat 660
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctctc gagggggggc 720
ccggtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 780
cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 840
ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 900
agcctgaatg gcgaatggaa 920

Claims (11)

1.一种CRISPR-Cas9核酸酶，属于CRISPR-Cas9系统，具有与野生型CRISPR-Cas9核酸酶相当的剪切活性，所述CRISPR-Cas9核酸酶是将野生型CRISPR-Cas9核酸酶的第180位到299位的氨基酸截掉后所得；所述野生型CRISPR-Cas9核酸酶来源于酿脓链球菌，其氨基酸序列如SEQ ID NO.7所示；或者所述CRISPR-Cas9核酸酶的氨基酸如SEQ ID NO.16所示。
2.一种多核苷酸序列，其编码权利要求1中所述的CRISPR-Cas9核酸酶。
3.一种表达载体，其特征在于含有如权利要求2所述的多核苷酸序列。
4.一种宿主细胞，其是经过权利要求3所述的表达载体转化后得到。
5.一种制备权利要求1所述的CRISPR-Cas9核酸酶的方法，其特征在于,包括以下步骤：首先,构建含有权利要求2所述的多核苷酸序列的表达载体；将所述表达载体转化宿主细胞，筛选并挑出单克隆；将筛选得到的单克隆细胞诱导表达，并通过亲和层析或/和离子交换方法从表达产物中分离出所述的CRISPR-Cas9核酸酶。
6.权利要求1所述的CRISPR-Cas9核酸酶、权利要求2中所述的多核苷酸序列或权利要求3中所述的表达载体作为基因编辑工具的用途，所述的用途不包括疾病的治疗与诊断。
7.根据权利要求6所述的用途，其特征在于，所述编辑是单点编辑，或者是编辑位点大于等于两个的多点编辑;所述编辑方式选自基因的删除、突变、插入、倒位、移位、重复或易位。
8.根据权利要求6所述的用途，其特征在于，所述基因编辑工具还包括与靶标DNA片段匹配的引导sgRNA。
9.根据权利要求6所述的用途，其特征在于，利用权利要求1所述的CRISPR-Cas9核酸酶与能够介导它的sgRNA组合，对基因进行编辑。
10.根据权利要求6所述的用途，其特性在于，将权利要求3所述的载体和与之匹配的引导sgRNA一同转入宿主细胞，对基因进行编辑。
11.根据权利要求7所述的用途，其特性在于，所述的单点或多点的基因编辑是利用权利要求1所述CRISPR-Cas9核酸酶对双链DNA进行剪切，并通过宿主细胞的修复系统对断裂的缺口进行修复。
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