CN108239642B

CN108239642B - Adipocyte differentiation related long non-coding RNA and application thereof

Info

Publication number: CN108239642B
Application number: CN201611203679.XA
Authority: CN
Inventors: 杜权; 易凡; 张沛
Original assignee: Peking University
Current assignee: Transsion Biomedicine (Suzhou) Co.,Ltd.
Priority date: 2016-12-23
Filing date: 2016-12-23
Publication date: 2021-07-30
Anticipated expiration: 2036-12-23
Also published as: CN108239642A

Abstract

The invention provides a long non-coding RNA slincRAD related to adipocyte differentiation, wherein the RNA is combined with DNMT1 protein and utilizes a PPAR gamma and other signal paths to regulate adipocyte differentiation and adipose tissue development. The expression of slincRAD can be inhibited by using siRNA or shRNA targeting slincRAD, so that the differentiation process of fat is specifically inhibited. Transgenic mice knocked-down slincRAD show better blood glucose tolerance and have positive effects on blood lipid metabolism.

Description

Adipocyte differentiation related long non-coding RNA and application thereof

Technical Field

The invention relates to the field of physiology and biomedicine, in particular to a long-chain non-coding RNA with an adipocyte differentiation regulating function and application thereof.

Background

Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with transcripts greater than 200bp in length and relatively long nucleotide chains. LncRNA has the characteristics of multiple types, multiple action modes, multiple quantity and the like, so that the LncRNA has abundant information, has various molecular mechanisms participating in expression regulation, and realizes the regulation of gene expression mainly from three aspects of epigenetics, transcription level and post-transcription level. LncRNA may have functions mainly in several aspects: (1) interfering with the expression of downstream genes by transcription occurring in the promoter region upstream of the protein-encoding gene; (2) affecting downstream gene expression by inhibiting RNA polymerase I I or mediating chromatin remodeling and histone modification; (3) by forming complementary double strands with the transcript of the protein-encoding gene, the cleavage of mRNA is disturbed, thereby generating different cleavage patterns; (4) forming complementary double chains with the transcript of the protein coding gene, further generating endogenous siRNA under the action of Dicer enzyme, and regulating the expression level of the gene; (5) by binding to a specific protein, lncRNA transcripts are capable of modulating the activity of the corresponding protein; (6) forming a nucleic acid-protein complex with a protein as a structural component; (7) altering the cytoplasmic localization of a particular protein by binding to the protein; (8) as a precursor molecule for small RNA molecules, such as miRNA, piRNA. Just because lncRNA can participate in the regulation of protein coding genes from the transcription and post-transcription levels, the genes comprise disease occurrence related genes, development related genes and the like, and the lncRNA can influence both intracellular signal transduction pathways and signal transduction pathways in the development process of organisms by participating in the regulation of the protein coding genes. It has been reported that lncRNA can affect the occurrence and development of various diseases, including nervous system diseases, cardiovascular diseases and various tumor and cancer diseases.

In recent years, the research on long non-coding RNA has attracted much attention, and the research on lncRNA related to adipocyte differentiation is very limited. The exploration of new lncRNA playing an important role in fat differentiation not only reveals an important mechanism for regulating and controlling the function of fat cells, but also provides a new detection or treatment target point, and is an important scientific problem to be researched and developed urgently in fat metabolism-related diseases.

Disclosure of Invention

The invention aims to provide long non-coding RNA slincRAD related to the differentiation function of adipocytes, and the slincRAD is used for regulating the proliferation and differentiation behaviors of the adipocytes, and a mice model with the slincRAD knocked down is established and is used for the research of screening of drugs for fat metabolism related diseases and the like.

Knocking down the expression of slincRAD in cells and mice leads to the reduction of the expression of adipocyte differentiation related genes, the influence on the development of adipose tissues and the weight loss.

The invention firstly provides application of long-chain non-coding RNA slincRAD in preparation of a preparation or a medicine for promoting adipocyte differentiation, wherein a long-chain non-coding RNA sequence is shown in SEQ ID No. 300.

Secondly, the invention provides an inhibitor for inhibiting the expression of long-chain non-coding RNA slincRAD, which comprises small interfering ribonucleic acid (siRNA) and small hairpin structure RNA (shRNA), and the sequences are shown in a table 2 and a table 3.

Further, the invention provides application of shRNA and siRNA serving as a long-chain non-coding RNA slincRAD expression inhibitor in preparation of a medicament for inhibiting adipocyte differentiation.

Again, the invention provides an oligonucleotide for detecting long non-coding RNA slincRAD, selected from the group consisting of:

detection primer combination 01:

01 forward primer: 5' -ATCCATCTCCCCTACCCATC (SEQ ID NO:200)

01 reverse primer: 5' -TGCAGAGGACTCACAACTGG (SEQ ID NO:201)

Detection primer combination 08:

08 Forward primer: 5' -GGTGCAGTTTTGCACTGAGA (SEQ ID NO:202)

08 reverse primer: 5' -GATGGAGAGAAAGGAAGGGG (SEQ ID NO:203)

Detection primer combination 16:

16 forward primer: 5' -CCTAGTGCCTTTCTGCCTTG (SEQ ID NO:204)

16 reverse primer: 5' -TGGGGTGATTGTGTAGGGAT (SEQ ID NO:205)

Detection primer combination 31:

31 forward primer: 5' -CAAGTGGTTCCGGATTCTGT (SEQ ID NO:206)

31 reverse primer: 5' -CCGTGAAATGAAGTGGTGTG (SEQ ID NO:207)

Detection primer combination 54:

54 forward primer: 5' -GCATTTAATTGGTGCTGGCT (SEQ ID NO:208)

54 reverse primer: 5' -GACCCTTCTCCTAGCTGCCT (SEQ ID NO:209)

Further, the invention provides application of a reagent for detecting the expression level of the long-chain non-coding RNA slincRAD in preparation of a fat differentiation promoter or inhibitor.

In a fourth aspect, the invention provides a transgenic mouse model for knocking down long non-coding RNA slincRAD, a preparation method of the mouse model and application of the mouse model in fat metabolism-related disease drug screening. The fat metabolism related diseases include but are not limited to metabolic syndrome, obesity, hyperglycemia, hyperlipidemia, diabetes.

slincRAD promotes differentiation of mouse 3T3-L1 cells, probably via the PPAR γ signaling pathway.

Therefore, slincRAD can be used for promoting the differentiation of fat cells, and can also be used for preparing or screening medicaments for treating fat-related diseases, such as obesity.

The invention has the beneficial effects that:

Drawings

FIG. 1 shows the differentiation process of 3T3-L1 adipocytes induced in vitro. (A) Oil red O staining results during adipocyte differentiation. The samples were taken on

days

0, 14 and 21 of the in vitro induction of differentiation of adipocytes, and oil red O staining was performed to determine the differentiation of adipocytes using the results of staining for fat droplets. On day 0 of induced differentiation, the proportion of oil red O staining positive cells was 0%; on day 14 of induced differentiation, the proportion of oil red O staining positive cells was 24.2%; on day 21 of induced differentiation, the proportion of cells staining positive for oil red O was 54.9%. (B) PPAR γ and CEBP α expression during adipocyte differentiation. And detecting the expression conditions of PPAR gamma and CEBP alpha in the differentiation process of the fat cells by using a real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Calculating the expression condition of the genes in the induced differentiation process by taking the expression level of each gene at the 0 th day as a reference, wherein the expression value is expressed as follows: mean ± standard deviation.

FIG. 2 shows that knocking down the expression of slincRAD gene can inhibit the differentiation of adipocytes induced in vitro. (A) Oil red O staining results 2 days before induced differentiation, first siRNA transfection was performed on 3T3-L1 cells; on the day of the differentiation-inducing treatment, a second siRNA transfection was performed. On the ninth day of induced differentiation, cells were harvested, stained with oil red O, and intracellular lipid production and accumulation were observed. Control: cells that were not treated for induced differentiation and were not transfected with siRNA. Mock: cells that were not transfected with siRNA were treated for induced differentiation only. NC-siRNA: cells that have undergone induced differentiation and are transfected with a sequence-independent siRNA. siRNA1-siRNA 17: cells transfected with slincRAD-specific siRNA were induced to differentiate. (B) Quantitative analysis of oil red O staining results. The oil red O staining results were quantitatively analyzed using ImageJ software (https:// Imagej. nih. gov/ij /). To eliminate background staining, the staining value of Control cells was first subtracted as background staining. Then, taking the staining value of Mock as a reference, and carrying out normalization treatment on the staining values of other cells; the resulting staining values were then normalized to cell number to reflect the effect of siRNA treatment on cell differentiation. Statistical analysis of the results of the experiments was performed using t-tests (.: NC-siRNA as control, P < 0.05).

FIG. 3 is a graph of the regulatory effect of slincRAD-shRNA on adipocyte differentiation. (A) The gene structure, transcript structure and shRNA target location of slincRAD. (B) Inhibitory effect of slincRAD-shRNA on target genes. Wild-type, slincRADSRNA-8, slincRADSRNA-93T 3-L1 cells were hormone-induced according to the protocol described in example two. After 6 days of treatment, cells were harvested, total RNA was extracted, and expression levels of slincRAD were measured using qRT-PCR. A sequence-independent shRNA targeting the GFP protein gene was used as a control (CTL, control) for the experiment. Experimental data are the average of three biological replicates and are presented in the following manner: mean ± s.e.m. (C) Inhibiting the production of cell lipid. On day 7 after hormone-induced differentiation separation, cultured cells were stained with oil red O, and the effect of inhibition of slincRAD gene expression on cell lipids was analyzed. The upper diagram: oil red O staining of the plates; the middle graph is as follows: local staining images of the plates; the following figures: quantification of oil red O staining using ImageJ software. The control group (CTL) was wild type 3T3-L1 cells.

FIG. 4 shows the construction process of transgenic mice. (A) The constitutive process of the transgenic mouse construction is shown. (B) Genotype identification of transgenic mice at litter 11.

FIG. 5 shows the establishment and identification of transgenic mice. (A) Pedigree profile of transgenic mice. Two first generation transgenic mice (accession numbers: 13,15) with shRNA8 +/-genotype and one first generation transgenic mouse (accession number: 11) with shRNA9 +/-genotype were mated with wild-type C57BL/6J female mice. Artificial insemination was performed using the first generation transgenic mouse numbered 9. A total of 20 litters of 143F1 generation mice were obtained, of which 81 were transgenic. The mice in the figure are marked as follows, as indicated by 9# (M3, F5) with 3 male and 5 female mice in the 9 th litter. Mice numbered 1-6 were sacrificed prior to birth and used to study embryonic mouse development and gene expression. (B) The expression level of slincRAD gene was down-regulated in transgenic mice. The gene expression level in mouse organs at birth for 10 days, 15 days and 30 days was detected by qRT-PCR. In PCR, U6RNA was used as a detection control. The expression level of slincRAD gene is expressed as a fold relationship relative to the wild type gene level. Experimental data are the average of three biological replicates and are presented in the following manner: mean ± s.e.m. (C) Mouse genetic pattern map. The table shows the genetic information for each mouse, e.g., 3 male mice and 5 female mice for the 9 th mouse, 1 of the 3 male mice being transgenic mice, and 5 female mice being transgenic mice. In the developmental stage column ED15 represents day15 mice at embryonic stage; day15 represents postnatal Day15 mice; 7Mo indicates mice at 7 months after birth.

Figure 6 shows that knockdown of slincRAD affects development of mice. (A) After birth, body weight control of transgenic mice and wild type mice (, values with significant differences). (B) Body weight profile (, value with significant difference). (C) Body morphology maps of transgenic and wild-type mice.

Figure 7 shows that knocking down slincRAD gene expression affects development of mouse adipose tissue. (A) Body fat and muscle measurements. Using a small animal NMR instrument, we examined body fat and muscle content (n.gtoreq.4 in each group) in 1-month and 7-month old mice. White column: a wild-type mouse; black bars, transgenic mice. (B) The weight of the major organs of 5-month-old mice (n.gtoreq.4). White column: a wild-type mouse; black bars, transgenic mice. (C) Histological section of adipose tissue. The upper diagram: hematoxylin-eosin staining of white adipose tissue. White column: a wild-type mouse; black bars, transgenic mice. The scale bar on the figure is 50 μm. The following figures: size distribution of adipocytes. Hollow circle: a wild-type mouse; solid circle: knockout mice. Data representation mode: mean ± s.e.m., each group n is 3,. p < 0.05; p <0.001.(D) histological sections of the major organs of 7-month-old mice. The scale bar on the figure is 100 μm.

Figure 8 shows the regulatory effect of slincRAD on key factors for adipocyte differentiation. We analyzed the expression levels of the key adipocyte differentiation factors PPAR γ and CD36, and slincRAD, in liver tissue of wild type and transgenic mice using qRT-PCR. The expression level of the transgenic mice (fold relative to wild-type gene expression) was calculated with the wild-type mouse gene expression level as a reference. Data representation mode: mean ± s.e.m., each group n is 10.

Figure 9 shows the effect of knockdown of slincRAD on blood glucose and blood lipid metabolism in mice. (A) Comparative study of blood glucose, plasma total Triglycerides (TG), Total Cholesterol (TC), Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL) levels in 7-month-old wild-type and transgenic mice. Hollow column: a wild-type mouse; solid column: transgenic mice. Data representation mode: mean ± s.e.m., 14 for each group. P < 0.05; p < 0.01; p <0.001. (B) Sugar tolerance test. Hollow circle: a wild-type mouse; solid circle: transgenic mice. Data representation mode: mean ± s.e.m., 14 for each group. (C) Insulin resistance test. Hollow circle: a wild-type mouse; solid circle: transgenic mice. Data representation mode: the average value is +/-S.E.M., and n in each group is more than or equal to 3.

Figure 10 shows the effect of slincRAD on key factors for fat differentiation. In the adipocyte differentiation process, qRT-PCR is utilized to detect the expression level of the differentiation key factors day by day, and a curve chart is drawn. Blue line: gene expression levels in wild type cells; red line, slincRAD, knockdown gene expression levels in cell lines. The internal reference is beta-actin. Data representation mode: mean ± s.e.m., each group n is 3.

Figure 11 shows flow cytometry to detect changes in cell cycle during differentiation of wild-type slincRAD cells and slincRAD knockdown (shRNA8 and shRNA9 knockdown) cells.

FIG. 12 shows the analysis of expression profile of Dnmt1 gene. Changes in the expression of the Dnmt1 gene during adipocyte differentiation were analyzed by qRT-PCR. The internal reference is beta-actin. Data representation mode: mean ± s.e.m., n-3 biological replicates per group.

FIG. 13 shows RNA co-immunoprecipitation. Cellular RNA fractions interacting with DNMT1 were obtained using anti-DNMT 1 antibodies and co-immunoprecipitation techniques. And then detecting the content of slincRAD gene transcript in the immune complex by utilizing a qRT-PCR technology. The negative control of the experiment was immune complexes captured by anti-IgG antibodies. Data representation mode: mean ± s.e.m.

FIG. 14 is a Wien plot analysis of promoter methylation levels in wild type and slincRAD knockdown cells.

FIG. 15 is a Venn diagram analysis of DEP gene.

FIG. 16 is DEP index of differentially methylated proliferation-associated genes in wild-type 3T3-L1 cells.

FIG. 17 is Heat-map analysis. DEP index of methylation level of gene promoter was analyzed in comparison between (-2) th and (+2) th days of adipocyte-induced differentiation. Red represents genes with high methylation levels; green represents hypomethylated gene, and the graph was drawn using a Heatmap Illustrator.

FIG. 18 shows the change in expression of proteins such as p21 in western blot. The cell lines studied included wild-type 3T3-L1 cells (WT), slincRADSRNA-8 (sh8), and slincRADSRNA-9 (sh9) slincRAD low expressing cells. The study time points were (-2) to (+4) days of adipocyte differentiation induction in vitro.

Figure 19 is a graph of the effect pattern of slincRAD.

Detailed Description

The invention is further illustrated below with reference to specific embodiments and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

The first embodiment is as follows: laboratory animals and materials

The mice for test are purchased from Beijing Wittiulihua laboratory animal technology Limited company and bred in Beijing university laboratory animal center. All experimental protocols and procedures were approved by the ethics committee for experimental animals at the university of beijing.

DNA oligonucleotides were purchased from Invitrogen Beijing, and RNA oligonucleotides from Ruibo Biotech, Inc., Guangzhou.

Example two: in vitro induced differentiation of 3T3-L1 adipocytes

The 3T3-L1 preadipocyte in vitro induced differentiation is a common adipocyte differentiation model, and is induced to differentiate into mature adipocytes under the action of an inducer (1-methyl-3-isobutyl xanthine (IBMX), insulin and dexamethasone). To study the differentiation process of cells, we first examined the formation and accumulation of fat droplets in cells using oil red O staining, and found that 54.9% of the cells were positive for fat staining at the end of induced differentiation (day 21). In addition, the expression conditions of cell differentiation key factors PPAR gamma and CEBP alpha are detected by using real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The expression regulation of these two factors in cell differentiation was found to be consistent with literature reports, indicating that the induction of the differentiation process was successful. The study protocol and experimental results are as follows:

1. 3T3-L1 preadipocyte culture and in vitro induced differentiation: definition of time points for induced differentiation: the day of starting the induced differentiation by adding the induced differentiation medium was day1 of induced differentiation. The day before day1 of induced differentiation was day 0. The day after day1 of induced differentiation is day 2, and so on, other induced differentiation time nodes are defined.

3T3-L1 mice preadipocytes were routinely cultured in DMEM liquid medium containing 10% fetal bovine serum, with 1 change over 2 to 3 days. On the 1 st day of induced differentiation, the conventional medium was changed to a differentiation-inducing medium, and in vitro induced differentiation was performed. Induction medium was supplemented with 167nM human insulin (Sigma), 0.5 mmol/L1-methyl-3-isobutylxanthine (Sigma) and 1. mu.M dexamethasone (Sigma) on a regular medium basis. On day 2 of induction of differentiation, the medium was changed to DMEM medium containing only 167nM insulin. On day 4 of induction of differentiation, the differentiation medium was changed back to the conventional DMEM medium, and the culture was continued until the differentiation process was completed.

2. Dyeing with oil red O: preparing an oil red O reagent according to a conventional method. At the key time node of the differentiation process: pre-induction differentiation (day 0), mid-induction differentiation (day 14), and final induction differentiation (day 21), samples were taken for oil red O staining.

The experimental steps are as follows: 1) gently rinsing the cultured cell samples obtained at each time node for 3 times by using Phosphate Buffered Saline (PBS) subjected to ice bath; 2) cells were fixed with 3.7% formaldehyde for 2 min; 3) incubating with oil red O reagent for 1 hour at room temperature, and staining cell fat; 4) rinsing with deionized water for 3 times; 5) observing and photographing under an optical microscope; 6) the results of the oil red O staining were quantitatively analyzed using ImageJ software (https:// ImageJ. nih. gov/ij /) against cells that did not undergo induced differentiation.

Oil red O staining results are shown in fig. 1A. On day 0 of induced differentiation, the proportion of oil red O staining positive cells was 0%; on day 14 of induced differentiation, the proportion of oil red O staining positive cells was 24.2%; on day 21 of induced differentiation, the proportion of cells staining positive for oil red O was 54.9%. Mature adipocytes are round with large numbers of lipid droplets aggregated and oil red O staining appearing orange-red.

3. Expression assay of PPAR γ and CEBP α: and analyzing the expression changes of the PPAR gamma and the CEBP alpha in the differentiation process by utilizing qRT-PCR, wherein the internal reference gene is beta-actin. The sequence information of the primers for detecting gene expression is shown in Table 1. The experimental results are shown in fig. 1B, and the experimental procedures are as follows:

1) total RNA extraction: cell samples were obtained on day 0 (pre-induced differentiation), 14 (mid-induced differentiation) and 21 (final induced differentiation) of induced differentiation, and total cellular RNA was extracted and quantified using Trizol reagent.

2) Reverse transcription of RNA: the reverse transcription reaction of the RNA samples was carried out using a reverse transcription kit (TIANCcript M-MLV, cat: ER104-03) from Tiangen Biochemical technology, according to the method described in the kit instructions, and the following steps were carried out: taking 1 microgram total RNA sample, adding 2 muL (10 muM) 6-base random reverse transcription primer and 2 muL dNTP (10mM), mixing uniformly and then placing

Water bath denaturation for 5 minutes; after standing on ice for 3 minutes, the mixture was taken out, and 4. mu.L of 5 XFirst-Strand Buffer, 0.5. mu.L of RNase inhibitor (40U/. mu.L), 1. mu. L M-MLV (200U/. mu.L), in a total volume of 20. mu.L, were added; mixing, centrifuging briefly, and performing reverse transcription reaction in an Eppendorf PCR instrument with reaction parameters of

10 minutes;

50 minutes;

5 minutes; then is arranged in

And (5) storing. Wherein the RNase inhibitor is a product (cat # N211) of Promega corporation.

3) Real-time fluorescence quantificationAnd (3) PCR: the HotMaster Taq DNA polymerase reagent (cat #: ET106-01-01) from Tiangen Biochemical technology company is used for detecting the expression condition of the gene in the sample, and the experimental steps are as follows: mu.L of the reverse transcription product was taken, and 2.5. mu.L of 10 × HotMaster Taq Buffer, 0.5. mu.L (10. mu.M) of the forward primer, 0.5. mu.L (10. mu.M) of the reverse primer, 1. mu.L of dNTP mix (2.5. mu.M each), 1. mu.L of SYBR Green I (5X), 0.2. mu.L of HotMaster Taq DNA polymerase (2.5U/. mu.L) were added, and finally 17.3. mu.L of deionized water was added to make the reaction total volume 25. mu.L. Mixing, centrifuging briefly, and performing PCR amplification reaction in an Eppendorf PCR instrument with reaction parameters of

The pre-denaturation is carried out for 2 minutes,

the mixture is denatured for 15 seconds and then is subjected to the treatment,

the annealing is carried out for 15 seconds, and the annealing time,

extension for 30 seconds; the number of cycles was 40 cycles. Each reaction was set up in 3 replicates.

4) And (3) data analysis: the expression of the target gene and the expression of the reference gene (beta-actin) in the same sample are respectively detected. And normalizing the expression of the target gene by taking the expression level of the internal reference gene as a reference. The expression level of the target gene is then quantitatively analyzed using the delta delta Ct method commonly used in the art. Specific methods and procedures can be found in the literature: livak KJ and Schmittgen TD. analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta Delta C (T)) method. methods.2001 Dec; 25(4):402-408.

TABLE 1 RT-PCR primer pairs

Example three: inhibition of adipocyte in vitro induced differentiation by knockdown slincRAD

To investigate the effect of expression of slincRAD genes on adipocyte differentiation, we obtained 6 siRNAs specific to slincRAD genes, including siRNA1, siRNA9, siRNA12, siRNA14, siRNA15, and siRNA17, by screening (table 2). Using cell transfection reagents, we introduced these sirnas into cultured 3T3-L1 cells, respectively, to knock down the expression level of slincRAD gene therein; and then, by using oil red O staining, whether the generation of cell fat is influenced or not is identified, so that whether the differentiation process of the adipocyte of 3T3-L1 is influenced or not is judged. We found that certain gene-specific sirnas significantly inhibited cellular lipid production and accumulation compared to control cells that were not siRNA-treated and control cells that were scramble siRNA-treated (figure 2); in particular siRNA-17, the treatment of cells with this siRNA reduced the lipid content of the cells by 70%. In addition to studying cellular lipogenesis, we also studied the effect of knocking down slincRAD on the expression of PPAR γ, a key adipocyte differentiation factor, using real-time fluorescent quantitative PCR. The research result shows that: the use of siRNA (e.g., siRNA-14) to knock down the expression of slincRAD gene not only affected the production of cellular lipids, but also significantly inhibited the expression of PPAR γ gene in cells (fig. 2), which is consistent with its inhibitory effect on cell differentiation. The experimental procedure is as follows:

1. 3T3-L1 cell culture: 3T3-L1 mice preadipocytes were routinely cultured in DMEM liquid medium containing 10% fetal bovine serum, with 1 change over 2 to 3 days.

2. siRNA transfection: using BLOCK-iT^TMThe RNAi Designer (Invitrogen) program designed siRNA molecules specific to slincRAD genes, with specific sequences shown in table 2.

The day before transfection, 3T3-L1 cells were seeded in 6-well plates to achieve a confluency of 60-70% after cell attachment. Lipofectamine available from Invitrogen corporation was used according to the manual^TMRNAiMAX transfection reagent cell transfection experiments were performed. For each well of the 6-well culture plate, an RNAiMAX/siRNA mixture was prepared by adding 100pmol siRNA to 400. mu.L of Opti-MEM (GIBCO) cell culture solution, and after standing at room temperature for 20 minutes, the RNAiMAX/siRNA mixture was added to the cell culture solution6, in a hole plate; after transfection for 4 hours, normal cell culture medium was added for culture.

After the cells continued to be cultured for 2 days, the DMEM medium containing the differentiation inducer was added to the cells until the cells became confluent, and the differentiation induction process was started. And a second siRNA transfection is carried out on the same day, and the RNAiMAX/siRNA mixture is directly added into the differentiation induction culture medium for siRNA transfection.

3. Inducing differentiation in vitro: definition of differentiation time node induced in vitro by 3T 3-L1: the day of starting the induced differentiation by adding the induced differentiation medium was day1 of induced differentiation. The day before day1 of induced differentiation was day 0. The day after day1 of induced differentiation is day 2, and so on, other induced differentiation time nodes are defined.

4. Dyeing with oil red O: on day 9 of the in vitro induction of differentiation of adipocytes, cell samples were obtained and analyzed for oil red O staining. See example two for experimental procedures. The results of the experiment are shown in FIG. 2.

TABLE 2 siRNA sequences

Example four: construction of slincRAD shRNA transgenic mice

To identify the in vivo function of slincRAD gene, the effect on adipocyte differentiation, cellular lipogenesis, and expression of key differentiation factors, in the case of knocking down slincRAD gene expression in mice, was studied. To this end, we constructed slincRAD shRNA transgenic mice using specific shRNA targeting slincRAD. The method comprises the following specific steps:

screening for slincRAD-specific shRNA (slincRAD-shRNA). 1) Design slincRAD-specific shRNA. Using an online design tool, we designed different shRNA expression sequences for the slincRAD gene sequence, and obtained 4 specific shRNA expression sequences (shRNA-8, shRNA-9, shRNA-15, shRNA-17; FIG. 3A; see Table 3 for sequences) by screening. 2) And (3) constructing an shRNA expression vector. A shRNA expression vector specific for slincRAD was constructed using the pSD31 retroviral vector purchased from Invitrogen. According to the protocol described in the kit specification, we first synthesized a double-stranded shRNA expression cassette, subcloned it into a linearized pSD31 retroviral vector, and identified by sequencing to obtain the shRNA expression vector for slincRAD gene. 3) Cell transfection and in vitro induced differentiation. The shRNA expression vector was introduced into cultured 3T3-L1 cells using Lipofectamine 2000 cell transfection reagent. The shRNA-introduced cells were induced to differentiate using the in vitro induced differentiation protocol described in example one. 4) And (3) identifying the active shRNA. On day 6 of induced differentiation, cells were harvested. The expression level of slincRAD gene was measured using real-time fluorescent quantitative PCR according to the method described in example one, and the primer sequences for detecting expression of slincRAD gene are shown in table 4. It was found that slincRAD-shRNA8 and slincRAD-shRNA9 were able to efficiently inhibit the expression of cell endogenous slincRAD. Wherein slincRAD-shRNA8 reduced the expression level of the target gene to 1% of the level of the wild-type cell, and slincRAD-shRNA9 reduced the expression level of the target gene to 3% of the level of the wild-type cell (FIG. 3B). Oil red O staining revealed that slincRAD-shRNA8 and slincRAD-shRNA9 significantly inhibited cellular lipid production while inhibiting the target gene (fig. 3C).

TABLE 3 shRNA sequences

shRNA8(SEQ ID No 21)：

5’GTGGAAAGGACGAAACACCCGGCCTATAACCAGTACAGTTAATCAAGAGTTAACTGTACTGGTTATAGGCTTTTTTGGATCCCGAAT 3’

shRNA 9(SEQ ID No 22)：

5’GTGGAAAGGACGAAACACCCGGCACCAACCTCTTCCAATATCTCAAGAGGATATTGGAAGAGGTTGGTGCTTTTTTGGATCCCGAAT 3’

shRNA 15(SEQ ID No 23)：

5’GTGGAAAGGACGAAACACCCGGGATTAGTGTGGCAGATTATCAAGAGTAATCTGCCACACTAATCCTTTTTTGGATCCCGAAT 3’

shRNA 17(SEQ ID No 24)：

TABLE 4 slincRAD amplification primer sequences

01 detection primer combination:

01 forward primer: 5' -ATCCATCTCCCCTACCCATC (SEQ ID NO:200)

01 reverse primer: 5' -TGCAGAGGACTCACAACTGG (SEQ ID NO:201)

08 detection primer combination:

08 Forward primer: 5' -GGTGCAGTTTTGCACTGAGA (SEQ ID NO:202)

08 reverse primer: 5' -GATGGAGAGAAAGGAAGGGG (SEQ ID NO:203)

16 detection primer combination:

16 forward primer: 5' -CCTAGTGCCTTTCTGCCTTG (SEQ ID NO:204)

16 reverse primer: 5' -TGGGGTGATTGTGTAGGGAT (SEQ ID NO:205)

31 detection primer combination:

31 forward primer: 5' -CAAGTGGTTCCGGATTCTGT (SEQ ID NO:206)

31 reverse primer: 5' -CCGTGAAATGAAGTGGTGTG (SEQ ID NO:207)

54 detection primer combination:

54 forward primer: 5' -GCATTTAATTGGTGCTGGCT (SEQ ID NO:208)

54 reverse primer: 5' -GACCCTTCTCCTAGCTGCCT (SEQ ID NO:209)

Constructing a transgenic mouse: obtaining DNA fragments respectively encoding expression frames of slincRAD-shRNA8 and slincRAD-shRNA9, wherein the DNA fragment is 285 bases in length and comprises a U6 promoter, an shRNA coding sequence and a 6-nucleotide termination sequence TTTTTT. The DNA fragment was introduced into the pronuclei of fertilized egg of C57BL/6 mouse by microinjection, and then the fertilized egg was transplanted into the pseudopregnant female mouse to develop into mouse (FIG. 4A). To identify transgenic mice, we designed two independent primer pairs capable of amplifying shRNA expression cassettes (table 5). By amplifying the shRNA expression cassettes integrated in the mouse genome, we identified 4 male transgenic mice, first generation transgenic mice, that incorporate slincRAD-shRNA8 and slincRAD-shRNA9, respectively (fig. 4B, 5A).

TABLE 5 amplification primer pairs for shRNA expression cassettes

Establishment of transgenic mice: mating the transgenic mouse with a wild female mouse to obtain a progeny mouse. Extracting the genome DNA of the tail tissue of the progeny mouse, and performing PCR amplification on the shRNA expression frame integrated in the genome DNA by using a specific primer pair (shown in table 5), wherein the progeny mouse integrating the shRNA expression frame with positive amplification signals is obtained. Thus, a total of four slincRAD-shRNA transgenic mouse lines were obtained (FIG. 5A). The genotype of the three mouse lines was slincRAD-shRNA8+/-, and was therefore designated slincRAD^shRNA8Mouse, one mouse line was genotyped as slincRAD-shRNA9+/-, and thus was designated slincRAD^shRNA9。

To examine the situation where shRNA inhibited the expression of the target gene, we sacrificed mice at

days

10, 15, and 30 after birth to obtain mouse tissues, and examined the expression level of slincRAD in the organs using qRT-PCR (fig. 5B). We found that the expression level of slincRAD in other organs was significantly reduced, on average to 60% or less of the wild type tissue expression level, except for the tenth day brain tissue, compared to the wild type mice in the control group.

In order to obtain a sufficient number of transgenic mice for subsequent studies, we used natural mating and artificial insemination methods for breeding transgenic mice. In artificial inseminationIn breeding, we first removed one slincRAD^shRNA8The mature sperms are separated from one side of seminal vesicle of mouse and transferred into wild pseudopregnant female mouse. Thus, we obtained 143F in total₁Mouse surrogate, of which 81 is slincRAD^shRNA8Positive mice, 62 were wild type mice only (fig. 5C). The ratio of positive mice to wild type mice in the obtained offspring mice was essentially 1:1, following the mendelian segregation law. This indicates that knocking down the expression level of slincRAD did not have a serious impact on the viability of the mice. For convenience, slincRAD^shRNA8The transgenic mice are also referred to as slincRAD knock-down mice, or KD mice.

Example five: effect of knockdown of slincRAD expression on mouse development

To investigate the effect of knockdown of slincRAD on mouse development, we performed comparative studies on the physical development status of wild type and transgenic mice, from day15 of embryonic phase to 7 months after birth. During embryonic development, the transgenic mice had no significant difference in slincRAD expression levels from wild-type mice, and were very similar in individual morphology and birth weight. After birth, the body weight of the transgenic mice began to become significantly lower than that of the wild-type mice, although the same feeding conditions were maintained (fig. 6A). At 5 days postnatal, transgenic mice (n ═ 11) were weighing-12% lower (p <0.05) than wild-type control mice (n ═ 9). This difference was maintained until the adult stage of the mice (fig. 6B). Morphologically, the transgenic mice did not have the abdominal bulge of wild-type mice (FIG. 6C), a phenomenon that may result from normal development of abdominal fat in mice being affected.

This example data shows that knocking down the expression of the mouse slincRAD gene does not affect the embryonic development of the mouse, but significantly affects the postnatal development process of the transgenic mouse. Although the expression level of slincRAD gene showed significant reduction both at embryonic and late birth compared to control mice.

Example six: effect of knockdown of slincRAD Gene expression on mouse adipose tissue development

Based on the above work, we speculated that the effect of slincRAD on mouse development may be due to its interference with normal differentiation of adipose tissue. To verify this concept, we examined the body fat, muscle, and moisture content of mice while awake using a small animal Nuclear magnetic imaging device Nuclear spectroscopy (EchoMR-700, mchomar, Houston TX). The specific operation is carried out according to the scheme provided by the equipment manufacturer, and the scanning interval is 0.5-3.2 minutes. Each animal was subjected to 3 independent measurements.

The study of 1 month old mice found that the transgenic mice had a body fat content 12% lower than that of wild type mice and a muscle content 9% higher than that of wild type mice. Studies with 7-month-old mice found that transgenic mice had 22% lower body fat content and 6% lower muscle content than wild-type mice (fig. 7A). Furthermore, we compared the weights of the major organs of mice and found no significant difference (fig. 7B).

To analyze the effect of knockdown slincRAD on mouse adipose and other tissue development, we prepared tissue sections of the major organs of 7-month old mice. Firstly, obtaining organ tissues of a transgenic mouse and a wild mouse, soaking the organ tissues in 4% paraformaldehyde solution, and fixing the organ tissues overnight at 4 ℃; paraffin embedding, and continuously slicing at 5 μm width; hematoxylin-eosin staining and microscopic observation were then performed. We found that more deeply colored islets appeared in adipose tissue of transgenic mice compared to normal tissue of wild type mice (fig. 7C). High power microscope observation revealed that these regions consisted of smaller adipocytes. In 7-month-old transgenic mice, the average size of adipocytes was 19.5. + -. 4.8. mu.m, while in wild-type control mice, the size of adipocytes was 58. + -. 10. mu.m. The mean size of adipocytes was 21.4. + -. 4.6 μm in 9-month-old transgenic mice, and 74.7. + -. 14 μm in wild-type mice (FIG. 7D). The tissue structures of all organs except the adipose tissue were not significantly changed (fig. 7D).

Example seven: mechanism of action study of slincRAD

To elucidate the mechanism of action of slincRAD, we investigated its regulatory role on two adipocyte differentiation key transcription factors PPAR γ and CD 36. PPAR-gamma is a nuclear receptor that regulates almost all aspects of fatty acid metabolism, such as fatty acid transport, synthesis, storage, mobilization, activation and oxidation. CD36 is a class B scavenger receptor that promotes adipocyte differentiation and adipogenesis.

We detected the expression levels of PPAR γ, CD36 and slincRAD genes in the liver on

days

10, 15 and 30 after birth of mice, respectively, using qRT-PCR (figure 8). On day 10 of mouse birth, the expression levels of PPAR γ, CD36 and slincRAD in the liver of transgenic mice were reduced by 50% compared to wild-type mice. However, as the development process continued, the expression levels of PPAR γ and CD36 were restored to wild type mice at 15 and increased to levels higher than wild type mice at day 30, showing a dose-compensating effect. These data suggest, on the one hand, that slincRAD acts through the PPAR γ signaling pathway and, on the other hand, explain why the regulatory effect of slincRAD on mouse development is mainly manifested in the early stages of embryonic development.

Example eight: effect of knockdown slincRAD on blood glucose and blood lipid metabolism in mice

White adipose tissue is an important link in energy metabolism of the body, and the excessive accumulation of white adipose tissue of the human body can cause abnormal metabolism of the body. To investigate the role of slincRAD in energy metabolism, we used 9-month-old wild-type and transgenic mice as study subjects to study the effect of knocking down slincRAD gene expression on blood glucose and blood lipid metabolism of mice.

And (3) detecting the blood sugar content: blood was collected from the tail vein of mice, and the blood glucose level of mice was measured using a blood glucose meter ACCU-CHEK Performa Glucometer (Roche, Denmark) from Roche, according to the specification of the blood glucose meter. And (3) detecting the blood fat content: the mice were fasted for 6 hours, and blood was collected from the tail vein, and the plasma components were separated and assayed for triglyceride, total cholesterol, and LDL-C, HDL-C3 levels using a Roche analyzer (Cobas 4000 analyzer).

As shown in fig. 9A, plasma triglyceride levels were significantly reduced and HDL-c levels were significantly increased in the transgenic mice compared to wild-type control mice. Plasma Total Cholesterol (TC) and LDL-c showed an upward trend, but no significant difference.

In addition, we performed glucose and insulin resistance experiments. The baseline blood glucose levels of the experimental mice were measured three consecutive days before the start of the experiment. The mice were fasted overnight (about 16 hours) before the experiment, and then D-glucose was injected intraperitoneally at a dose of 1.5g/kg body weight, tail vein blood was collected at 0, 15, 30, 60, 120 and 180 minutes after the injection, blood glucose levels of the mice were measured using a glucose meter of rochon, and changes in blood glucose levels of the mice were analyzed to plot a glucose tolerance curve (fig. 9B).

Before the insulin tolerance test, the mice were fasted for 6 hours, followed by intraperitoneal injection of human insulin at a dose of 1IU/kg body weight, tail vein blood collection at 0, 15, 30, 60 and 120 minutes after the injection, and changes in blood glucose content of the mice were monitored to plot an insulin resistance curve (fig. 9C).

Analysis revealed that fasting blood glucose levels were significantly reduced in the transgenic mice compared to the wild-type control mice (fig. 9A). In the glucose tolerance experiment, the transgenic mice exhibited significantly better glucose tolerance than the wild-type mice (fig. 9B), as shown by the GTT curve. However, there was no significant difference in insulin resistance between the transgenic mice and the wild-type mice (fig. 9C).

Example nine: effect of knockdown of slincRAD on differentiation of 3T3-L1 cells

To analyze the role of slincRAD in adipocyte differentiation, we constructed cell lines stably transfected with slincRAD-shRNA8 and slincRAD-shRNA9, named slincRAD, respectively, using psd31 retroviral vector and conventional puromycin screening, which were established in example four^shRNA-8、slincRAD^shRNA-9A cell line. In addition, we also transferred a shRNA targeting green fluorescent protein and unrelated to slincRAD sequence into 3T3-L1 cells to construct a stable transfer cell line used as a negative control, named shGFP 3T3-L1 cell line.

Referring to the user manual of retrovirus vector and the conventional technical method, we first produce retrovirus containing shRNA expression cassette, infect the harvested virus to cells, screen the infected cells for puromycin, and establish shRNA stable transfected cell strain. The experimental procedure was roughly as follows: culturing 293T cells in a T25 flask, allowing the cells to grow to an almost complete confluent (sub-confluent) state; cells were co-transfected with 3.25. mu.g of shRNA lentiviral plasmid, 2.88. mu.g of pRSV plasmid, 1.44. mu.g of PRRE plasmid, and 1.44. mu.g of pMD2G-VSVG plasmid using Lipofectamine 2000 transfection reagent. The medium was changed 6 hours after transfection and virus particles were harvested 72 hours later and filtered through a 0.45 μm filter. Subsequently, the obtained virus particles were added to 3T3-L1 cells cultured in a 6-well plate in a medium containing 8mg/mL polybrene, to infect the cells. And (3) screening by using a culture medium containing 4mg/mL puromycin, and screening to obtain a cell strain of the stably transfected shRNA.

Using the detection method established in example two, for slincRAD^shRNA-8、slincRAD^shRNA-9And wild type 3T3-L1 cells were induced to differentiate. We named day when the inducer was added day (0), day before day (1), day after day (1), and so on.

At day 4 day (+4) of induced differentiation, cells were harvested and tested for expression levels of slincRAD gene. We found slincRAD with reference to wild-type cells^shRNA-8And slincRAD^shRNA-9Expression levels of slincRAD gene in cells were reduced to 32% and 55% of wild type.

Further, we analyzed the lipid content of the cells using a high content analyzer (Columbus Image Data Storage and Analysis System) of Perkin Elmer. The lipid analysis kit used was the Bodipy 493/503 kit from Life Technologies, Inc., a reference kit for the specific procedures and a user manual for a high content analyzer. The experimental protocol is generally as follows: 3T3-L1 cells at different differentiation stages were fixed with 4% paraformaldehyde for 15 minutes and punched with 0.5% Triton X-100 for 15 minutes at room temperature. After washing with PBS, the cells were stained with 1:1000 Bodipy 493/503 solution and 1:10000 Hoechst 350/461 buffer for 45 minutes.

The observation and recording of cells was performed using a Columbus Image Data Storage and Analysis System high content analyzer under a 20-fold objective lens. Fluorescence intensities of Hoechst (nuclear) and Bodipy 493 (lipids) were detected using Hoechst 33342 and Alexa 488 channels, and the number of cells was recorded using bright field images. The fluorescence image obtained was analyzed with a software system attached to the high content analyzer.

We found that in slincRAD^shRNA-8And slincRAD^shRNA-9In the cells, the lipid content of the cells was reduced to 27% and 30% of the lipid content of the wild type cells.

Example ten: effect of knockdown of slincRAD on cellular Gene expression

To elucidate the mechanism of action of slincRAD, we compared gene expression of wild-type and shRNA-stable cell lines (slincRAD-knockdown cell lines). During the 6 days after the initiation of differentiation, we performed day-by-day analysis of the changes in the expression of cytokines involved in adipocyte differentiation, including C/EBP α, PPAR γ, pre-adipocyte factor-1(pref1), CD36, and Fabp 4. C/EBP and PPAR γ are the most critical transcription factors in adipocyte differentiation, and other genes play a role in fatty acid transport.

During induced differentiation of 3T3-L1 adipocytes, the expression level of slincRAD gene began to be up-regulated on day (2) after exposure to inhibition (FIG. 10, top line). Throughout the subsequent "contact inhibition period", the expression level of slincRAD gene was continuously up-regulated, with its peak expression occurring on day (0), i.e. the day of addition of the differentiation inducer. Expression levels of slincRAD gradually decreased after induction of differentiation began. In sharp contrast, the up-regulation of C/EBP α and PPAR γ occurred at day (1) and day (2) after induced differentiation, and then the expression levels of pref1, CD36, Fabp4 were up-regulated.

The above data indicate that expression of slincRAD gene is upregulated significantly earlier than other cytokines, suggesting that the gene may play a role in the initial stages of differentiation.

In addition to wild-type cells, we also investigated the regulation of gene expression in slincRAD low expressing cell lines. Knocking down the expression level of slincRAD gene was found to significantly suppress the expression of adipose differentiation-related factors (fig. 10, the lower line), which is in sharp contrast to the active expression regulation pattern in wild-type cells. The expression levels of C/EPB alpha and PPAR gamma are only significantly inhibited or retarded by 1/4-1/2, pref1, Fabp4 and CD36 of wild type cells. These results indicate that knocking down slincRAD affects the expression of key factors for adipocyte differentiation, as well as cellular lipogenesis, and ultimately leads to a decrease in cellular lipid synthesis.

Example eleven: effect of knockdown of slincRAD on cell cycle

Modulation of the cell cycle is an important aspect of the regulation of cell differentiation. To investigate the effect of knocking down slincRAD on cell cycle, we compared wild-type 3T3-L1 cells, slincRAD^shRNA-8And slincRAD^shRNA-9The differentiation process of cells focuses on the changes in cell cycle behavior. During the induction of differentiation, we selected three time nodes, representing the proliferative phase cell day (-2), the growth-arrested phase cell day (0), and the terminal differentiated phase cell day (+2), respectively.

Cell cycle detection system: flow cytometer FACS Calibur system from BD immunocytometer Systems, inc (San Jose, CA); analysis software: MODFIT Software package (Topsham, ME) from Verity Software House, Inc.; all tests were done using the same instrument setup. Before the analysis on the machine, the cells are suspended in a 35mm culture dish and fixed overnight at 4 ℃ by 70% ethanol; staining with propidium iodide (BD Biosciences, San Jose, CA) for 30 min under ice-bath conditions; subsequently, the cells were analyzed on the machine, and at least 10,000 cells were counted in each sample.

There was no apparent difference in cell cycle behavior between wild-type and slincRAD knockdown cells at the cell proliferation and differentiation stages; the difference between the two occurs mainly in the growth inhibition phase. In the cytostatic phase, 31.8% of wild-type cells are in S-phase and slincRAD in S-phase^shRNA-8The number of cells increased to 42.1% in slincRAD^shRNA-9The number of S-phases cells in the cells further increased to 51.1%. Correspondingly, the proportion of wild-type cells at G2-phase was 28%, in slincRAD^shRNA-8This ratio dropped to 18% in slincRAD^shRNA-9Of which only 10% (fig. 11). These data indicate that knocking down slincRAD affects cell cycle behavior. We also found that there was a clear dose-response relationship between the level of inhibition of slincRAD gene and the effect on cell cycle.

Example twelve: effect of knockdown of slincRAD on apparent Regulation of cell differentiation

As an important aspect of the effector mechanism of long noncoding RNA, the apparent regulation and control in the differentiation process of fat cells are systematically studied, and the methylase DNMT1 is found to play an important role in the regulation and control.

Based on these and our work, we speculated that DNMT1 may also be involved in the signaling pathway of slincRAD action. To test this hypothesis, we first investigated the changes in expression of the Dnmt1 gene during the induction of differentiation in adipocytes in vitro. It was found that the expression level of Dnmt1 gene was reduced in day (-1) and day (0) at the initial stage of induced differentiation; after the addition of the differentiation-inducing agent on day (day +1), the expression level of the gene was abruptly up-regulated to a higher level (FIG. 12). These results indicate that, similar to slincRAD gene, the expression level of Dnmt1 gene is tightly regulated early in the induction of differentiation. This co-regulation suggests that the two genes may also be functionally related.

To investigate the possible interaction between slincRAD gene transcripts and DNMT1, we performed RNA co-immunoprecipitation experiments. The research protocol is referred to essentially in Keene et al (Keene JD, Komisaw JM, Friedersdorf MB. RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein compositions of ribonuclear protein compositions from cell extracts. Nat. Protoc.2006; 1(1): 302-7). On day of differentiation induction, 1X 10⁸3T3-L1 cells were cross-linked with 1% formaldehyde. The cells were washed with a Phosphate Buffered Saline (PBS) solution containing 1mM PMSF, lysis buffer was added, and sonication was performed to lyse the cells and release the nuclei. To remove non-specific binding, 50. mu.L of protein G D was added to each sampleynabeads (Life technologies), incubated at 4 ℃ for 1 hour. After removal of the Dynabeads and the proteins bound to them, the remaining lysate was incubated with 5. mu.g of anti-DNMT 1 antibody (Abcam, China) and incubated overnight at 4 ℃. Add 100. mu.L protein G coated magnetic beads and incubate with antibody-cell lysate for 3 hours at 4 ℃. After thorough washing, the proteins are degraded by proteinase K, releasing the RNA component into solution. The RNA fraction was precipitated, treated with DNase I (NEB) enzyme to degrade the genomic DNA therein, and the finally obtained RNA fraction was resuspended in 50. mu.L of RNase-free deionized water. Negative controls for this experiment were samples incubated with anti-IgG antibody (Santa Cruz) and samples without antibody treatment.

And (3) carrying out reverse transcription and qRT-PCR amplification on 150ng of the RNA co-immunoprecipitate product, and analyzing the content of slincRAD gene transcript. The sequences of the primer pairs used for PCR amplification were as follows:

slincRAD-A forward:5'-TCTGAATTGCCCATCTCTCC-3’；

slincRAD-A reverse:5'-CGTGCCTATGTTCCAATATCC-3’；

slincRAD-B forward:5'-GCCACAGCACTAGGGAAGAC-3’；

slincRAD-B reverse:5'-ACAGTCATGCGTGAAAGCAG-3’；

slincRAD-C forward:5'-TACCATGTCGGTCCCATTTT-3’；

slincRAD-C reverse:5'-TGTGCCAAGTCTTCAGGTTG-3’.

on Day (0) of differentiation of 3T3-L1 adipocytes, we used anti-DNMT 1 antibody and RNA co-immunoprecipitation to capture the RNA component interacting with DNMT1 protein and with it. Using qRT-PCR, we found that slincRAD gene transcripts were enriched in anti-DNMT 1 antibody complexes compared to the negative control group, indicating a physical interaction between slincRAD gene transcripts and DNMT1 protein (fig. 13).

The physical interaction between slincRAD and DNMT1 suggested that we involved slincRAD gene transcripts in the epigenetic process of adipocyte differentiation. To identify downstream signaling molecules regulated by slincRAD, we entrusted shanghai kangcheng bioengineering, inc, to investigate the effect of slincRAD gene on downstream factor Promoter methylation levels in wild-type and slincRAD knock down genes in 3T3-L1 cells using a gene Promoter methylation chip (NimbleGen meCHIP, Arraystar Mouse RefSeq Promoter Microarray).

The experimental procedure was roughly as follows: on days day (-2) and day (+2) of induced differentiation, cells were harvested and genomic DNA of the cells was extracted using DNeasy Blood & Tissue Kit (Qiagen, CA). 1 mu g of genomic DNA is subjected to ultrasonic treatment, and subjected to co-immunoprecipitation with an anti-mouse 5-methylytosine monoclonal antibody (Diagenode), thereby obtaining a 5-methylytosine-containing genomic fragment. After labeling and purification, the hybridization was performed with an Arraystar Mouse RefSeq Promoter Microarray, and the hybridization chip was scanned with an Agilent Scanner G2505C Scanner to obtain the chip hybridization results. Chip data are analyzed by an algorithm provided by NimbleScan v2.5(Roche-NimbleGen), a p value is calculated by using paired t-test, a threshold value of methylation degree up-regulation or down-regulation is set to be that the multiple change is more than or equal to 2.0, and the p value is less than or equal to 0.05.

During the in vitro differentiation induction of 3T3-L1 adipocytes, cells were in a proliferative state on day (day-2) and entered a differentiated state on day (+ 2). Therefore, by collecting cells at these two time points and comparing the expression level of the gene with the methylation level of the promoter, the apparent regulation of gene expression during differentiation can be understood. We first analyzed wild-type cells and found that the methylation levels of 7509 genomic sites in wild-type cells were altered during differentiation compared to cells during proliferation, and these sites corresponded to 2334 mouse genes (peak score > 2; p < 0.05). Of these, the methylation levels of the 582 gene promoters were up-regulated in differentiated cells, and the methylation levels of the 584 gene promoters were down-regulated in differentiated cells. Further analysis of slincRAD knockdown cells revealed that methylation levels of 941 genes were up-regulated and 469 genes were down-regulated in cells in differentiation compared to cells in proliferation.

To visualize the methylation status of gene promoters, we performed Venn diagram (Venn diagram analytes) analysis on these data. In wild-type and slincRAD knockdown cells, we found that the expression of proliferation-related genes was generally suppressed, while differentiation-related genes were generally induced. The number of proliferation-associated genes inhibited in slincRAD-knocked-down cells was lower than that of wild-type cells, while the number of differentiation-associated genes induced was also lower than that of wild-type cells (fig. 14). Wherein. 9 proliferation-associated genes were specifically methylated in wild-type cells, while the other 4 proliferation-associated genes were specifically methylated in slincRAD knock-down cells. For differentiation-related genes, the opposite is true: 20 differentiation-associated genes were specifically methylated in wild-type cells, and 94 differentiation-associated genes were specifically methylated in slincRAD-knockdown cells. To further analyze the status of each methylation site, we performed differential expression peak (DEP Promoter) analysis on the differentially regulated genes, finding 550 DEP sites in wild-type cells and 913 DEP sites in slincRAD-knockdown cells, with a total of only 49 DEP sites.

Subsequently, we performed Gene Ontology (GO) analysis. The GO analysis classified the genes differentially regulated into 18 groups, including the protein metabolome, lipid metabolome, proliferation-related groups, etc. (fig. 15). Considering the effect of knocking down slincRAD on the cell cycle, we further classified proliferation-related genes with reference to the following criteria. 1) In wild type cell differentiation, the methylation level of the gene promoter shows an ascending trend; 2) in slincRAD knock-down cell differentiation, the magnitude of the increase in promoter methylation levels was reduced compared to wild-type cells; 3) the gene promoter has DNMT1 binding site. The first two criteria were used for screening to obtain 12 genes, including the cell cycle regulatory gene CDKN1A (p 21). Reported research work indicates that the CDKN1A gene is closely related to the differentiation process of adipocytes. When data from wild-type cells were analyzed, it was found that the methylation level of the promoter of p21 gene was increased 2.64-fold in differentiated cells compared to proliferating cells (FIG. 16). However, this was not seen in slincRAD knockdown cells (fig. 17). In addition, we identified multiple DNMT1 binding sites in the promoter region of the p21 gene, suggesting that this gene may also be involved in the epigenetic regulatory pathway of slincRAD/DNMT 1.

Further, we examined changes in expression of p21 protein, cyclin-A, cyclin-B and cyclin-dependent kinase 1(CDK1, cdc2) using western hybridization. The western hybridization was performed according to a conventional experimental protocol. Specifically, differentiation was induced in vitro on days day (-2) to day (+4) of day by day in adipocytes, and cells were lysed to obtain total cellular protein. Total cellular protein was treated with RIPA buffer (1% NP-40, 0.5% deoxycholate, 0.1% SDS,150 mM NaCl and 50mM Tris [ pH 8]) with the addition of a protease inhibitor (Rocktail protease inhibitor, Roche). After sonication, the protein sample concentration was determined using bicinchoninic acid assays from Pierce. The protein samples were then loaded and separated by SDS-PAGE electrophoresis. For hybridization, a polyvinylidene difluoride membrane from GE Healthcare was used, and anti-DNMT 1 antibody from Abcam, anti- β -actin antibody from Transgene, anti-p 21 antibody, anti-cyclin-A antibody, anti-cyclin-B1 antibody and anti-phosphorylated cdc2 antibody from Shanghai CST (Univ-bio) were used as primary antibodies. The secondary antibodies were horseradish peroxidase (HRP) conjugated antibody, rabbit HRP antibody and mouse HRP antibody from CST. Hybridization blot imaging was performed with the BIORAD ChemiDoc XRS system.

We found that expression levels of cyclin-A, cyclin-B protein in slincRAD-knockdown cells did not change significantly compared to wild-type cells, whereas levels of p21 and phosphorylated cdc2 protein were significantly different. On day (0) of differentiation, expression of p21 protein and phosphorylated cdc2 protein were significantly increased in slincRAD-knockdown cells (fig. 18).

Example thirteen: hypothesis of the mechanism of action of slincRAD

Taken together, we propose the mechanism of action of long noncoding RNA slincRAD to regulate adipocyte differentiation through the slincRAD/DNMT1 apparent regulatory pathway, see in particular fig. 19. In the early stage of cell differentiation, slincRAD gene transcripts alter the methylation state of promoters of downstream factors (e.g., p21) and regulate their expression levels through interaction with DNMT1 protein factors. For p21, down-regulation of the gene expression further down-regulates the phosphorylation level of cdc2 protein, thereby activating the cyclin-A/cdc2 and cyclin-B/cdc2 protein complexes and causing the cells to shift from an active proliferative state to a "growth-inhibitory" (growth-arrest) state.

SEQUENCE LISTING

<110> Beijing university

<120> adipocyte differentiation related long non-coding RNA and application thereof

<130> 2016PKU006

<160> 27

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 1

caacuaggcu ucacaaauat t 21

<210> 2

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 2

uauuugugaa gccuaguugt t 21

<210> 3

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 3

gcguggaugu ggagaaagat t 21

<210> 4

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 4

ucuuucucca cauccacgct t 21

<210> 5

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 5

ccuagcagaa acuaaacuut t 21

<210> 6

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 6

aaguuuaguu ucugcuaggt t 21

<210> 7

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 7

ggaagaggau gacuaggaat t 21

<210> 8

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 8

uuccuaguca uccucuucct t 21

<210> 9

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 9

ggauuagugu ggcagauuat t 21

<210> 10

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 10

uaaucugcca cacuaaucct t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 11

ccacuacccg ccaugauuut t 21

<210> 12

<211> 21

<212> DNA

<213> Artificial

<220>

<223> siRNA

<400> 12

aaaucauggc ggguaguggt t 21

<210> 21

<211> 87

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 21

gtggaaagga cgaaacaccc ggcctataac cagtacagtt aatcaagagt taactgtact 60

ggttataggc ttttttggat cccgaat 87

<210> 22

<211> 87

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 22

gtggaaagga cgaaacaccc ggcaccaacc tcttccaata tctcaagagg atattggaag 60

aggttggtgc ttttttggat cccgaat 87

<210> 23

<211> 83

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 23

gtggaaagga cgaaacaccc gggattagtg tggcagatta tcaagagtaa tctgccacac 60

taatcctttt ttggatcccg aat 83

<210> 24

<211> 83

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 24

gtggaaagga cgaaacaccc gggattagtg tggcagatta tcaagagtaa tctgccacac 60

taatcctttt ttggatcccg aat 83

<210> 200

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 200

atccatctcc cctacccatc 20

<210> 201

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 201

tgcagaggac tcacaactgg 20

<210> 202

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 202

ggtgcagttt tgcactgaga 20

<210> 203

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 203

gatggagaga aaggaagggg 20

<210> 204

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 204

cctagtgcct ttctgccttg 20

<210> 205

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 205

tggggtgatt gtgtagggat 20

<210> 206

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 206

caagtggttc cggattctgt 20

<210> 207

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 207

ccgtgaaatg aagtggtgtg 20

<210> 208

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 208

gcatttaatt ggtgctggct 20

<210> 209

<211> 20

<212> DNA

<213> Artificial

<220>

<223> artificial

<400> 209

gacccttctc ctagctgcct 20

<210> 300

<211> 102074

<212> DNA

<213> Mus musculus

<400> 300

ttgcaggtct cctcctatgg tcaggtttca cccagtacag aagagagctc cctactttct 60

ccctaaagga gaaagtcttc cactggtggc ctttggatca agatgtagaa ctctcagttc 120

tttctccagc atcaagtctg cctagagcag ccctgcttcc tgctgtgatg ataatggatg 180

aatcctctga aactgtaaac catccccaag taaatgttgt cctttataag catgtcttgg 240

tcatgctgcc tcatcacagc aataagatca aactaagaca gcagatatca ttaattgttg 300

cttatattta tgaaccaaat gcaagacctt ctaggttata tagcccacaa ggttatgtaa 360

acccatctag gaaagtttat gatggtggat agacaataca actagaacag tctctgtccc 420

tattttaact gttcacctga tgaataatgg attcaggtga ctaagagcct tcttgtaggg 480

ttcagcaacc ttctaaaaat atttcattct tcatataaaa agttaaattt tacttggctc 540

acattgtttc ttatttgccc cagtatcagc taaactccta tgatctaata actccagatt 600

ttctttggtt tctatttcat agtactaaaa gttgggcatt ctcataggac ttaagaaata 660

tgggaacatg aatgtttaaa ttttttttct ttttaagaaa aaaaacaatg atgttaaaag 720

agtaggttat ttttacttta atttacctta gatgtgtcta aaatccaaag ttaaattaaa 780

accattagcc ttggagtaat aatttcctca agaaaaaaaa ttctttaaaa ctttattctt 840

tcttgttcat ttattcatgc aagttgtttt atggccacga gaattgtgat gatgttaaca 900

cgaacagtga tgataagcag ctaaaagcac tttgtcacac atgagtgtga ttaataggac 960

tcaggtaaat atgaaacaaa ctgtgtcaac ccttctctca aagctcaggg atcctcatgg 1020

gtgaagaaat gaaaatattg aaagacccaa ctgtagtgga ggactgcctt gaaccagtgt 1080

tttctagtca tgactgggcc attgcacaca cgggtcacaa tggctgtgac tatacacata 1140

aagcctgtac aagatcaaac cagccaaata tccagcacgg actggagagg gcctcacaga 1200

gtcctaccta tacctgaaga gcttttggaa gttaacagtt ggtgagggaa ggagagtcag 1260

atttcttcag ggatgtagca cctggaaggt taccaattcc tcagtggaga ggcccacacc 1320

cacatacata gtaacaacac taaatggtct taatgtttgt aggttgggtt gtttcttaaa 1380

aagtcaacag gggaagaatg gtggttagat tgtgggagga gttggagagg atgaactgag 1440

gagttggggt tgttccatgc tatatacatg aatggaattc tcaaagataa taaaagatgg 1500

aatgagaaat gactatacca atttttgtct ctaactttca catgtatgcc ttggcaccaa 1560

tgcacgtgta aacacacaca cacacacacg cacacacaca cacacaagta cacatacaca 1620

caggtacaag tacccacatg cacacatgca cacaattata tgtgtgctga ggtgggggca 1680

gttttctata tgttaaggga tctgaactca taggaaccac agatatgaaa caggaaatca 1740

aaggaaagtc aagtaaactt gaaaatttca aaagatgaaa tgaagggttg tgcagggctt 1800

gggggaagtg gttcatttta cattgacaag ttgacaggac aattcttatg tcatttgcat 1860

gagataggta cacctattac aatataacct ccttcggaac tacatctgtg ctacacagaa 1920

taaagaagct aaataaacaa cagtagcaaa gaacagaaaa gtgctttaaa aaagatatta 1980

gtagtttgag gtgggggtag ggaaacatat agtaagccaa tttgacccca gagagaggat 2040

ctgtttggcc cctctaatgg ttctaccaac cactaacgtg taccttctcc agtattcagt 2100

cttgccacaa gtccaattat aataatttcc atgagaagca caacagggtt ttagggatga 2160

tgagaatcac actgccatct gattctctgt ttttggctct tagcagtgtt ccaggcacgg 2220

agatgtacaa agtcaattca cttcagcttg aaaatgtcat ccctctgaca ttcaccaaaa 2280

accctccttc ttaccacagt gtttttgctg actgatcaaa ggggaattat tcttattaaa 2340

accaaattta aagtgtcagc cagatagaag ctccagtgcc ttgtggcatc aaagataacg 2400

ctaatgacca atgatacagg agaagctaag tgcaatgcaa attatgcagc aattctcaca 2460

cacagtgagt ttgagcttct tgcctgtgca aggatgggct attcttgagc cactatgcac 2520

aggagactaa atatgttccc tcttcatctc tcctacatgt taaggcaaag gaaggatcct 2580

acgctgacat gtacagtcca aaggagtaga gggaaagtgg acaggtttct ttagaacctt 2640

cgttgtgttc ctcagacaag ctggatagtt cccatttcta tacccttcca tgtcagaaag 2700

acataattat tggagcactg gtctctgtct gtccaagcag ccttggatca tgttctcagt 2760

tattcttcag tatttgcctg gacacagacc tggaggctcc ccaacaaatc tgtgcagtta 2820

atcagtacag gaaagcagtt tgcagaatcc tacaacagag aaagtgcctc ttctggtggg 2880

tatttaacta gtcacaaccc acatctgtgc tgatttgaga gggacagcca atgtgttaac 2940

tatgtgtaaa ttatacatat atgtactctt ctttagaaga gccaaaggag caaataaaga 3000

aaggactttg tcatcttcac ttagattcaa gaaaacaaaa caacaacaaa accagtatgt 3060

cagtaaagaa actggagtat tggtgctgag aagggatgaa gagcagaggt aaatgctggg 3120

ccatgagttg aaagcatcct agggaccccc ttaggcaaga acataaatta aactgctccc 3180

caaggccagc agtcagagat cagtcattct cttaacaaag gatttttgag gttctttatt 3240

tattgtcaag ggtttttcct gggcctattg ttgttgatgt tgttgttact aaaatatatt 3300

ggatggtcct ccctcacacc ttttgatttg agccaaggtt ataaaagtaa tttgaattgt 3360

atatgagccg tttcacatcc tcatgaagcc tttcaatatg acacatttga aagtgaatga 3420

ttgtgagagg gatgttgaga atagtaaagc cttaccagga aatgtgaatt gcattgctga 3480

catttaccca taaatgaatt actcacagac tcatgcatat gcagaaacga ttgaaatttc 3540

tccattagtt aattgttgat caaaatttta ttagaattta ggtacatatc atttgggcat 3600

attattttct taggtgactg tgtatcgtgt aaaataaaat gagtctccag agtaactgaa 3660

aaaacaaaca aacaaacaaa aaaaccaaac aaacaaacaa aaatcactac attttacaat 3720

tatcagataa ggtgtagggt gataaataca acctttacaa catctgggaa accattattt 3780

ctagaaaata aactgtaaac taagcatgat ttcattatgc atggctttaa agctacgaag 3840

aaacttatgg tttcctctgt tcctataagc tattgtcttt catatcgcat gtttctaagg 3900

aagtgacaca caaaacctca ctgtgccttc taggtccatt gtgaaggtag tgtgataata 3960

taatgaagat actctgaagt acttggaagt gtgtgtgtgt gtgtctgtgt gtgtgtctgt 4020

gtttatatag tcatgtaagt tgtagtgaca ttatggcttt gaggaactac agaatgaaca 4080

tctaatgaga tgtttccctc ttatttaaat agaatgccat aaatgttttc caataaagaa 4140

atcagtgaat tattattcct tatgattcag gttatttggg aatactactg aatttttttt 4200

cctcccaact tcattatttt ttaagttgct ttattatgtc atcaagaacc atgattcatt 4260

ggcacatgca ggaaagtgtt gggcctgagc tcagaaacac aatgtcttat tccacttaag 4320

tcaacagctg tccagagcta acaaactaca ttcttagcaa taatttctaa aagccatttt 4380

gttttttttt tctatcttaa tggtgctatt caattctcta ttccatccct aaacaaagag 4440

gggcaatgat gtaagttatc agaagctgca gacaacccat tgatccctcc attagaataa 4500

acagttgagt gctgtctgga cagatgtgag gattgcagac atgtggtaaa agtaactgag 4560

ttgggttttt ctccccttgt ggctttagca cattattgaa ggggagttcc agctgagatc 4620

agctctcaag ttgaattgtg tctatttgtg gttccaaggt catcagtagt cagagagcga 4680

atacagatta atgtagggaa ttgaatttaa ttctcattac cctggaggaa tcactctgaa 4740

tggatgctgt gcccatttgt ctaacagtcc cttctgcgtt cctcctgcac cttatctaca 4800

gtaatacttt caccgaggca agtaaataat ccagaaaacg acatatcgtt gtaaaagacc 4860

acaagtgtgg attgtgcagt tttaccgcca gtgactagaa taatagctgg gatggaacag 4920

atgtggttta aaatcttcta caaatgagtc acctagaaaa acaccttaca ttaacccttt 4980

caaatggtca gtttttgaaa gaagtcagat agaattagag ggtggagctt gtaaagaaac 5040

cgagataaaa tggtgtaatg gagaccatag gttcaagctt gtttggctgg acaatgagtt 5100

ttttttaggt atttgcttac acacaaataa agcaaggatc cggcctcctc cttgctcttg 5160

agggaaatca tgtgtgcctt ttaattacga actttatttc tgcatgcttc cctttggtcc 5220

aaatccatct cccctaccca tctttctctc cagcaggcta tgcagtactt tcctaatgtg 5280

gagcgagtat tcctgtttca tcagttgctc ctgaaaaaaa ggatctggcc tctgttacag 5340

acactttcgg ggagcatcct ctgagctcta cttgggaatc cagttgtgag tcctctgcat 5400

ttcataatca ctgaccaaag ctcgggaagg gctcctttca gccctgtcct tcatttgaag 5460

agaattgtct ttaagcttct gcagaaagtt gagtgatgcc ctactttaaa aggtagaggg 5520

ttgctttatc cagccttaat gccatagtgt gtgcctggtc ctattgtggc attattatgt 5580

tgtgtttggc tgatcttctt gggggaaggg gtacctgggg gagaggaaag gatggtgttg 5640

agagattgca gggagtccgg caaggggaaa ctgagactgg gatgtacaat acgagagaag 5700

gaaaaaaaaa gaataaaaaa taaaaaaata aaaataaata aataaataaa agatgcagac 5760

tgtgtggtta aggtgagatg cttgaaaaac aaacctacca gatcccttcc ctcagatatg 5820

caataaagtt ttgatgatac cgttttgttt tgttgttgtt gttgttgttg ttgttgctgc 5880

tgctaataac aagaacactg agtattggct tgctttcaat tcttttaaag acacttttaa 5940

aatgctgtac aagtgaaagt tggaagcatg aaatattatg tgtgtgtctg tctgtctgtc 6000

tttgtgctac atgcacgtct gtgtatatgt gtatacgggt gtacatgctg aagtgtggag 6060

gccagcagtt aatgatcagt gccttcctat aatgttttcc acattacatt ttgagacagg 6120

tgatcacgcc taatctaagg cttagcagtt cagttacacc agctctccag taagctgcca 6180

agatctgcct cacatcccaa ccccaccgag cactgcagaa ccacttgtgt gctgatttta 6240

tgtgattagc cagattttgt gtgagtgcta tagaacctga ctcagagctg caaagtgaga 6300

gctttatcca tagagacatc ttcccagccc tgataatggt ttttgaaata tcaaaataaa 6360

tatcaaatgt catcctctag ccattctcag acaaattata gaagtgtcaa ttgagtgcta 6420

caaaaattag atacacaaag agggttaaga aaataacaga agtttctcaa ggatgaaagt 6480

caagccacac tctgaactca aaagatgctg aggaagctga aacttgggca ggtgagtctc 6540

ttttataaaa gatttgtgga gtctctgaca aatctctgaa ttctgtgtta aaaaaatgaa 6600

ccaaactatt ttcttgcaaa acaatgtcca tatgttttcc tggagaaatc cgaaatcgct 6660

gagactctgt cataaggaca catttctcct ttgaccagtt ctgggacaga tctgagaatc 6720

ttatgggatg tgttccagta tagaattgca tttttaaaaa ttgataaaaa tggtcttatc 6780

actgcattgg gactttgaag tttcaatatt tatcaagcta gataggcgaa acgcactttc 6840

ctaaaaggaa ggactataac tcggccaaac aaagaaaggt cctgtgtgcg tgttaacact 6900

ttaataagag attacaccaa aggtcacact ggttcaacac attataaaga ggacaaaagt 6960

atgtatactc aaaggaagat aaaagtgctg aaaacattgc taaattaatt accagaccct 7020

taaatgtctt ctagcaccaa gaccatagag agtgaccttt gggttacttt ttttcctgtc 7080

tctcagaaag aaccatttgc atctcaactg gataaaaatc atttgcattc taacaggttt 7140

ctaaaggtta aacttaatta tacagtctgc aatgctatca gtagtaatat aaatcaaacc 7200

aaacggacgc ttcttagaga atttgataag gttagtgaag gatgggtata tggccaccat 7260

cttctctcac caccaacgtt tgcatacttt ttcactgtag aagaaataaa ataagatctg 7320

agaaatccca tctcaatctt gaatgaaaaa aaatgctttt tctgactaga acaagcatcg 7380

caagcaagga tggtgacaag tacagactca gagtcattcc atgtatcacc tgaccccagc 7440

tatttagggg aaggccagaa actaagtctg actgaggact cacgaatgcc cttttgtttt 7500

cccagagtct gactaaatta tggccaggtt tttatagcct cttccttact ggtgttttac 7560

aaaatgaaag ctagggaatg ctccacttgg ggatgaggca ttatgatcat aaacagtggg 7620

cgattatcat gtagaagaat gcgaattgat ccattaaaaa gagtgggata ttgattgttt 7680

tgtagtgtag ctccctataa tggaaacata ataatggaat accttagggg aaggggtctg 7740

gaagaatgaa acaaatatgg aattatttaa cttcactgtt gagaacccaa agtagaagca 7800

ccacctggat ccagcttggc catgaagagg caaatgaagg tcatgaattc tgtgagatag 7860

cagggagatg catatcaaaa aataaaaata aaaataaata ttgggcgaag acctgaaaaa 7920

gcttggaacc ttttcttcag ttaagcaaga gagcacatgg ggtaagtgca tgctggattg 7980

tggaatattt tttgaaaaat gaacactgca tttactctat gctgtgttta agtcttagct 8040

ctacatctta gtagctatgg gaacttagtc aaataacaaa accatttatg cttcagttac 8100

ccaatgatta aaaatatgga ctaaagcact tctgccacaa agcttgtgga gaaaattgaa 8160

tgaatatcat gtatgttcct ggtgggctat cagtttatat ctgccacatg tcttcgaata 8220

cttaaagaca aatacttcaa atacctgagc acattttgtc agtttttctc taattataag 8280

ttccatgtct tccttttttc ctaatatgca cagatcacct ttaagttaca ggcacgatgg 8340

tttgcagact gcatggtatt tatgatttgc tctgagattg gaaagaagaa ggaaaaatgg 8400

tacctattac ccactttcct tgccaagtca tttggcagat gtaatatcag gatagatgta 8460

tcagttccca cgtgcttttg tgatgctatt atcacatgag cagagtcttc aggcaagtag 8520

tggtaaagca ggactgagcc ttgataatat cagttctgga ccaacagttg atttcatttc 8580

tcttttgtaa ctaattcgtt gcagcaaacc accctacagt tgaagtacct cgtttctgtt 8640

gttcagactg tacacagcag cttccactat tgttaatatc cattgcagca gcattgttgt 8700

atcccacaga aatactttct agggatggaa acatctctca gcataacctt cccacatagc 8760

ttttgcaatc tatttctccc atgctgtttc acagaaaccg tccttaactt agaagtgtgt 8820

acattgggta attaatggtt acatgaacgg atgcttcatt taactctcct ctaggtttat 8880

tttcatctct catcaatggt ctaatgcgag gagagattta gtttatttaa aataagtcta 8940

cgatcactgg gtcactggca ttttggagta cagatttttg gacaattaga aaaatgtata 9000

atcaataaaa gaagagtcct tctgtgctac aaggcaagct tgtctagaaa tattgtgtga 9060

tttacctcta aaacgttatc ttttcagctc tttattgatt agtcgtgact tataactgtt 9120

tgaagggagg aaatatatac tccacagata acactgtctc acagcaaagc aaaggtaatt 9180

tgtttctaac agctggtctg tctgggtaaa aaacccatgg gtcaatgcat tagatatcca 9240

aaactataca ttcatgcaag attttgtcat tgtgaggact tacaactaaa gaatagttca 9300

aagagtgttc cagaaaagtc agtaatgaga agagggagta tatatgtcat aacgatgaat 9360

ttagtggaca tttgtgattg tggtgaccag tctatttgtt aaattttctt gattcccatt 9420

ctcctctttc ttgacggctt taattgtgtc tttaaattgt catgtccatc agtcttcatt 9480

gtctttccct gttaggctct gaagagcaca tttgctgaac ttgtcacctt cgatgctggc 9540

ttgtgctttc agacaacagg ctccttgtta tgcagttaga cctataaata gctgggcgtt 9600

gaacctgcta gctagaagat gtctgtactg aacaattctt tttcaggaac attttttaga 9660

aaataagctt ttgggtaagt ttatcatttt cctagtgaac tagagcatat aactgcacag 9720

tgcgttaata taaataaaga agatttgata ttaaagtact gcatgtagaa ttacagtcat 9780

tgtccccatt ttacctcttg gagtatgaga ttagtataga cttgagatag tctatcactt 9840

aaaaagaaaa gtacatttag ctcttgggat gtaatttgaa gagtcattga tacggagaga 9900

gcaaaataac agaacgagtc ctaaaatgag agcatttgaa ttcaacctgc aactctgaat 9960

tgcccatctc tcctgtcccc cctgcactgt cctcttcttg tcccttcatt ttcccctgcc 10020

ctcattctca tctccatctt tgttccaata gatcccctca tgcacccaca gagaagccac 10080

agaaaagtat cagggaagca catgtggaat gggcatccaa cgtaccatgc tattgatttc 10140

ataataaaaa ggatattgga acataggcac gtctattcgt ttacatacta aactggccat 10200

ttctgctgct tctaaagttg tttagtcatg gagacaaaaa ccacgtggct acatattcta 10260

tgataattac catctgacac tttacaggaa acactggctg acctctgata tagattatgc 10320

tagaaactac aggtctttag attttagtta cttatttatc tacttttttt ggtttgaaat 10380

acacaatgga gtacctcatc aaattttgct agcagcatga acaagaaaga aactagacat 10440

ccctggggtg gagtggggac gtgggaaaat acctgatttc agaagcagga taaaaacttt 10500

acaagatcca atgttgaggt actctgtgtg acattgtttc tctgtccttc ctcacctacc 10560

aaagacattc tcagatttcc ggtagtaaac atctaataac attagtaaat gtctagttag 10620

ctgggcagta agtggaaggt ggaacttcct aggtcagaga aaggaactgt gggaagaaga 10680

ggaaagcctc tttcaccaga aaacatgaga gattttggac atgactaagg accggaaagg 10740

tatagctaga atagattagg tgttcaagtg aaatgaacta gttgagagtc tgcccagcta 10800

agacctaagc tttgaaattg tgcttttttg aggaaatagc tggttaagta ataactgtca 10860

cccatataaa attccagcat taatatgcac agaatattaa tcatcacttc caacctgcct 10920

gtaagactcc tgtgaagaag taaagagcat gctacaaaac aagagaaaaa atatatagca 10980

aatataaata tagggatatc aagaagacaa atgaggccga ttatgatttt caaggtagtc 11040

gaagctgaga aaggtaagaa atgcataagc ccagtgatat tgggttctaa ctcaaaatat 11100

aaatacatcc ttatgatcct actgtgaggt tcccatcttc ttcagggtct ttccccaaac 11160

aattccatca gagtccccag cctctgtcca atatttccgt gtggttaccc gcagctgtgg 11220

gtaaagcctc tcagagaata gttatgttag gttcatgtct gtaagcataa catagtatca 11280

ttcctagtgt cagaggctgg tgcttggcca caggatgggg ttcaagttgg aacagtgatt 11340

gaatataaag aaatgactga atgaagaaat aatggagata gcagacacac tggtgaggga 11400

atctgatagc atttacatag ctcatctaac ttctggaagg tgagtcacca ccgtctactt 11460

ctgtctgaac tgtacacagt catttcctta gaatgagcaa agcaggatgg aaaaaaaaac 11520

agtaactttg cagttctaga acccaacaga cacttcctta gccaggcaac taaggctaac 11580

actaggctga caataacgtg taaatttgac ataaggcaaa acttaggctg acagtgccta 11640

cccttgaaat gatgattaag atagctactt tactttgtgg tctttctcca caaaatacat 11700

aattcaggtg ttttgtttgt ttttatttgt tttttttttt ttttttgatt tccatcaaac 11760

attaccccct cttcctgctc ctgccccaca caaagaccct acctctaccc cctcccattc 11820

ttatctgaga gtgtgcccca cctccatgta acctcccata ctcacaccct ggcacatcaa 11880

gtctctccca ggataggcat atcatttctc actgaggcca gaagaggcaa ccctgccggg 11940

aaatggatac cacaatcagg ttacaacttt aggtaggacc tccgctccag ttttgggggg 12000

cccaaaatgg agagtgagct gtctatctgc tacatatgta cccaggtcct tattcaagac 12060

catgtgaatt ctttggtttg tgactccgat tctgagagct tacaaaagtc caaggtagtt 12120

tgctctgctg gccctcctgt gagattacca tccccttcag ggcccttccc ccaactcttc 12180

cataagagtc cccgacctct gtccaatgtg ggtggattgg agtccttatc cctctcctgg 12240

ggatatagct caatggtaaa atatttgcct aggatgcaag agatcctggg ttcaaacgct 12300

agtaaccccc caaaaaaaac ccatttatag cataatcaag tttaattcaa cagaaaaata 12360

ttagataaat cctaattgag aaactctgca aaatacctgc caattactgt tatagcttga 12420

atctgtggta tctcagtgcc tcatctacta caggattgtt ctctggcagg tggtgctatt 12480

aggaggcggt gcctcaagga aggatacagg tcactgggga aattcatcca aaatgtattt 12540

tctcctgggt agtttcccca ttctcttgct ttattcttcc tggttgtcac aaaataagtg 12600

attcacagtc ctattctctt ccttgacact tctgtaacac taatgcttat gtgctatata 12660

acaatgaaca aaatccctga aatgatgaag cacagaaaaa acaaatattt atttctttga 12720

tttttttttc ctttttggta ctttttaaat gggaaacaat gtcaaagtca ttcaaagata 12780

gtaaaggtat ctcaaatcta gcattatggg atacatgaat ggaacagttt tattattagg 12840

gtaaatgtga aacctggata tccctggcac ctgagttctc cgatgtcagt ttaagatata 12900

tgtataatat gttacatgta ttttaattta tgtatttact tatagtacat ataattttaa 12960

ttcaaatact taatgctgtt gattaatgtg actctcatta ctgttttatt attctagaga 13020

aaggatttga tagtatagat ctcagcttaa ctatattcta tagctatgct tgcctaaaac 13080

caaagtgcca gatggtgatg caataaaatt ctaagccaaa atgcagaatt ctggtctttt 13140

tgaagtagaa actgatactt catgattgct gtgtcgctgt gtcaggttcc attgccatca 13200

aagacaagct aagagatccc taactcagcc tgatttcttg tttggcaatc atcgaggagt 13260

cattatctga aaataatata tgaatcttga taaattttag ataataaaat agtgacaata 13320

ttttatcttg ttcattgtaa caaatgtatc ataaaaatat aaaatgctaa taaggaaaag 13380

tggctgtgag gtagatctaa attattattt tttgtgtggc tgcaaagtta caaaacattt 13440

cactggaaac cctttgccgt cttcctattt attactcatg ggctcctttc ctacaagtgc 13500

aagaaaaatg aggtagtcta tttcaagaca ggacaaaaag agtgggaagt cacagtcacg 13560

ctctggctac actggagtcc ttcagaagct ccacataata acaatataga aagaggagag 13620

gctgtgggta aagggtgtag tctactgggg acacagttaa ttcaaggact gtgttttgga 13680

aataactgaa aagcaacaaa agtgctggta caacctctgg gactttatag tgagcaacac 13740

agcgattcaa cagctcattt gtattttgga gggaaatcag cataagaaag agaaaattcc 13800

tgtatttaaa ggggggaaaa cattttaaag cacagtgtat ttccaaagaa attgttactg 13860

cctggtagaa aatgtgtggc tcataaaaca aggacatttc tatgccaaga atagatctaa 13920

aattgacaca tagttcaacc tatgcattgg tgaaatttga aagctttcaa gactcaagga 13980

aaggtgcgta caaagctact gttcattata acatacataa agcaccgatt gtgaaccagt 14040

taagcatact gtagtcattc tgacatggaa actgatactg tttgaggggt gggtcaagtt 14100

ctgtccgtcc atcaggacag ggagtcattg gcatctctgc cctctactgt cagatacaaa 14160

gagatggatc ctcattcaat cctcaggaaa tcacattttc aatctacaga agcataagta 14220

agcaatctaa gggactcact gttacgaata ttattatcca aagatcaata ctttcaaacc 14280

ttctccagca taatagaaca tcatcaaaag tcacccggac tgtgatgtga aaatatattt 14340

aaagaacact gttagtcttc atgtggctca cgatttgggg gaaaaaaagt ggtttcacct 14400

atgtatttat gtattttata atattagacc tgagatatat aagcaaacag atagtttcca 14460

ttgatttaat atgcttgtct caggatagca tgggcacagt tcaattagca atatcctacc 14520

ttgcaaatac ccaatgacct ctacatttgc accaggcttt gttcaaatac tttgaggcca 14580

gaactgtcat gaaagccagt ctgctttatc acagaactag tagtctgata tactatgttg 14640

gtattaatta agtattcatc caaaccaagg tgaaattgct taaataataa tctagaagag 14700

gcaaaccaag gtgaaatcgc ttaaataata atttagaaga ggcaaaggat gcttaccatt 14760

ctcttaagaa aataggaagg cattgaaaac ttaaagcaca aagggctatt tttgtttttt 14820

tgaaactccc aaggtctaca gagtgtttaa atggtagaat agtctggtag gggaacaggc 14880

aggttacaag gtcagtaaag aattggggag taaaataatg aggtcctcag cgagactgtt 14940

ttccctgagg acaccaaaaa gtgggcaaat gtgaggatca tgtaaagaat catttgtacc 15000

tagtgataga ctgagcaaaa gggaagagaa ataaagacaa acaaatgtaa aagttgcctc 15060

aggaatcctg gctactgaac cataatcttt gtcgctgtcc tttcaggtag actagaaaaa 15120

tcaatatgat gtaaactctg acaaggtaga atagaatgca tctattttag aaggttaaga 15180

taccaccgaa acaagagtga aggcacatct gggtgtcaga ataaaacaaa acaaacaaca 15240

acaacaaaaa aaacacaaca aagcagaaac caaaaactct gaaatcatcc actcaggaaa 15300

taatgagaat acaaacaatt acctaaatta gctactttaa aacttggaaa atcaacccaa 15360

atcttaaaac tgcatggaga agaacgatgg ctgaaagtca gcaaagacaa tgagcactgt 15420

gacattttgc ctcacactcc actccctcca tcattgtctt ccgaattcca taaccgctct 15480

gacagtcagc aagttcacaa tatgccagag gaacagctag catagcaaca gacaagaaag 15540

acatcaagtg catttggagg attctaaagg gcccatccag gaaacacatt tcatttttct 15600

cttcctagca gctcccaatt aaggcttagt ctcagggctt gcctttattt gatacaaacg 15660

cagaattctt cctcaatggc tcttacctta gggtatttgg agaaaacagt ggaaatgcac 15720

tatattgcag gtggctgaaa caataaagtg attaaaagct aacagggaat ttaccaaaac 15780

tatttacaag ggaaaatgga aatgacttgg cctcagggga attagacaga tacaatgtat 15840

cactgaacgt tatatcatgc tcctcgtgaa gagctataca catgctgggg atgaccttag 15900

aaaaccccaa tctcgaactg caaccctata ggtggaacaa caatatgaac aaaagaaaac 15960

cccaatctct cacctgtgat ctatactcct gctcttctta aacacggaac aaagactaaa 16020

gatgttttga atgggctaga accctgaaag tatacactac atgtaaatat agcaaagatt 16080

tggcttattg gtttaaggct ggtaaggaaa ctcccattta attattagtc aaatattaag 16140

ctaacagaga ataaatttca tagctgaaca gaaccaagaa cacaaactta caaaaattag 16200

gtgagaaagg tattaaatag gcaaagagaa attttagcaa tgattttttt aaaagtccac 16260

aaatagcaaa agagccaagt tacaaagtat tctttcaaat ctgaagtgtt actacattac 16320

atgattttac atgtcaaatt ttatcagaaa aaaatggaac ccggaggagg aggaggagaa 16380

ctaggagaaa gaggagaaag gggaataaaa aggatgttca cataaaatag tcaataggaa 16440

cttgcatatc gtgtactggg ccataaatgt tcctcaagat tatctctact gcaggaatga 16500

tggtgctctg ggagaatgca aaagctgtag tagcccaggt aaaggaagca aggtcattct 16560

ggataatatt tccctaaaaa gcctggttgg acctgggcta tttcctctct cactttctgt 16620

tgcagatact aggatgctaa gtgtgtgtgt gtggtatata gcatgtattt ggaaataaga 16680

ggacaacttt gagaagttgg ttcttatttc taccttgttc agataaggta tctgttattg 16740

tccctcttat ttttccctag ctggtttgaa agctttcagg caatagttct atctctgctt 16800

cctatctaga ggcatcagaa ctgggatcac aggtgtatac aaccacatct gaaattttat 16860

gcggagcttc acaaatgaag ctcaggtcac aaggcatgta tgatgaatac ttttccccac 16920

taatctatcc ttctgccctc atccaaaaac ctttaaattt atttagttgg agtattttgt 16980

cacagcaaca gaaaggcaat taatgcagaa aattaataag gataataata acaccatgcc 17040

atatgatcca aggtctttcc aacttatttg caggaggtgt ttggggaagc ctggataaga 17100

agactgggga aagtaaggag tatgacagtt taatgggtgg ttttggtctg tgttcaaacc 17160

agaatgatga aaactgtgga caataaaggc tctgctcatg aacttccaga ttgaaataag 17220

gccatacact gggcaccttg aacagaagtc atttgttctg gagtagaatg agttaagtct 17280

gttcatgcct tcagtcttta aagagttaag tttaaggtga cagctagtta ataggatgga 17340

aggtatgtca gggccgcaca gtactcaaga ggtgccacag gaatggctgt ctcctttcag 17400

ttagacctag ggtaagaatc aagagtaaaa aaccatgagt ggaaaaattt gagaatcttt 17460

ctctggccag aagaggattt ctttgaaagt tggagccaag gaagatgtgg tttctaaaga 17520

tcagtatcac tagaaaggag ccagttctgt aactttagct actatgtggc tgatgcaaga 17580

tgattaccag ttagagacct tctttggcta cacaggaaga aagccagaac aacataatgg 17640

ggtcttgctt caaaacagaa agtcaaaagg aggctgggtc ggtaggtcaa gagcagaaca 17700

cttggcaaaa atacacacag tgggccctca ttttagaaaa actcaagcat ttttttcatc 17760

ttgagaacag cttttaaata ggtttagggc tataaaaatg tagacatttc agtttcaatc 17820

tgagataagg acttctggat gccctgctac aatggcacca ctcagggatg tcattcctcc 17880

agatttgtgt ctcggctgca gcctggtgcc aggcagagga gaaacttctc tcccaggaga 17940

agtgattaca catatctgat gatctgccat atgactttgt aggacaaact gagcaggaga 18000

cgatggtggg tcaatgaaga ttaggactgc accttaaata agactgctta gttattgaaa 18060

gctatttaaa actaaaccat attttatttt ataacaccaa agatattagg ggaggaatca 18120

gtggggagga atcattctct tcttgttacg gctacaatga atcaatctcc tttttctgct 18180

tgtcaacaat actttgtaac tgccacactg aggatggagg gctgagccca gttgaatagg 18240

gaatgccaaa acctagcttt gaccccagca tctccagtaa cagcactgct cctgcaatac 18300

tgcttttgca aacacaaagc ctggaagaat tacagtgtca cacatttcct ctcatatttc 18360

aaacacagac aaggaagaag cagcagttgg agctgggtta gagtccctga cacttgcatt 18420

gagtgggcag tgtgtaaagc tgtatgagtg aaattggtgt tggagtggag actccaagaa 18480

actgggcagg ccagtaacat tgaattcttg ccacagctac cagtgacagt atagcagtaa 18540

ggccataaaa gcaggaatag ctatatactt tgtgtggcac tttaaacata ataatgaact 18600

aagtaaccaa taatgcacta ggttcacatt aaggaaaaaa aaatccaagt acttcccctt 18660

cgagttgttt aacaatgatg gagcccaaat ttaaattctc tactttgcac tacctctgtt 18720

ttccaccatc catattggcc tgttgtatga atctaaatct gctcctgcct gaaggctgcc 18780

atgcatttat gtccccgttc ttctgtctat gcctgttact ttctgtctga gcatacttgt 18840

ctctgacaat gttctctttt attaaacagc acataaaata gcaaatgggg aaggaaggag 18900

ggatgcttca tagaatttta caattattaa gccactgact ccaactgatc ccaactgacc 18960

ccaactgact ccagataagg aacagataac taatagtact acaaatatca gtgtttatca 19020

agcaatatgg aggatttatg gtagactttc catttatggt agatattcca ttcataggat 19080

tgctttaaac ctatacttga tatttgctaa cctatatttg atattattaa tgattgtcat 19140

ttatatatat acatatatat atatatatat aatttaaggt taaatttatg tttcatttat 19200

gtttatatag gttgtgaatg ttggttacat gggaaatcct tagacaagta agattggttg 19260

ttttaaagtc agctaaaaca aacaggctga gtactgaata ggattgccat cttaagtgac 19320

ctccccttat gtcatttact ctagcctgga gctccatcca aagtacaagc tcctttgaat 19380

gcaagtgaaa gctttatcat atggcatgaa catagctttg gaatgcccat gatactgtga 19440

tcactatgta atcaatccta gtgtcagaaa gagacgtgaa gatttaatat ttatatatat 19500

ttgcttcttg gtttgatcaa attccttcat gctgtttgtt tttcttcctt cttgaaatag 19560

gcatatttac tttacaaaat tatatcgtgg aatgctttgt ttttctgcga tctcacagat 19620

aagtttgctt tgtgtctcag aggagaggtt gactagagtt tttggcccaa ggctagacat 19680

atcatcacaa tagatcttga agctggactg ggtgcagttt tgcactgaga aagacatgac 19740

cctcttgggg ggtcaatgtt ggatgattta aatgtaaatg tctccagaag ctcatgtgtt 19800

gaaaggctgg tacccaggaa atgcaactct tgagaggcag ttggacgctt tagcggggag 19860

cacagggcta tgaggagctg tcaaacacta tgtaagacat tgaccccttc ctttctctcc 19920

atctgtactg tgtaaggtga acagtgttct ttaacagttt ttctcatcat cagaagctgt 19980

gctaagtatg agtggaaact tctgaaagtg gaagcgaaaa taaacatttt cttcttcatt 20040

ctactttttt aaagattata aaacacaact ttctctcttc cctttccttc tgccaagccc 20100

ttctatagaa tcccttttct ctcttttata tatatatgtc ctctttgtat taattgtatt 20160

cacacacaca cacacacaca cacacacaca cacacacaca cacatatcct atccagtctt 20220

tacaatgtag gacagacaaa aaataatgag acaaggatgc aagcagccat tgctagcctg 20280

atctatgatt taaaagattt caaactaaaa ctaatcagaa gagataaagt gaggcatttt 20340

attctaatca agaaaacagg taacagttca ccaaaaacac attagtatcc ttaacataca 20400

tgtgacaaac tctagagtat gcaattcatt taaaacaaaa acctactgcc aggtttaaag 20460

acatcagtta acaactactc attattagta ggtgattttt ttaaattagg tatttatttc 20520

agttacattt ccaatgctat cccaaaagtc ccccataccc tcccctcccc attcccctac 20580

ccacccactc ctttcaacaa tggatacagt aaccagacag gtggaaggca aggaacaaaa 20640

tgaaacacta aaaggaatac ttttagaaca aagatagaaa tataaacctc aaaccaattg 20700

aagactcaaa aatgatgaca aaacgttgga cttaatggac acatgcagac tacttcaagt 20760

agtagcacaa tatacattta tttcaagtaa aaagcaaatt tccatcagta tagattgtac 20820

tttatactat caaataattc tctgtatagc tatggattga tatactgatt ttgcatatct 20880

acctaaacat aattgaaaag caataggaaa atacaggcaa atgaggagac atacaattag 20940

aaaaatgtta aatagcacac tcctcagcaa ctgttgacac aaatgaaatc ccaaaggaaa 21000

ttattttgag acaccagaaa acaaaaaaca cacaacataa ggaaacatat gagattcaga 21060

caaaatgttc gtcagtgcta cacttgcatt cataaaatcc ctgttggaaa agcacaagcg 21120

tctcaagtga agagcctatc tttttacttt tggaaacaat acagaaagtt aaaactaaaa 21180

taaccaggga aaaacctaca gaaaggaaat taagagtgca aactcaggag agaatagaaa 21240

agcagtactc caaagcaatg aatgaagttg ttggttcttt gaaactatca acaaaacatg 21300

taactttagc cagatgaact agaattctga agagtcaaat gactgaagtc aaaattaaca 21360

aggagcattc taccacttcc ctctcaaaaa taaaaataga tacataacaa atactaaaag 21420

cattgagata ttaaattaaa tgacatacat acaaatccta ggaatataaa aattattaaa 21480

atagacatag tataagataa aaataaatag aaaatatgaa tggacatgta acgagccccc 21540

caccccccca aaaaaaccct tgaattaacc ataagattca tttgcattag taaccatttt 21600

cattatcaag tgtaaccaac atcttgacat tacaacacgt ctcagtcttt ttgctattat 21660

tgtgataaaa taccctggag gaaaatactt taagacagat tttaacaatc ttacaccttc 21720

aggtaacaac agtccatcca tcactaaagg tgaatcaaga aggaagtctc agcagacaat 21780

cacatccgta cacagaagcc cagagggagt gagggtgggt gtgcctgtca gtcagctcac 21840

tttctctagt cttgtatgag attcccagtt ccagggagag atgctgttca cctttaggct 21900

gaggctgtcc atgtcaatta acatcataag gctaatccct tgcagacatt cccatagccc 21960

agtttgagaa tccctgatca agactcgtat cataggtgat tctagaccat gtcaagttga 22020

cagttaaaac caacatcata catgactttt tcatctacat ctgtgctagg ttatgcttgg 22080

taacttactg tcctgggaca cctgcagctt gccatagact aactacagtg tagacattcc 22140

tggtaaagct agacattttt acttatgaat cgtgaatgca aagacatgca aggatacata 22200

gtaatgaatc ctgaagctct tccctattaa gtaacaacag cctcctaaat tgttctacat 22260

attcattgaa agttctctca ttatcagagc cgcctcatca gaaggattta acaaaggacc 22320

ctaacattcc ctactgaaat acggagggaa cagaaaaatg aaaacaatct tgaaaaagaa 22380

tgaagatgaa catattgtcc tttctgactt tgaaatttac tgcacaactg tggtaatcaa 22440

atgctgggat gctttttgta taaagcagaa aatacaggta agtggagtag aaattctagc 22500

aggaaaaagt aaattttgga cttacgttca atttattttc aacaagactg ctaggacaat 22560

tcaatgcaaa gaaaattttt ttgaacaaat agtgcatgaa aactcagtgt ccaatctttt 22620

ctgtctacca caatgacaac tgcaaaggta cattaataag tacagatatt ggagtcctgt 22680

atgttgtgag ggagaaatta aactcacctt gaaacggttc ttcaatgctt caaagtattt 22740

ggtgtagagg tcatacagaa aaaaatgatg tggaaagaac tcttccaatc accttggtat 22800

cacacttaag tgttcttaaa ccagagtcct gtatgtgaca acggaactta taaatctctg 22860

agaggaatgt tcaggagtaa atctttgtga cctttggtta tacaacagtc tcttagacat 22920

aatatcaaat tcatgaacag gaaaagaaag aaattaaagg aattcatatg ggctcagaac 22980

accccaagac caagttctca gcacaaagga gatattgtct gccccagagg gatatagaac 23040

agggaataag agagaaagac agtagaagca tgacaaggga gaaggggaaa ggaacaagga 23100

gagggagagg tcaggggtat ttgtcccttg gggacaaagg actgcctttg gatagagagg 23160

agacagactc aacccaaagg caaatggtgg tttataacag taaaaggaga aaccccatgt 23220

taggatgagg tgctaaattt caattaagca tgttaattag gagagtcaaa ggggctttcg 23280

atgagtggac tataatgctt tgatagctga actttggtag tcaatcttag gaagaggaag 23340

tagccaaata agggaataga ccttggcagc tagctctcgg gatgtaatct aaaggctttt 23400

agcaaggcag agggaacagg ggagaaaagc aaggcctgcc aaggccattt ttgccatgct 23460

gaggctgaca agagaccctt ctgaaaaaca aagagtaaat ttaactacat caaaaaaaaa 23520

tgtgtttcaa aagcaaccac caagaaaata aaaagagtgt gtgtgtgggg ggggggggga 23580

gcgttagaaa attctgtaag aaaactgtat tttgatatgt aaagaaactt ttatagacaa 23640

cagaagatga gattcatatt tatagaactt gaagtgattg gatatatatt caaagaagat 23700

acacaagtaa tcaatacaca caggaaaaga tgctcaatgt cacttgttac taaaaggtac 23760

aaataggaag ggctacgtgg taccacacca tcaccagtca gtgaataaaa taagcagaag 23820

acagcacttg ccagtgctgg ctaggaggtg aaaaactgtg atttcatatg ttgctgatga 23880

gctatgaagt aaaatggtat aactcatcaa gttgtgttac aacacaatga tgtcactctg 23940

ttttatggtc aagggaagta aagatgcatc cacagaagaa ttcatacatg gatagtcata 24000

gaagcaggct tgtgacaaca aagactccgg aaactataaa tatccatcaa ctgaacagta 24060

gattgaaata atgacatatc catacaaagg agtatgtttg gtctcactaa ttattcagaa 24120

tagtgaatct aggtagagag attgattatg ggccagggtc tattgacata agaagggggt 24180

gctgactgga atgagctccc ctctgggaat aatgaaacat ctaaacgtag agacagtaaa 24240

aatcctataa ctttatttgt gcttaaaatt agacactgga catttcaaat atttgatttt 24300

gagttaataa ttatcaataa acatcatgaa aataagaaac cacaataaac ttcaatgcat 24360

tatgcttata ggacagatga taattccaga tacaaattag aaaactggtc aatttccaaa 24420

tgttattaag tagaaatgtt cagaataaaa ctataaagta caaattttcg aagtagggaa 24480

gacacagtca cagagtccag agactgtaaa agcaaatagc tgcaaacaaa cttctgatac 24540

gctatgctta tgctcatggt cctcaccggc ttatagctat cattttagag aacaaaaaca 24600

tatgccaaaa gttttttttt tttgagacca gacgagagat attactgaga taaaaggagc 24660

cagctgagat gaaagtctgt ggtcctttgc tgtttctgaa catctcaaac ttttcaaaga 24720

catttggaaa ccagaacaaa gcttcaagac agagcaaaaa ctaaagggag ccattatctg 24780

taatagaaat tgccaaaaat aaagtggaag tactatgtga gcccttgggt ctatcctctg 24840

aaaactcttg gaaatttttc atgggaacct aaatagaggc acaatttaat gaaaaggaaa 24900

cattacttgt tttcatgtgt gtgtatggag tgctcaaacc tccacttggt tcttagagta 24960

attacgtata catgtgagaa cagctaaaga aaatatttgt gctaattttc acaagaaata 25020

tagtggaaca tcaaataaat ctaacagcgg tgtcacaaat ctaattatgg cctcattgtt 25080

tcagttagat tactttgttg ccgttagaat tttaatcaaa gtattgtttg tataaatttg 25140

aaaactcttg gcaattgaca agcttgagca atcatatctg acaaactaac caaaaagata 25200

aaataatggg gtcaaatgaa gtggcttctg aaaagtgtgt tcaaacctgt atcatccata 25260

gttgacattt cattgaacgt gaattcaact gagggatgca cctctggatg tgttgatgag 25320

cttgtttcca taaatgagag gagaagagag aagaagaaac tgagaggaga agagtccccc 25380

agagtgggca gccttttcca tcagtagacc agatataaag aggaccaaag aaaaaggaac 25440

gtagggttct tggcttgcca tcttcacttc tttatggcaa gcactggctt taggtatttt 25500

gctatttttg ttttgttttg ttttgttttg tttttctgtt gctgagataa taaccatcac 25560

ccgaagtgac agaaacccaa agagaagcat agaggatgga gtaagtatat tccataaagc 25620

cagcagcatc tgctcagggg tggtgctgcc cacagtgacc aagctagctc agtcagtcaa 25680

ggggctctcc aggggggtag caaggattaa aaagaaaaag acagttagac aacactgtag 25740

catgacccca gcctgttctg aggctgaagc aggcttattt ttctcagtct gcttttatac 25800

cattttaatt acatgcaaga aataaggtca gttctaggtt aaggaacaag caaggcaata 25860

acaatgtccg gtgatcagtt tctaggggct tatcaggata accaaggtat ctttttttgt 25920

cccagcccaa tgtcaaattg ctgccaagtt tctagaagcg gcccccaaag ctctccacaa 25980

cacagttgta tcggccccac tctatgagtt agcgatcagt aaaacgcccc catagatatg 26040

ctcataggtc agtctcattg aaaggagtcc tcagttaaag gttccctctt tccagatgtc 26100

tctagtttgt agcaagttga caaaataata aacgcagctc agcgtctact tctgctactg 26160

gtgccaccat catcactgac accaggaccc tgcttcttca acctcctaag acactttgga 26220

gtccaggagc tcttgaggaa tctacaggtc attagtgccg agttaggatt ggaaagatac 26280

tcagcttcat cggctgagca aggaccgggt tcttcgcctt actgctggga aaacactcac 26340

ccttaagact actcagcctg gatcctgtat ttcaacctaa aatctccctt ttcacagtaa 26400

tattgacctt gtttggttcg ttttctttag agtgcccaga caatacagtg cgccagtaga 26460

tttttaaatg taattttcta aaagatacga tggggaagga taaggaaaat aagatgagct 26520

tggggatgta actccgcaga gaatatgttc ctgtttccct gatcgcccac atcctgcctt 26580

tcttatcttt aactatagaa tgttatcttc ctcgcagata ctcaagaatg gttaaataaa 26640

acgatactct gaaacattcc ttgtgtagct ggcacacact gtaagtcctc agtaactatg 26700

gtgcttgtcg cttctatcaa taatgaatct gattttataa gataacaatg taacccagga 26760

taccaaagga acattgctgt gatattttaa agcgttctaa tatttattag gggttttaat 26820

actctttagc tgttataggg tcttgtctta ctcattagca ccagtaaaaa tgatgagaaa 26880

ataacaattt gtatttaaat atgtggcaag aaggtcacct tctgagaaac aagtttttcc 26940

tcttcatcct gtttaaagta tggaggaaag attgacccca gagtggctgc gtgacaacct 27000

ggcagaatgg gaaaaataca ccagacgtct tcctgagctc cctcctgcac atacagaaat 27060

gggagaggaa agagtggtta cttttcaaat cgctgtagtt gtcacgcaaa tgagtgtact 27120

gtacatactt gagtgcacac aaatacttca tatttagcta ggatgtgctt ctttcttatc 27180

aatctacaga gtaggtaggc ttcagattca taccagaggc atacaggaca acaaactttc 27240

cttaattatt ttaaaatttc ccttccagat cagcaatgtt ataaacttgg tgtgttaagg 27300

ctgctactat ggaatctctg gggttgtagt actgagaggc atcagatgga gactggttat 27360

gccagtaata taatacgggt accattactc tttcagcagg aaaagagaaa catgaccctt 27420

acaaaaactc taatatatac attgatacat acatagatac atagaaagat ttatatatgt 27480

gtgtacatat atatgtatat atgtatatta catacacata tgtgtgtgta tacacatgta 27540

tgcatacaca ttcacacaca ctgacataga ccttggtagc tccgcagcaa tatagaggaa 27600

tcactccttt ggagatttca catccataat atatgaccag ataaaatgta ggagctaaag 27660

agatttgcag cagcacacag ttaatccatc tcagggaagt agagccaccc taccagtata 27720

atttaatttt ggactggatt gagaatctaa cctgaattca ggtgcatttc atacataaaa 27780

ccatgggaca ggttcctgtt ctgtaaggtc tcgaaatagt actgaaaaga tcaggtgcaa 27840

gcagcctagg gcatctctcc attgtttcta ggaatagtgt tgacatcgat ctactcaagc 27900

aaaatttaaa tacacgccca gtaagcacca cctactgtgt gctgggcaac gtgtatctcc 27960

tttattacct ctttccaaga aacatcatat ttgtgtacct caagtttaca gtggttggct 28020

tactacaata gagtggagtc aattcttcag gcataagaaa tgttttaaaa ttctcatcta 28080

tcatttgtgc accattagga attctatcgt catcataaag ttgtgaggaa tagcaaacac 28140

atctattgca tggcaaatac tgcttcagat cttttacatc atctcatgtg atgccccccc 28200

ccaaaacgac ttcttggttg caatcacatg tataggtgca atcacactgt agtttcacag 28260

atgtggaaat gaagcaagag atagcaaatt gtttggctaa tattacaagg ccactaaatt 28320

atggaccagg gactcacaaa aaagcatgct gaattcaaac taaggactct gttaaagcta 28380

cactttcact gagtgatggg taaattatta gccagggatt taactttgct tttacctaaa 28440

cttattgtag tgtttctcaa attttaatgt attcatagat tcttatacaa aggtgtacat 28500

gttcaactca agtttgattg gcagccttag gggatttcaa cagcaatttt gccttactaa 28560

cttgagaaat taaacctcct gtgaagcaat cttaccctaa tacacagaat tgcatgcagg 28620

ctgcaatacc agtgctcctt agcgatgcaa ttgtatatgt taatcacaga tgaattacta 28680

actcatcggt ggtcagacat tttagtaaaa ttaatctgca taaggcatat ctgctcattt 28740

gctttgtctt tcctgttccc tttcttcaca ctggttattc atcactcatt catatccatg 28800

gactaacttt gtgaaccaaa caatacaaca cctgtacatg gttctgttaa tgagcaccat 28860

gctcactttt gctgagcaac aaccttttat agcactcagg attgtattaa ctcaaaggaa 28920

aaaccaaaca gaaaggacta agaggaaaca gcaacagaaa atgaaaaaca gtcacaatga 28980

cagccttgga gagatcagag aaggaagagc aacataatga gcaggggaca aaataagtct 29040

tggtgtttat aaaaacgcat ttcacactga gagactgcac actcactcct gagcaggaag 29100

cccagagcag tggcaagaag tctctgctgt gcggtctgca gctgctgagg aggctgggta 29160

ggcaccacct acctatcagc caacacgcta gggcagagcc cggtgcctct gaaggaaaac 29220

ctccgctggg cagcaatcac cacgggaatg tttcagacat gaccgagaca gaatgaagct 29280

agttggaggt ccccataata catacacaca tataaaatcc acaaaccaat cttagcaggc 29340

agttcctcaa ttgagggtct cattttccaa gtaacactga cttgtgtcaa atggacaaaa 29400

actaagcagc caaaggctta ccaaagtcca ttagagagtc aggaagaagt ggtcaggtgg 29460

acctgctggt ctgtgacaca tctacctact ttcaaagata caagagagct tctctccttt 29520

ttcacctatg cggccatacc acagcagcaa ccgcattcca gggttcaaaa tgacgtgcaa 29580

gaaaatgggc agctgtctta gttccagtta caaatcaaag gagagagcaa ctaatggtct 29640

catttttaag tagcatatat tcattgtaaa aactatgaga tttgattatg actttttctt 29700

tttctttttt ctttttctgg tttttcggtg attatgacat ttttatacac aaatatcata 29760

tactttgttt tgttttgttg attttacgag acaaggtttc tctgtgtagt cctggctgtc 29820

ctggaactca ctatgtagat cagactggcc tcaaactcag agatccacct ctctctgact 29880

cctgagtgct gaaattaaag gcgtgcacca ccatgcccag cctaaattgt ttttaatatc 29940

tagattccac atatgatatg agcaaaaaca gaatatttga ttttcaaatc tggctttttt 30000

ttttttttat taatgtgacc tacaactgtt ctcctgcaaa tgacataatt ttgttcttcc 30060

ttatggccaa ataaaactcc tttgtgttca ttatattttt ctttatcctt tcttttttaa 30120

cattaatttt ttaaagaatt atttatttat tttatgttta tgtagctgtc tccacacaca 30180

ccggaatagg gcatcagatc ccattacaga cggttatgag ccaccatgtg gttgctggga 30240

atcgaactca ggacctctgg aagagcagtc agtgctctta accatggagc catctctcca 30300

gccctttctt tatcctttca tctgttgagg gatacttagg ctgatgccat aacttagcta 30360

ttgtgagtag tgatgtactg gggtcccctc cgctatgacc ttgccagtat ttgttgttat 30420

ttgacttctt gatgacagcc attctaatgg gtatgaaata gaattcctat gtaattttaa 30480

agctctcatt agtttatctg gggaagatgg agtgtactca gaggcaggtc atggacccga 30540

ggagctggct cacccacttc accctgtgga ttttaggggt cgaactcagt ctgttagcct 30600

cagcggcggc aagtgtcttt acctaccatg ataacttgcc gacctcccag aatgtaattt 30660

taatttgcaa ttccctagtg gctaaagata ttgagcacat tttttcacat atttagatac 30720

tacttgtata tcctttaatt ctatgaattc tttctgtatt ctaaatatta attccaagta 30780

agataaaaga actaggaagg tttgccgccc attctgtcag ctgcctcttc atccacactt 30840

ggctctttca tttacggcac aaaagctttt ttattttatt caagcctata tgccactctt 30900

gcttttattc tgaggatgag agtccttttc agaaagtctt tttgaaccct gtatctcgaa 30960

gtatttccct ctagtatttt tgtaggttca gctcatactt aaatgaaggc ctctaatagt 31020

ttggaattga ctttcctata aggtataaga caaggatgta attgtcctct tctgtaggtg 31080

aatattcatt ttttcccaga acggttggtt tggtgacaaa tctgtctttt ctcccctgta 31140

gggtttttgg catctttgtc agagtcagat ggctgttgga tttgcatctt tatcccattt 31200

tcccccttca ggagtggtgt gcatcaattc aaggacctgg tcacccggtg cctgtctaca 31260

gggagccaga gctccaatct agtataatag tatccagtaa aaaaaaaaat ttaaaaaaat 31320

atatgtgtat atatatatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtatac 31380

atatcctcca ctgagaccca gaatatggca cacaagtaag tgttggctcc tccaggggag 31440

ggttgcagaa atctgtttct accctcagtt tctatttctg aggtcaccca ctccacagaa 31500

gtctaatttt ttcattgcag atgtctttct cctccttggt tatgtttatt cctagggacc 31560

ttttcttttc cttttttaca tttggaggga ggtatgtgtg gcaattttaa atagaattgt 31620

tttctaactt ctttctcaca agtttattat tggcaaagaa gaagaggaag aaggagaaga 31680

aggaggagaa ggaggagaag gggaagaaga agaagagaag gaggaagagg aggaagaaga 31740

ggaagaggag gcggcagcag cagctacaga ttttggagct ggccagttag cactgagcct 31800

gacaatggat ctaaaagggg agcaggggat ctgacacctt cacatagacc tctattcagg 31860

caaaacacca gtgcacacag aatacaaaat aaataagttt taaaaatagg agaatgactt 31920

attgttgctg ggtgtgatgg cacatacctt aatcctaaca cttgagaggc agaagtaaat 31980

ggacctctct gagtttgagg ccagtctggt ctacataaag agctccagta tagccagggt 32040

gacatagact ctctatgtac atcatagaaa gaccttgttt caaaaggggg gaggggtggc 32100

ttttgcttct ttcttgatct tgctcttcct ttcttgccac ctcacctctt tcccattccc 32160

tcccccatca tgtgctcatg gccagcctct atctctacac tctctttctc tctgtgcctt 32220

tctctgcctc tcctaccctg ttgactcaac gtcccatgcc ctgaataaac tctattctat 32280

acttgctctt tgtttctgtt tttattctgc tatcatctaa taagatagta aaaattacac 32340

acgcctctaa cccaagcaca tgggaggcag agacaggcag ttctctggaa gtttcaggcc 32400

aacaaggcca cacagtgaga acatgtctcc aaaagcgcgc gtgcgcgcgc gcacacacac 32460

acacacacac acacacaaaa tgaaaaaaga cattattctt ttgtatttgt taaaacttgc 32520

tttctcgcct agaatgttgt atatttatga taaagttcca tgagattctg agaagaattt 32580

gtaaccaata gctgttagaa tattctgtgc gtgtctgtta aatccctttg atctgtgggg 32640

cggtttagct gagggctatt catttttaac gggatgattt agatgtagag atgaggtatt 32700

gtcgtctccc actatcactg aatctggacc cgactgtcct tttatatcca gtagtgatca 32760

ttttatgaat ttggccatac caacatttgg tgtatacata tttataatca cgtcctatgg 32820

ttcagccagg tttttactcc cagatcctca aagttgcaac cattctctgt ctacagagca 32880

gctcagtttc aaacaataga agcccacccc agacagcatc tgactgttgt actccagagc 32940

aaagggtttg cttgccacac aggagaagcc actatgacaa caaaccacct cccaagatgt 33000

aagtcgcctg ttgtccttcg gctaggaatg tcagtctctt ctccagcaac tcctgactca 33060

ccttgtgttt ggctttggct ttccctccct tcctaccttt ctgtttttct ctgcttttca 33120

ccagccatca tttcatttct tttgtaattc cagatgtttc tgtctttctt tggaataaag 33180

tttaatcttt gttacttaca ctcttcttct tcttcttctt cttcttcttc ttcttcttct 33240

tcttcttctt cttcctcttc ttcttcttcc tcttcttcct cttcctcttc ttccttttct 33300

ttcttcttta aaaaacactc ttctttcttt aaaagattat tgtattagtc agggttttac 33360

cgctgtgaac agacaccatg accaaggtaa gtcttattaa aaaaaaaaaa aaaacaaaaa 33420

catataattg gggctggctt acaggttcag aggttcagtc tattatcatc aaggtgggag 33480

catggcagca tccaggcagg catggagcag gcagagctga gagttctatg tcttcatcca 33540

aaggctgcta gtggaagact gacttccagg cacctagtat gagagtatta agcccacacc 33600

cacaatgaca cacttactcc aaccgagtca cacctattcc aacaagacca cacctccaaa 33660

tggtgccact ccctggtcca agaatataca aaccatcaca attatttatt tatttagtgc 33720

atatgagcaa ctgtagctgt cctcagacac accagaagag ggcatcagat ctcattacag 33780

atggttgtga gctaccatgt ggttggttgc tgggaattga actcaggtcc tctggaagag 33840

cagctagtgc tcttaaccac tgagccatct cttcagcccc tacttacact cttctaattc 33900

ttaggagttc ctccttaggc taccttcctg acaggcacgt tcacaccacc ttgtaatgct 33960

cttccatgtt ggtcaaactg ggacagttcc tgaaactaaa gtaaactggt ttccagaact 34020

tccagaccca agacaacctt gaattataac tttatttttt gtttttttca aggcagggtt 34080

tctctgtgta gccctggctg tcctggaact cactctgtag accaggctgg ctttgaactc 34140

aagagataca tctgcctctg caccccaggt gtgagcatta aacgtgtgtg ctaccattgt 34200

ctggtgaatg ataactttat ttccacctag tgcctttctg ccttgtaact tctggcacct 34260

aggctaggtc acaaggtggg caaacgtcct cccttcaaat ctgtaggagc aataaactcc 34320

tcttcattcc tcggaactga agggaattgg aggctctttc cctccaggcc cctgtcatgc 34380

tgtgcatggc tgctgtctct tctaatgagg ctcctcttta accaggtttg cactctgggc 34440

ttcaaggccc atcatgactg cctacagctg tgatgggtgt gttcatctaa tccctacaca 34500

atcaccccac tcaacacttc tgtgtctttt cacaaggata cctaaggttg ggacccaggc 34560

cagtcagaga tacacagtaa ggccccgtct tagacaaaac caagccaaac caaccaacca 34620

accaaccaac caaccaaaaa ttccttggtt attttctgat gccatattta tagtccacaa 34680

acctttactg tgattattca ccattatagt gtcattccat gtattttaca taatctacta 34740

taaattcaat gtattgttct taggtctcac aggtcatcca aatatccaga aatattaaac 34800

tgatatccag atcttaaact ggactgtaga attacagtgg ttagataaaa gcctgaattc 34860

ctcaggaatt catgaaactt gttcctatct gccaaaagga gtcattcctg ttacctctgg 34920

aggaagggac aaatggctct ctgtgacacc actcagcatt tcaaggctaa tgctggcctc 34980

taggctccct cgccagcaca agcctcttaa ggtgactctt tcacatagtc atattattgt 35040

accacctgga agagtctaaa ggaaagagag agagagagag agagagagag agagagagag 35100

agagactggg aatggagaaa tggctcagtg gttaagattc aatttccaac aaccacatag 35160

tggctcacaa ccacctgtaa tgggatccaa tgccctcttc tggtgtgtct gaagagaaaa 35220

acagtgtact tatatacata aaataaataa atctaaaaaa acaaaaggaa catggcaagg 35280

gttaggaaag caggagagaa tagtggagat agcaagataa caagaaaagg agactacaat 35340

gtgtgtgtgt gcgtgtgcgt gtgcgtgtgc gtgtgcgtgt gcgtgtgcgt gtgcgtgtgt 35400

gtgtgtgtgt gtgtgtgtgt gtgtgcatgc gcgcgcgccc gcgtgcgcat gtgtgaactg 35460

ttgttgggaa tctcaggtag gagcggttgc catgattatt aacccagctg ctgggctgac 35520

tcccgcagct cccgtgtttg ctgtgagaat ttcttccccg agaaggtata gacaatattt 35580

acccttcttc accaacaagc caaagtacaa ttcactgatg ctcagcctga tgttaatggg 35640

tttacataca gaccatgagg gagggcttaa tggcagaagt gtgagggacc ctaaagtaga 35700

ggcattggaa agtcctgacc ggatccccat agctctttgg gcccctttaa atcaacctcc 35760

ggaacttacg tacggcggta cagtggtcct cacgttcccc aacgctgccc ctctttaata 35820

cagttcctca catggtggtg accccacctg taaaattatt ttcaccgcta cttcataact 35880

gtaatttttc tactgttatg aatcatgatg tgaatatctg ataggcagga tctctgatat 35940

tcgatcccca ctgaagggtg cagcccccaa agcagtcagg gcccaaaggt tgagaagcac 36000

ggtggcacct cctgagaccc taaggcagaa gaatcacatt caaatagaat aggagtggct 36060

ggagtcccgg gtgagagtcc attgggccat cccctccccc ttgcctgagg ggatgcaaat 36120

agtccgtggg tcactgctgg taaggctcta acaagcaggc acagctgatc tgagggagtg 36180

ctcagaggaa cactctgtcc acagcaggga catagtgtga gctgcagatg ctcaagcagt 36240

tataaatact attctgtaga aaactcgttt aattgttgta tgtgacagac gcggtaataa 36300

actggccaca atggctgttc cacgatgggg ggtaggcatt gtctacaaga gagagaaaat 36360

atccagctga gaagagatgg gggataagcg atctgggatc ggccagggcg atctgggaga 36420

gaaggaagga cagctggaga gggaggagga gaaggggatg ggggagggag gagggaggag 36480

ggacttggtg cactttgggt aaattaatag gacgcaatct ggtggtttca atgaaaatgg 36540

cccccagagg tgtgtcacta ttaggaggtg tggccttgat ggaacaggtg tagccttgtt 36600

ggaggaagtg tgtcagtggt gtgggcttta aggtctcaga agttaagaca agcctactgt 36660

gtctctctct tcctgctgcc tagacatcca gatgtagaac tcttagcttc ctctccagca 36720

ccaggtcagc tcatgcttct caccatgacg ataacggact aagcctctga actgaaagcc 36780

agccccaaat aatgtttttg cctttataag agttgccacg atcatggtat ctcttcacag 36840

caatgaaata ctaaaacaaa accatattgt atattttctc cctcaacata tataattact 36900

aaaaccacat agaaatgata aaaaaaaaag gggggggggg ctggagagat ggctcagtgg 36960

ttaagagcac caactattct tccagaagtc ctgagttcca ttcccagcaa ccacatggtg 37020

gctcacaacc atctgtaatg gggtctgatg ccctcttctg gtgtgtctga agacagctac 37080

agtgtattta tatataataa ataaataagt ctttgggaaa aaataaatga taaaagaatt 37140

tgttcacttt actattctgt aagtatttta ctgtaatgtt agcttggtct ggaccactat 37200

gtcaataaga ttccattatg actcagggag atctggtgtg caaagatgtg ggttacacct 37260

tgctttgagg tactcctctg ctgctcttcc tggagggcgg gggttgtttt gttttcattt 37320

atttattttt gaacgcaggt ttcactgtgt tttccagact gatttctttc tttctttctt 37380

tttttattag atattttctt taatgctatc ccaaaagtac cctataccct ccctccaccc 37440

tgcttcccta cccactcact cctgcttctt ggccctggca ttcccctgta ctggggcata 37500

taaagtttgc aagaccaagg ggcctctctt cccaatgatg gccaactagg ccatcttctg 37560

ctatacatgc aggtagagac acgagatatc cattgttttt aataactgag tagtactctg 37620

ctgtgtaaat gtactacatt ttctgttccg gactgatttt taactcttgg gctgaagtaa 37680

tcttctctca gccaacgacc taaggcgaca ggcaggagtc gccacactca cttcccagct 37740

gtgctgcttt aagcaagagc tgcttaaccg cagttgacct gtttgaacgg gggggggggg 37800

gggggggggg ggaaataata gagagataaa aacacctccc tcaagtggag tatttattgg 37860

tgacaagggg atggaaaaga gccaacaatt attgagtaca aagcaattta taccggtccc 37920

tgaggtaagt gcttcgcaag catcaactca aattacaaac tatgtgttca gagaactgat 37980

aacccttgag cagagtgtgg atgttgcttt gaaaatccct cggaagttat gtcagccaaa 38040

agctggcagg aagcaaacgg tcctgtccgt ggggtgaact agaggagact ttgacgatga 38100

cgtaaccact taggaagatc tatgtcatgt ctaaaagaaa gtaaacggaa gatgacgaaa 38160

cgctgcagga cgagcaacca ccgccaagga gcctgcaggg acaaagagtg tcctcaagtc 38220

atcaagtaac tgtgagggga gaaagtgggc ggagggcggg ggggggggag cgaggggggg 38280

gggttgcaag gcccgggtca catgacccgg ccgtgcggag aacctaaggg agtaaaattc 38340

ctgcatcact ctgctcacca gagcacatct atagctgaag agaggagctc gacacgctta 38400

gaattctgct aagagcatac tgagaatatg tttttttgct gtgttgttgt tgtcgaagta 38460

gttacgttct ttttcttttt cggttttcga gaccgtttct ccgtgtagcc tttgctgtcc 38520

tggaactcac tctgtagacc aggctgacct tgaactccca gagattcacc tgcctctgcc 38580

tcccgagtgg tgggcgtaaa ggcgtgcacc acctctacct cgcctggttc agagactatt 38640

tttaccgttt taataaatag agataaaagt tttgatcccc tcaatccctt ttactgcctt 38700

gaatgttaca caacagagtt ataacaaaac atgtacacaa acaccaatat gctgaagatg 38760

gcagaataaa tagaactaag ctatacactg gttgttacag acataatata cagccttcac 38820

atcaataaag acaggggtga gaatctttaa tcctagcaca tgggaggcag aggcaggtga 38880

atctctgtga gttcaaggcc agcctggtct acagagtgag ttctaggaca gccagggcta 38940

cacagagaaa ccttagctcc aaaaaacaaa acacacactt aacaggttcc tggggggcgg 39000

ggggggggcg ctcagtggca cactgttgtt cccagtaact tagggcattg gtgcaagagg 39060

ctcccacagc cagcctaggt agcatgccag tgcttcattt caacactaaa ggggagagga 39120

ctgggtacaa aaaaaggtgt agcaagcaca taccaggtaa aatggaaata gctcagctat 39180

accaacagta agtcaaagag aatttaggag aaaaaagtca taaataagaa agaagccatc 39240

ggtgtatctt tctcaaaaaa ctgtttaaac acatgaaaaa acacagccac agtgtactca 39300

tataagtaaa ataaataatt ctttttaaaa gaaagtcatt gtgtggcaat aactggtgtt 39360

agcatgtgta tcttcctgag taattccctt tatgcctcca cccacgggaa ataactcttt 39420

atctatattc tataggagac taatatgcaa taattataga gtatatgtat cctaaaaagc 39480

aaaaggaagg atggcattct aactaatact tcctatctct ccttccaggt actttattgg 39540

atctgaataa atgtgccatg tgttttttgg caaaattgta tcctagccag atatataacc 39600

ccatgttcat aaccccagcc cttaagaagt ggagacagga agacagcaag cttgtcgtta 39660

gcttcagcca catagagaaa ccctatctta aactccccag aacactaaca agaacactaa 39720

aaggagcaat ggactcagaa ggacacgtct tgtgtgttct ctcttaatct gagtgtggga 39780

tgtgtggaag taggaagaag ctgggagtgc cagggacagg ccagaaggtg atggaggttg 39840

ttatgctcat agtatttgcc tgcctgagag aactccagcc ctattatttt gtatacttaa 39900

tatccaataa taaaatgttt ctaaatgaca tcattataag tatcttaaaa aaactcaagt 39960

agatcaagag cagtgaccat cgaatgatcc aggatgcgac tgagttggag aaggcacagc 40020

gggctcggtt cagcagcagc atcacctctg tgacattcag aagagcaaca taatcaccac 40080

tttggaggtc ggtcacctta caggaacagc tccataaaat atagaaatag gtaaggaatc 40140

atgtaggcta tcgggtttcc gtacaggatg tggagggcgg agcaggagaa actgggaaga 40200

ggaggagtgg ggggggggga ggggggggag cagcttaggg tggagccctg tcctggagcg 40260

catctgtata cagtaggaaa tgagacagtt ggcagataca atagatctaa aggtgttcga 40320

aaagaagcct caaactcagc cggttgtggg agtgcgtctg tctcactgga cacaaccaga 40380

aagagttaaa ctgaacggaa attagtctgt gtgacattcc aggtgggata acagaaatgt 40440

actccactac cagagcagac ttcgttgtcg ttgcagcaat tacaccgtgg gatttgagga 40500

ctgtaacaaa tcagaggctg ctctgcactt cgtagtcttc ttctaaacag taagcagtac 40560

tagctgcagc agcagcaaaa gacacccaga cacatgcggg accacgtggg tgtgcacact 40620

ctgatggagt gacaccctac tacgcaacaa cctggaacct cgatgctttt atggtggcca 40680

gcacagaccg gaaaagttag cttgcttctg catgggaaca agtctcaggc tgaaaaccgc 40740

tgaaagaagg aagctagttt tttcaaacac atgatccatg gtttgttaac aattgttgga 40800

tacattgtta ccatttgaac ttggaccaaa ggaagggttt gacatccctc tcaaccttac 40860

cctgggtgtg agggcagggc tttgccatgc ctgtgggtct ggtgggcctg agtagttggt 40920

tctagacatg gctgtgacgt tggttcagga cttcaccact attattagta ttattattat 40980

tactattatt attaatttat ttaatatatg tgtgtacact gtagctgtct tcggacacac 41040

cagaagagag ctggaaggac tgaatggtgg attgtttggg cccagaagtt tgagataagc 41100

ctaggtcaca cactgaaact taccttaaaa aatttttttt tcttcctatt tctccttttt 41160

aacttaggga aattggctgg aggctgagga atcatgtgga ccttataagg aaaacagaca 41220

taatgtgctt acttctcgtg gctgcgacac aacccctaag agtaaaccca tgtaaaggag 41280

aaagggcata tttggctcgt gtttccagaa ctttcagtcc atggcacgct ggttcggctg 41340

ctttttggcc tgtgaggagg cagagctgca caaccaggag catcgtggag caaaaccatg 41400

tagctcgtgg tagcagggca gcagagaagg aagggcgggt tgatgcggaa acaggaaacc 41460

atagtattct ctaagtgggc ctcgcctcct acacttcctg cctctcctcc cccatgagat 41520

cgggcttcgc taatccatga acccatcatt ggaccaatca ttgatgatgt ttcagtcctt 41580

agggtgtgct ccctgcccac aggtcccact gaacactgcc gctcagggga caaaaggctc 41640

cgtgaacgta tagggtatat ttaagatgct gtcttagtgt cgctactgct gtgataaaac 41700

accatggcca aagaagcatg gggaataaag gacttacttg gtttaagttt ctatgtctct 41760

ctgtacatca ccgaaggaaa acagggcagg aaactggagg caggagctga tgcagaggcc 41820

acggaggggt gctgctttct gcctcacacc ctgtggcttg ctcagcctgt tctcttagag 41880

aatcaaggac caccagccca gggatagaaa cacccacaat ggggtgagtc ttccccagga 41940

atcactaatt aagaaaatgc ctcatgccta tagcctgatc ttgcaaagac actttctcag 42000

tcgaggctcc ctcctctcta gcttgtgtca agtgacacat aactgggtgg gaagacggaa 42060

acctgcagat attgtggcat atgcctccaa tcccagctct agggacactg ccacagcagc 42120

atggtgaatt aaaggcctac ctgggcataa agtgagcata aagctagcag aggcagtata 42180

agaccctgtt ttaacaacca aaagctatgg tctggattca gaaactggat gagatttgat 42240

tattgttgcc cacaaaaagg aaaaagatat gggaagaaat ggatattcgg gcgaagagaa 42300

tcacaaactg agccaggaac tgtcagttag cactcactgt ccctttcttc aaaaaagcag 42360

gtactgtggt ttatgcctgc agttacactt ggactcctcg ctgaggtcag aggatcactt 42420

gagccctgta agtctgaacc agcctgggca gcatagtgat actccaggtt ggcattttgt 42480

ctaggctctg ccccatagtt acctggcaac acccaggtgt attgggatca ctataaaagg 42540

ggctgcttac cccctccccc ctctctcttc tcttgccttt ttaccctctt ccctttctgt 42600

cccttctctt cttcttcctt cttcccctct ctctctctct ctctctctct ctctctctct 42660

cctatcccct ccccatgccc taaacaaact ctattccata ctatatctgt cctattgctg 42720

gtccctgggg gggggtgggg gggatgtctc agcatggtgg gcccgcagag gcaccccttc 42780

ccctacaccg tactgtgcct ccaccaaaca tacccctggc ttctttttct tttttataaa 42840

acacaacact ccatctttaa aacaaaacaa aacaaaacaa aacaaaacaa aacaaaacaa 42900

aacaaaacaa aacaaaacaa aacatagctt gctgcatcat tgtgggcaaa gaatagcttt 42960

gcttgcaact atgtgattaa tttctgagtt gaactcatgt tcatgacttt ggggtggcat 43020

tcttaaaagg caatagtatt tttttctccc tgtcttcatt ctggtcaaaa tgtgaaccat 43080

cttcttcttg cttttgcaga ggacttgggt ttagttttca gcacccacgc ggtggctcac 43140

aaccatctat agccccagtt ccaggggatc tgatgccctc ttctgacttc cttgggcacc 43200

aggcatgcac gtggtacata tatgtagaca aaacactcat gaacataaaa taaatcaatc 43260

taaaaacatt ttttaaaaaa agtacgagcc atctggtact tggaactgca caggtagaag 43320

gaacttgggt ccctggcacc acggagtact ctaccagagt tttgcataaa tttggtagca 43380

tgaatctcaa ttctttatct taatttgaat aaatggagct taagacattt tatagagagc 43440

ctggtgtggt ggctcacacc tttgagccta acacttggga gacagaggca agtggatctc 43500

tcaaggctgg cctggtttac aaagctatat gagaacctga ctcaaaaaaa attaatatgc 43560

ttcaattttt ttggtgacaa gtgttaaatg tatacagaag tggatacatt gatataaaaa 43620

ttgtgtgtat accaacaagc agctttatcc aattcattta tacttgtaat cacttccctt 43680

tccctcatta gactattatg aaacaaattc caaatatcag ttttttaatt ttaatttatt 43740

tttaactgtt acatgtatgg gtattttgac tccgtgtatg tctacagaac atgtgcatgc 43800

atgatgtcct caaaggctag aaggcataga atgtcctgga actgaagcta tagacaactg 43860

tgagccacca tgtgggtgtc tggattaaac ccatgtcctc tggaagggca gcaaatgttc 43920

ctaactgtcg caccaactct ccagttcaca ttacctcttc tgtgcggcca ttctcaaatg 43980

gagggctcct gattctttga gagaagatga gataactcag ggaaagtgtt tcccttggtt 44040

gaaaacagaa gtaacacaaa acaagggatt aggtaaagta ggcagttgtg gaagatgaaa 44100

gggtgtgggg taggagagta gagtgggtag tgttcccact tctcagactc cattttccac 44160

gctgactttc actgggacaa tcactctcag ctgcaaactc atttgctagg agtcagttat 44220

gtatcagtgg ctgcttgtgc aatttggtta tagctgaggc ccaaggggat gtcttctagg 44280

gtttctggaa aagtttgcca actcctcaga aatggcttgc atttacctgg gcaatagtct 44340

gtgatggaag gtgatggaac tacagccaag acgatcagag tagtgagatg gcaggaaacc 44400

ttgttctctg tgccccccac tctgtggttt gtcctgcaat tctaagccct cttaaagctt 44460

gccctcaatg gatctttgca tgacacgaac caccgttcca gaatttggct tcgttaaatc 44520

atttcctaca aaagaaaaac aattacatgg tgcctccctc ctccaattaa gaatattttc 44580

ttggtcctta ttaggtaaaa caaaacaaaa caacaaacca tatttttgac agcttttcat 44640

aaaacttatc atttccagcc atctctccag cccaaaactt atcatttcta cagattctgg 44700

cttgttctga gggcaatggt ggtgtctgaa gataaggaaa aatagctggt atggaatctg 44760

ttctgtaaag gtgagtatga gtattctgga taaagacatt cagtgtgttg cgtataaatc 44820

ccgctttcct tctgctaata gtccttcgta ggagcatccc gtgaagtctt atttacttaa 44880

tgtacccaac tccaaatagt atctcttaaa gctacatcct tcaaacagtc acatggggga 44940

acagagagat ggctcagccg ttaagaacat tgattgcttt tccagaggac ctagatttga 45000

ttcccagaac ctatatggtt actcacaacc atctgactcc agtcagaggt ggtcctatga 45060

ctccttctgg cctctgaagg taccaggcac gcgtatggta tagtcatgcg tgctggcaaa 45120

acactcacgc acagaagtta aatgaaagca accaaccagc tattggtcca gagggtggag 45180

agcttaaaca gacgcttaga gaagacctcc gtcttcaggg tagtgttgca gtcgtgggcc 45240

agcattgctt gggcgtggtt ttattttaca ttagcaaatc aaacaacttt gttatctaga 45300

ttccaaggta tagtgagtat taatcccaga gatgttacta gtgaagaatg atcactctgg 45360

gggaaaaaaa aaatcccaga aggtagaagg cattcctggc aacaagcgta gatcgccccc 45420

tgctggcaag gactagcaaa ggccagcaca aacggcagaa aatgcggtca agaatgatat 45480

acccagaata ggagagcaga agttacaggg aagtttgtat ttatcatcct aattgtcaga 45540

cagacaaact gggggcttcc atggttggag ggtctagcat gccccaaaga catatgtgta 45600

agtgaaggct gcaggtgact taaagaagca aagactgagt gtgaaagtga agctagtggc 45660

caagtcacaa gccagagtgg tacaagagtc agcttggtag atcccaacct tcctaaagcc 45720

gcgaccctcc tcaagctgtg tcagtgtaga aacagcaacg ctttccaagg tgccggcttt 45780

accaacactt tgtccagaac tagaggaact attcaaaatg tttgaactat tcaaactaaa 45840

caaattcaaa cttgtagaag ttcctggcta ggagtcacct cgtcgctacc aaccccctgg 45900

cttatttgtg agaagattga gagatctgac tttacgacaa ctgcggaacc tcagagatgg 45960

caccatttga aatcaccagc tgctgtcact tggacctcaa gcttttgggc agccctcagg 46020

catcctgttt tgttaaaggc aaacgcaacg ggctcaagcg ttctttcagc cgcgactgta 46080

ataaattgcc tagacataat agacgcttaa tccatgtgtg gaagcaaatg aactaatcat 46140

taaatcaggc gtattggagg cctataattt ggcgaggtct cagaagactg attcacttat 46200

atattcgtct gcttcttgcc ttataattat tatgtcttcc tccgcgggga tttgagaaag 46260

cttgtaatag aacatgcggc aaataggaaa tataaataat aaataaagcc agcatcagga 46320

gacacgtacg ttaggcaaac acaagttagt tgaatggaat ggaatgctca agcaataaga 46380

cagcacctaa ttatctaagc ccagctgcaa attggtacta gattttctgc tgaaagaaac 46440

ggaagcaggg catggttatt ttagtttctt aagatttata ggaaattaac tttaagatag 46500

agttttgatc tgctgttcta ttcttagaac tttatgggga tatggcaaac aaccctggat 46560

actgttctaa tgcaaattct ttttttcttt ttctcttttc tctttctttc tttctttctt 46620

tctttctttc tttctttctt tctttcttct ttccttcctt cctccctccc tccctccctc 46680

cctccctccc tccctccctc cctctccctt ccttccttcc ttccttcctt ccttccttcc 46740

ttccttcctc tctctctctt ttctcttttc ttttcctcga aaattctata gagctttctc 46800

acagctacgt gctagagtat gaagctttct cacagctatg tgctatagta tgaacctcta 46860

gcatagctgg ttctcagcct tcctaatgct gcaacccttt aatacagttc ctcatgctgt 46920

ggtgacccct ttaactataa aattattttc attgctattt cataactgta attttgctac 46980

tgtcatgagt tgtaatgtaa agatctgctg tgcagaatat ctgatatacc gacccctatg 47040

aaagggtcat tcgacactcc ccggggatct ctacccacag gtcgagaact gctgctgtag 47100

catggccttc atagctcact gaaagcaatt tagtagacat caaggaaata ttgctgagtt 47160

ggaaactttc aaagtgtgga cgaaatcaaa gaaatcacct gtacgtatcc cattccatag 47220

ttttctatca gcatcacctt gaaccagaca tttaatatgg gtgagaaaca tcatatactc 47280

tctactaagg cggaagcatg gttgttctat actgtccttt ggtgctaatg tgtgctttag 47340

ctagctggga ttaaaggcat gcaccacccc tgcctggctg taaagtttct tttaggagtt 47400

ttgaaggaaa agtgagttaa gcgtctaacg aaaaatcatc tttgggatgg atgcccagct 47460

acaacttcct gactggaaga cgtctgtgtt agtattctga gtgctcatgg ctatggtggc 47520

cggtggctgg tggctcagta agatagttca tggtcaggaa tgttcatgca actgcagttg 47580

gacagtggct ggaagcaccc aacctggaca cactcactca catgcctcct gccaagctgg 47640

gcagattcag tcagcgggag actggaacag gggagctttc tgggccatta aatatctatg 47700

gacctaacag gctcaggact cagctctaaa gtgctcttaa ccactgagtc atctctccag 47760

cccaacaccg actttcttag tgttaggttc agagcagtcc ccaaatggtg gtgctggaga 47820

caatgtcctt cccttctagc ttgcttatca agatgactgg tactaagtaa acagggggct 47880

aaaggtgggt ttctgtttac caggcatgtt ccttaaacca ccaggaccct ccttaaccac 47940

tccaaaggtc tactacgttg agcaagggca tctaattctt agcctgtatc agctcaatga 48000

cctcatctgt tgtcatataa aacctgcttg ctttctcatc attttgtgtg tctgtccgtc 48060

tgcccccacc ccatggcctt gacaagtttg atccccaata aaactttttt gtacccacag 48120

aagagccgtc tagttgagtg acttcacagt tccacataac agaagaattt atttagacta 48180

tgaataacag tggtgtcaga aaggcttcat ctgttgcttc atttaaactg aggctcctca 48240

ataaatgaaa agggaaggga aggtctccag cttgtctgtt tcagtctggc cttatctgtg 48300

ctcatgtggt tagttgcatc ccagatctcc attacgggct actgcaataa aaactccaac 48360

cccacatcac acccatggaa ttggatttca actctaaatc caaggagttc tgagttcctt 48420

ttatatgtca ttgaaggagc cacaaatgac cagaggtgtg tttgaccgaa ttctgagcgt 48480

ccaggagccc agctccacca gcattcctga gagcacactg gggtcctgca cacacacaca 48540

cacacacaca cacacacaca cacaccaggc tagcagggtg tagctggtta tgatgggaag 48600

tgctctgcaa cccccaaggc ttgtggcaaa tatggcccct cattctctaa acagttcaac 48660

tgtcaaaatt ttaaaatagg tacagagagg gacaatatac atgcaggatt agggttagga 48720

gtccagatga gaggagagag tttccaatcc cgctggggtc tctttggggc tgagagggat 48780

ggcgagtgtc gatgtcccaa aggaaacaca ctgagccagg agtcttgtcg aagcaggaaa 48840

taaactttat ggcatcagcc tcttgcttat ataagttcgg aacataggga gagggatttt 48900

tggtggggta agatgatgta agaggcaggg aaggttgacg gagggtatgg agtgactgac 48960

agggtcaagg gcaaagctct acatcagcaa tggcagggca gaggcagggc cagacaggtc 49020

ttgctgagtc tccccgatac ggaagtgact gagacaggtc tcaaaactca agccaggatt 49080

gggtttgtcc tcaaggccca aaccagggcc aaaatggggg gtccaacaca aaagccacaa 49140

tcctgagtgt tagcgttcat ttcttctagg ctctgcccca cagttacctg gcaacagcca 49200

gatgtgcctg actcactatg aactgggctg cttgccccat cctcactctc ttatcctctt 49260

accctctctg tcccttctct ccccattccc ctccctcctt cctggccggc ctttcctctc 49320

ccttcccttc tctctctcct tcttcctctt cttctccctc tctctgcctt tctctctctc 49380

ttcaactgcc ttcccgatgc actaaataaa ctctattcca tactatatct gtcatatggc 49440

tggtcccaca gggggaaggg atgtctcggc atgggcctgc agaggcacct cctttcccca 49500

tgctatactg tgcctctacc aaacatatcc tggttctttc attctttttt tataaaacac 49560

agtcctgagt aggttggttt gattccatgt gccttcacac acttgtgcca ccagcacaca 49620

gattcactgt gcgtcttctc agtctgtacc ccgcccccac tgggagtgaa ccatcagtct 49680

agtggcagtc accattgatt aggattttag acttctttcc cttctcttct tttccctttt 49740

cttctctttt tctctctctc tctttttttt taattggaga gtggatttta gtttccttgc 49800

ttgggtttgt tttagcaggg tcatatcatg gaaccccagc tgtccttgaa ttcacaaagc 49860

tacagctgcc tctgcctact gactgcttag gggattagaa gtgtttgcca ccaagtctgg 49920

tctactttgt tgtttgtttg tttgtttgtt tgttgagaca gggagagtat actttatgtt 49980

ggtaaactga catgtgccgt gtgagctcgc tgagattctg gttctggact tggggcttct 50040

tcttcctcca ttgcagaacc aaacatgtct atctctaagt attctccaag ggcccctgtg 50100

tggggacggg tggctgcctt aacactgaca gggagaaagc aacagtttaa gagtaaaact 50160

acgtttggcc agccctggcg gtacccacct agcacctcag aactcaggag gcagaggtgg 50220

gcagatccct gtaagttcga ggccagcctg gtctacccag tgaattccag gccagccagg 50280

gctacacagc tagaccctgt ctcaaaaaca aacaaatcaa caatcccccc aataacatta 50340

tgtccttgaa aatatctaga acattgacca tttccaacag aaattactat tgtttatcct 50400

gtgatataaa agtgcctgtg ctgagagaat ccaggtgttc agcccctcat gagaagccca 50460

tctgtggaga caaatgtcaa ccagaatctg ccctaacctc tgcagggctc tgtgagcaca 50520

tgacttgatg atgtcatttc tattcattac tcttctttta ttcataaact ttactcaaat 50580

ggacactctg ctttgcctgg ggattctcca tattaggaac tccaactgac cgcctcaatg 50640

gggcttctca ggtgatgtct gctaattgct ttatcttatg tgtatgtgtg cttgtgtatg 50700

tgtgttcagc ctgcatgtgt gggccacatg cgtgtggtgc ccttaggggc tagaagaggg 50760

cctcagatct cttgggactg gagttacaga tggttgtgag ccaccatgct ggtgctaggt 50820

tacgaaccct gggacttctg atagagcagc cagtgctctt aaccgctgag ccacttctct 50880

agcccctgct acatgctttg agtacgcaat ttgaagattc cttgccgtcc atctggctgt 50940

ctcatacctg ctatgtattc tttcccttta gcttgctata ttcttgacaa actcagttaa 51000

agtggacata tggctatata gatcatccac tggactgtaa agaatacgta gcccacattc 51060

aattcaaact tgctatgtag ccgaagatga cctctgcttc acagatgctg gggtcatagg 51120

tgtgtgctac catgtctggc cttcactgat tagttttgtc tacttctgga tttgtaaaag 51180

tggaattaca tggtatataa atatagattt ttaaaaaatt tttttgtttc ttatactcaa 51240

cttaatatct gtgcaaccaa tgtgttactt gtagcaatgt ttttttgttt tgttttgttt 51300

tgttttcctg cctcattcct gtgtactatc ccaatcttat aaataccctg cagtgcattg 51360

agctattaat ctgtagagaa gcacgaggct cttttttgtc tggctgatac gaaggaaacc 51420

ttgacagacc ctcctcctac ctgccaggcc cacttcacct tctgctttgt ttgtggacag 51480

tcatcccttt gtactttttg tttgtttgtt tttgtttttt ttgagacagg gtttctctgt 51540

atagccctgg ttgtcccttt gtactttcag gctaccttgc ttatatccct gtaagagtct 51600

caaaattgct gaaggaagac cccagactca gatagtatgc aaagacaaac aacatttttt 51660

ctgcagaaac aaccaggggt taaccattct ttaaaatggc gacccaagac aaaggcacgc 51720

agggcttata agaagggaac tgggggaacc tctagaagga ttgggtaatc taaaattgca 51780

ctgttgggtg ctagggggtc acagctggcc agcggtctgc attcttatgt catgaatgtt 51840

tagtcatagg atgagatagg gctgcccagc gcagatggtc catcctgaga taagacattt 51900

ccctacttct gtggttatcc ccaggctact ctttgggagg gggttttatg agcttactgt 51960

ggattttacg gtctgttcct ggagctggtg ccttacgtct tcttttggta atcaggtttg 52020

gtctttaagc ggagacaaat ggagtttatg tcaacctttc aaacccatgc taagaaacaa 52080

gcttagtttc tggccagtca aggtgcgaca cggtcgattg gtgaatttag gactgttgta 52140

tttccactga agtaaaagac agctgagaga ggtccccaaa ccctcccttt cctctcttct 52200

tatctctgga atgaggtttc gtgctagcgt ctttaggtct aactaggagt cttccgagaa 52260

ggctcagtga tcacccactg acagacccgt cactgctgac ctttttcgta gagaatgcgt 52320

ggagaccata tcgcccaggc ttccttgtag tttagtgggg tggagggact gagttttaca 52380

tcagtaagca gaagtccatg gcccgctgtg aatctcatga taatcctctt ctgcatggtt 52440

ccccgagttc tttctttttc tgccttctgg gtggtgacct tcataacaag ctaagatgga 52500

aaagccaacc ttggttctta agggactgtg gagaacggag gcttctcctc gattctcagt 52560

cctccagccc ccagctcacc acctagagct gtgctccctg agagtgaaaa atgaatgttc 52620

attgtgtttg ggcccttaga caatcttggg gttaactgaa tacagctgtc agtcatgctt 52680

acaggtatct ctgagataca ctacttatta aagagttaaa agttattata gtaaaagtaa 52740

tacagatgca aaaaaatagt ttaggacagt ctaaaataca attataaacc ttccacctcc 52800

ctttccacct aactggtagc taggcctaca tgtcctaaca cgttcagcta tatctatctt 52860

aattctcttg ttatttctgc ctaatctctt ctacttacca ttttatttat ttatttattt 52920

atttaatcta gtggcttctg actagactat gtatgatgac gagttcccga ggcgttttgt 52980

aacccagtgt gtttccagag tgctgtaaga gactagtatg gactgagtgg tttaagcagt 53040

aagcattgaa tttttgagtt ctggaacctg gaagtgactg ctgagaagtg tgggggcttt 53100

tctgggactc ctgcttgagg tttgtaataa agacccagag acgtttgtat accataaggc 53160

tgttggcctt tgctggaaca gaacctcagc taccatctga agttaacccc aaacccctgc 53220

cactgtgtcc cttcatgtgg ctctcctgtc ctgtccctcc atccccgctc ccccacccca 53280

agctgccagc tctttattct ccaaaaagcc acatttcaga aagagaagca gtatggtgtg 53340

ggggaagggg gtgcctctgc gggcccatgc taaggcatcc cttctcagtg agggacccta 53400

tatactatac tatatatggt atagtataga atagagttta ttcggagcat ggggagagga 53460

gttgagacac atggaggggt agggagggga gtggaggggg gaagcaggag caagaggaga 53520

gagcaagagc aagaaggcaa gagagcgcgg aggggaagag cagccccttt tatagttgcc 53580

aggtaacctt ggggcagagc ctagactaac aactccgttt gctttttaac atggtaatct 53640

cagagagcca gtcctttgag ctggtgaata aggtacacag agaaacccta tctcgaaaca 53700

aacaaacaac aaaaaagaaa agaaaaaaag aaaaagaaaa tacagagaca cacctatagg 53760

ccacttgacg caatggggcg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tcatggattg 53820

gcatcgaccc tgtgtgtagt cctttggttc aaacagaagc agcagagatt tctggagccc 53880

tttcataagg gcactaatgg cattcagaag gctccgttct catgaactgt ttggtttcag 53940

ctcctggaca aggggcagag gtgcatattt tacataacaa agcagacctg gcctccagga 54000

tctcccagaa tccctcagtc cctacctgtc ataccctgcc cccaaccctg aactctccag 54060

ctcagaggct gggctgccct tcccctacag cctcttccct atataatcca gacattttgg 54120

tatctcaagg tctttctctc ttcttctctc tctcttcttt ctctgtacgc aagccctttc 54180

ttcccatccc ccacccggcc ccatagcgac tccccggcct caatccttgg gggcagcctc 54240

gcgataaacc tgccttcaac acactctaat ctggtttgag tcggctcatt tcaccctcgg 54300

agaaatgacc tatcactaac cccttctcaa ggctccaccc cgtaacgcca tcacactgag 54360

gcctgggggc caacctagga attttagtgg ccacgggtat agatcttcaa tcttttaccc 54420

taaggaagtt ttaggcagag aggaaataac gtgtgagaag ggctatcaca ctttgtaatc 54480

acacgaagat gaacagttca atgtgggaca aataaaaacc gggaatcaaa ttagtgtgac 54540

ttttcacttc acactttcct ggcgaggcta ggaagctgcc gagatggagt gatgcttctg 54600

gtatgttggc catgggttca cctctagaaa tgttctcagg tgctcaaggg acagcaggag 54660

taaaggtgga gaaaaagagc tggcattggt aaattacctt ctctttcttt cttccccctg 54720

agggaccagt cacacaccgg tataattctg ggcatggagg ggagttgaaa gggtagtaga 54780

gacagagaaa gttagagaga gagagagaga gagagagaga gagacagaca gacagacaga 54840

cagacagaca gagaggagta gaggctaggg atgggcacgt ggaggggggg aggggatggg 54900

caaaaggacg gagtggagac agaaggcaag agagcaagag agagcagagg gggcaagcag 54960

ccccttttat agtgagtcag gcacacctgg ctgttgccag gtaactatgg ggcagagcct 55020

agacaaaatg ctaacaatgg tgcccccact tttagtgtaa atcttcctag ctctattaac 55080

ctgattaaca tattcattgg aggcacgccc caggatccaa ataatcccag agctccatac 55140

ctggatgtta agctcccaag tggttccgga ttctgtcaag ttgacagtta acacataaca 55200

cttgcctaag gtctcagaga agcaacatgt catcttcaca caccacttca tttcacggcg 55260

ggtgctaatt aatcttttgt ctgcaaaaat tgagatctgt gagctggtta aggtcaaacc 55320

cctctgtggg gtgacttggg caagccaagt gtcttgcaca tccagttttg taaacagata 55380

ttaggttacc aattaagcca ttccacctga gccagcagcc tctcttgctc caacccacag 55440

ggggcaggga gacagcagtg catttgtttc tagggcctgg gaagctatgc tttgaaacca 55500

tattaaaatc atatttgaat tgctgtcagc ttaacgcaga gttggcctga agtcccgaag 55560

tgagaatcga ctgtcccata acagatcaat aaagcgatag ccgcatctat agcccaccag 55620

agtccttgac aaagtgatac aaattttgtg ttagcttaaa aagaatcatt taaacactgt 55680

gcagccttca ggtatcagtc ctgagaagat tgtgggggga cagttcctat atagaaaaaa 55740

caaaaaaaca aaaacagaaa cgcatttctt tgttctctgc atcatgtcca agatgacttc 55800

tgtgttgatt cacgttacta agccaatgac atcatttaaa atccacccac tgaatgttta 55860

ccgaatcctt tctcctgtca gcaactccac agttaggttt gttttgtttt atttcagaca 55920

gcgatgagaa cagaaattcc tattggtgag ggcaggtctc atgactctgt cactgttggg 55980

gataaatcat ggggagaatc tgccttctac tggatgtact accattggtc aatgctgacc 56040

taccaggcta gcagttggga accccactta aaacaataat agatggtgtg gtgagggtgt 56100

tagacattca gtctctgcac ttgggagtca gaggcaagga gatccagagg gagtggcact 56160

attaggaggt gtatccttgt gagagtaggt ttagctttga tggaggaagt gtgtcactgg 56220

tgtgggctat gtgatcccct gtgcttaagc caggcctagt gtggtggcat tatccttcct 56280

gctgcctaca gctacctcac cagcaccagg tctgtctgca tgatatcagg cttcccacca 56340

cgactataat ggactgaacc tctgacctgt aagccagccc caatgaaatg ttttccttta 56400

taagagttgt tgtggtcatg gtgtttcttc acagcaatac aaccccccca caccccccac 56460

acacacaaaa gacactacta caaaccattc tatgaactta gctctgcaag agctaagatt 56520

tttctttaag aatagagatg gatgggctgg agagatggct cagtggctaa gagcactgac 56580

tgctcttcca gagatcttga gttcaaatcc caacaacagc acgatggctc acaaccatct 56640

gtaatgggat ctgatgctct tttctggggg catgaagaca tactcatata cataatgcag 56700

atgtaagtat gtactcatat gcataaaata aatctaaaaa aagtaaagaa tagagatagc 56760

tcagtggtta agaccacttg ttcttgtaga aggccctggt ttgattccaa gcacccacat 56820

gatggctcct agccattcat aacaacatcg gttccaaggt gcacatgata cacagataca 56880

tattgcaaga aaatacacat gcatataaaa taaaataaat ctaaaaaaac aatttaaatg 56940

ccatccttga aaaaaacaaa agaatggggg tgggggtgga ggtggactag agagatggct 57000

atgcagttaa gagcacttgt tgctcttcaa gaggaccctg gtttggttcc cagcacccac 57060

atggcagttt gcaactgtga ctacagctcc agggggaccc attgctctct tctagcttct 57120

tcaggcatca ggcatacatg tggtccacag acatgcatgc tggcaaacac ccatacacac 57180

aagcaacaac aacacagagt caggctggtg aggtgattca gtgtgtaaat ctgaatttac 57240

atacctgtcg ccaagcttgc caccccgagt ctgatcctca gagcaagagc cacatggtag 57300

gagagaactg agtgtcacga gatatattct aacctctgtg tacacaaaat aaattaatat 57360

atttttaaaa aaggagacag tctgtaccct gccccaacac catcagtagg gctcagtcca 57420

gacacagaca attgagttac gtcaacgctg tgtacccagc tagcatctct gttgagacct 57480

tctgaggtgc taattacagt tgggaattaa tcattattct aactgagcca gagtcttatt 57540

ttctactcaa gcttctcatt attaagtttg catgaattgc tgagcactgg tgtaaattac 57600

cattcagcga tgcacattaa acactcagag cggtcggttt ttaccttgca agcctatcgt 57660

gcgaatctgt ttgcaccaaa atcggttaac tgcttattgt aaaattccat ttttctttct 57720

ttctcctagc ttactttcac gatcccagga gttttagcgg gctgtttgtt cttccccttt 57780

cgtgctctga aacctaatgt gactccattg gaagttctgt cctttccaga aagtcaccta 57840

gctcagaaaa aaaagtgtcc ttacagattt ggtgtaagaa acctttgctc aaggaagggt 57900

gctcctgtct tttctgatgc cctcttctgg ggtatctgag ggcagctaca gtgctacagt 57960

gcacttacat ataataaata aatctttggg gggaaaaaat gagcaaggct tatctgaagg 58020

ggctgtgcag ggccaggctc cagcccagaa ggggacagaa tactgcggcg gaggtatttt 58080

agaacttctg cttgtctatt cagagaactc caatccctga cacatactgt ggcctttaat 58140

ctcagcactc aaggggtgga gtcagacaga tctctgagtt ccaggatagt ctgctctaca 58200

gaatgagttc tgggacagcc agggcatctc cattcccctg tatctccatt ctccttgtat 58260

gtatcccggt cgggatggtt ggcttactgt ctttctccat tttgcccagt ctgtgacctc 58320

agcgctcagc tcgtcggtgt acagcatact gagctcaccc gcccagtcca ggaggtcacg 58380

tgcccctacc ctgaccatag gcgtcagctt cctgccttct actccatttc tactccttca 58440

tccgtgcaat gtgatgtcct cacgcttgca ggggtgaagc gtggcacctg gggagggagt 58500

ggtgcttggg gactttctgt caagccatgg caaatgcatt cctctgaatt cttgctgggc 58560

acacacacac acacacacac acacacacac acacacacac acagagagag agagagagag 58620

agagagatca aaccacaaaa tggaatgtgt ctttcttcca ataggctcct cgtgtggtgg 58680

gaatgcggct ggcagggtgt catgtgacct gaagcatcct agcgcttttt aaaatgcaag 58740

cccactttta ttatctgcag cccttcagtg atttcctgtt cctctaactc agagcccaaa 58800

tccccccaaa tagtctctgc actaacgggt tcctcaggct ttctctcagc tccctacgct 58860

gcctcttcct cacactccct ctatctactt aacttctcat ttttagctct atgcttcagg 58920

ccattcattt ccttacagaa gcctccgtgg agttccttcc ggtagggctg gtgttccttt 58980

tccagagcgt ttcctccaac tcagtcatga acagtgttat tattggttat ctgtttctct 59040

aatttgactt gtaaatttca tgagcgtgag ggacacgccc acttcactca tcagtgagag 59100

cctaatagca acccataagg tgcgtggaat gtctgagtac gacagatggt gtgctagtcc 59160

aggaacgaaa aacagaacga gggagaggct ggggagcgcg gagggacccc gggtttgtga 59220

ctccagcaag aattcagaag aatgcatttt ccttggcttg acatgaagtc ccccaagagg 59280

tgaccaggaa aggtgatcca ctcctgtctt acttagcact gggtggggga gtggggggct 59340

aggtagtgga gcttatctgt ccctgcaaat gtggacccat ttcagaggaa ttaaatacac 59400

cctgtgggga attacacagt tggtcaatgg aagatccaag atccgctcct tttacagcaa 59460

gctgttttcc tcatgggtag gaaccaccaa ggttcagact ggtcattgcc agggacgtct 59520

gtactgcccc tgtctgtctg accagaatca gcaacatcta tcttagtctg ttattattca 59580

gtccatcctg gagtccgggt cgtgatggct gtcctggact tagtgcggac gccctcgttt 59640

ccgtgctttg ctcctggtat gtttcacttg ttcctgtagg ctgtcacagt gagaacactg 59700

acacagctgc cttgggaatc cagtggaagc tcagccacgt ggcagagata caggtggcaa 59760

agtgtcgggg caggtggcaa cggagctgag agacgattat ccctgtctta ggaggacttt 59820

agatggagca agggagcacg ttcagagact tgcctgggtg cttccgatgc ttagggtcac 59880

atgacaccct gacggccgca ttcacaccgc aggaggatcc tattagaaga catgttccat 59940

tttgtggttt gatatgtgtg tgtgtgtgtg tgtgtgtgtg tgtgcgcgcg cgcgcgcacg 60000

tgcgtgtgtt agagagaggg gagtgtggta taggaggctt taaggagttg gaaacaaacc 60060

cctgtctaca tagtggtatt ctattgatta agagaaaaat gactcttcat agccttcttg 60120

gttggcttcc attgatccca tctgtaatct ttaattgttt tgtaaagaca taattgattc 60180

aatgacaaaa tctttttcct ttcccccctt ttctttcttt cttttttatt ttttatttta 60240

tttttattta ttttatttta ttttcgtctt ttgagagagg agccactgcg tagcccttgt 60300

tagcctggct ctcattatat gcccaccagt ctggtcttaa attcacagag atctgattac 60360

ctctgctgcc tcctgggcac tggaattcaa aaataggagc tttcagccag gtggcttcag 60420

cctcaggagg cagaagcagg aggatatttt cgagttcaag gtcagcctgg tctacaaagc 60480

aagtttccga acagctaggg ccacacaggg aaaccctgtc ttgaaataca acagcaacaa 60540

caatgaaaag gggctttcat attttttgtc ccctttcaag caattgcaaa tacttgtttt 60600

cattgctagt tttatttatt tacaaaactc tgtgctaagt gacacaactc aggcccgtgc 60660

cttcaagaag tgaatggaca tactgaaaga gagaaagggt ccctgagtgt tcaggctgtg 60720

tgtgctcttc acgtgtcctt ggcttccttt tgtgcattct ggctttggat ttagatgggg 60780

gggggcgggg gatatctgca gtggcacgtg catatgttta gaatcgacct tgtagacatc 60840

aaacacggac tatcaaatat taaatcaatc atgcagctga ggcattttat aaatgaacct 60900

gttactccca cattcaaatt ctgaaagcct ttatacacat gagatggcct gtaagtaaag 60960

cgctgtattc atttatttca agtcatttat ccctaactta agtgtgtcca tcctctctac 61020

cttctccctc cctccctcct tccctccctc cttcctctct ccctccctcc cttccctctc 61080

tccctcctcc ctcccttcct ttttcatttt cattttcttt aaattttgag agaaggcttt 61140

actgtgcagc cttggctttt ctcaaacaag ccatcctcct gtctcattca ttccttgctt 61200

ttgtacgaat tcttgaagta aagaactatc aagttcacca tcgatccact gattctggtt 61260

tttgttttgc tgttgtttgt gaagtgagat cgtatacttc ccccctctct tatttttctt 61320

ttcctgaaag ttactatgtt taagggtcaa aatgtcatag attctcagga atcagagtct 61380

accttggggc tggtctggac agcgcaggtc tggttaagcc ttcgtacttg tccttgaacc 61440

tcagtgaaag cctcttgttg ctgtaggtag gagaatgggg gctcttcatc ttttctttct 61500

ttcattttag attctattta tttattttgt atatatgatg agtacactgt cgctgtcttc 61560

agacacatca gaagagggca ttggatccca tgacagatgg ttgtgaacca ccatgtgctt 61620

actgggaatt gaactcagga tctctggaag agcagtcagt gctcttaatc actaagccat 61680

ctctccagcc ctgccctctt catcctttct tatagtcaga cttcctatgc ctctaaagaa 61740

atgcatctaa agttgcatgc ttctgttgtt ctcactggcc tgctgtagaa tttgaagctt 61800

caggtttccc cgttttccag ctggctgtct gcagtggatg gagaggtgag aaaggcatag 61860

tcttgcttca tcaagcaaac ctgtttgctc tcggagcggt attgcataca cctacataca 61920

cacagatgtg ggcatatatc cctgtgtgtc tttgttgcag taaaagttaa cacttgattt 61980

ttctgtccct ggttcttggt tcacaggttc tggaatagac tgtctgtagt ggaaagagtt 62040

tccttataga tctcttttgg gtctcaggca atgctggaga agaggagaac tgggtagtgt 62100

gagaggggaa aagcctttca gtttctacca tctaacctga agacactttg aaactgcctt 62160

atgacttatg tgtatacata ttcgtgtgtg tgtgtgtgtg tgtatttttg tcattgttgt 62220

ttctttcttt ctttatggtg gtggagacca catcctcaca tatgttaggc aatcactgta 62280

cccttgagcc acattcatat atatttacta aaccagcact gtgcctgtta caaggcacaa 62340

aaccatctga gctgacagga aggcacacag gtacaagcat aattgatttg tgagttgggg 62400

gacagcagag gatactttga aagtgtcagg aaggctcaac ctgccatggt gctgcgtgcc 62460

tgtcgtctca gctaccggga ggctgaggca gggtctcttt agtgcatgga tttgaaggcc 62520

agcccagtga atcccaattt ttctttttat atgtaagtac aaagttgctc tctttagaca 62580

catcagaaga gggcattcga ttccattaca gatggttgtg agccactatg tggttgctgg 62640

taattgaact caggacctct ggaagagcag acaatgtgct taatggctgg caagaaagca 62700

atggagtgct tttcagctgt tctcagccac agctgtgtaa ggaatctcct ggaggcatta 62760

gaaagcatgc acactggggt tacacctcca ggaaaccagt tgtaattggt ccggcatcag 62820

tattttctcc agagctcctc atgcagtagg ctgagtggga agctacggtc caactgatgc 62880

tgctttcagt gtcctgtgca gtcatctggg cttcaaatgt aggatgtcag aaggcatctt 62940

agaacatcga gggacactga aaacagtaat ttccttatta tatgttacag gcgaacactg 63000

tgttattttt tcatgaagaa caaaattatt ctttatccac atgcacaaaa taaatatatg 63060

caatgaaatt tcaaagctgc ttgtttattt cagagttcct ttccttacca tgctaaaaga 63120

tatttctttg ctggtctttg atggcaagta cctttgatcc cagcacttgg gagtcagagg 63180

caggtggatc tctgagtttg aggccagccc ggtctacaga gtgagttcca ggacagccag 63240

ggctacactg agaagttaag tagataaagg gagtgtgagg aaggcggcgc tttaaaaaaa 63300

agaaagaaag gagtgaccat attattaaac atttttttaa aaaagattta ctttattcaa 63360

gtgtatgagt gttttgcctg catgtatgta tgtgtaccaa tgtgtgcctg gtgtcctagg 63420

atgccaaaca aaggtgttag actccctgat actggagttg tggtcagctt tgagctgttg 63480

tatgggtgcc aagaactgaa cccaggcctt ccacaagaga aagaagtgtt cttaaccact 63540

gagacgtcgt gtggctggct catatacctg cctgctgaac ttcattagcc agcaggccac 63600

atgcgcctct ggactctctg tcttgtacag ggattctgga atagatgccc taactcaatg 63660

gctaccaata ttaacctata ctctcccact cacgatggag tgctgaaggt caactgcttt 63720

tctcttcacc ttctgctatg atctgaatgt tggagtctct ccaaatttca tatacggaag 63780

agctgtgggg atggctggtg gctgggaacg cttgctgggc aagcacgagg acctaagttt 63840

gaattccaga acccatgtct ggcatggctc tgtgtggggc tgtgggtata ggggaagatg 63900

gcggagggag agagcccgca tccggccaga gttccagggc tctggacagt tggatgcagg 63960

aggactgctg gatgctttcc acgtggcctc gatgggcatc tggctgtgtg aatccactga 64020

ccctacacag cagagggtgg gcaaggggca gctcctggta ccaggttctc agtctccagc 64080

atcccacact ggatacgaca gaggagctga gaactgaata ggggctctgg ggcggcactc 64140

gccccggaaa tacgggaaga gagggcagga ggggagggga gcactgggtg gttcccatgc 64200

aggcgagagt ccttagtctg gaccttgatg aacagagaca gtctatggtt ttagagcttt 64260

actgtagaaa gacagggggg aaggggagaa ggtagaaaga gagagagaga gagaccagcc 64320

atggccacgt agagagaggg ggagaagaga aaaggagcta gagagtaaga aaggtgaagg 64380

cttagagaga gaggtaaaag cttagagagt aagagagtga ggaggggcca aacagcccct 64440

cttatcttgc tgttgctagg taactgtggg gaggagtcta gccagaaggt cagaagcttg 64500

ggatgttgtc tatgtgactg atatccatat gcccctcctg tgggggctgt gggggtggta 64560

acttcgacag cagccaggcg ttcaggagac atgatcaaat gcctcccatt ccatgtaggt 64620

agaaatcacc accacagggt ttcaccaggg ttcaagacct cagctcaact ggagaccagg 64680

gtgcctgccc cacagctctg catgcctgtt accccagcat tgtgggaaga taggaagagg 64740

ttgatacaag catagcctga ggctcactgg tcagccagct tagccaaaat ggtgaggctc 64800

tttccaagag ggaccttgtc tcaaggcaac tgtgatggtt tggatgaaaa tggttcccag 64860

aggttcatag agagtggcac tattagaagg tgtggccttg ttgaagtgtc ttctcttctt 64920

cttgttcttc ttccttcctt ccttccttcc gacttagact tggttgttta taggaataaa 64980

tatacattat agaatacaaa tcaacttcta ctctttataa atataactct ttagcagcag 65040

gaacagagca agatatttta tatccatctc atatctctcc tcgatcaacc tcaacactgg 65100

atgacagttc agggccccaa gattcaggct gaggattaag gttagaactg aaaatggagg 65160

agtgagaaca caagtagaga aggagatagt gcattccatc agcaggaaaa gtatctaagt 65220

ataacagtta cagtattggg gggggggatg gaaacggtta tataaaccgc ccgtggctct 65280

gagtacctga aatctcagca ctcaagggat agagttggaa ggctgattga gtctgaggcc 65340

agcttggcta cgcagtggaa acttgcctca aagaggggtg gtcagaagaa aagaaagaaa 65400

atcttcattt atttttatta cttaagttac tataagtata ctttcaaaaa cataatatat 65460

tggttgcatt agtatatcaa ttacacaata taaatgtgtt ttctgtttcc aaataataga 65520

gaacttggag actttttggc tgaatcattt tccctttaac aggcttgctc ttcccctccc 65580

atggcttaaa tcacattggt aatatatttt cccacaattc ttttctttgg ggcctcctaa 65640

agaataaaac caagaaggcc aattcaatga agttctctta tctggaaatg caaatatttt 65700

cgaaagatag aagtatgtga cgtggggggg gggagtacaa cttagggggc catcaaaata 65760

aagagaattt tccaacaaaa gaaaaaaaaa atgttgccaa tatttaaagt agccctagct 65820

aaattgaaaa gacccccccc cttgaaaaat ctcctttcat atactctccc atttctaaat 65880

ataaattgtt ggcaaataat tatgaggaat aataacacat catttagttt cctgccagcg 65940

aggtctggcg gagtagaagc gcactgaacc ttaggagcac gcagttttaa aaaacgaaca 66000

attggttcga gatgttggga atagaggtta ccatggcaac cgcatctcac agctctgtgt 66060

gaggccctat tgtgttggtg tttgagtggc agacactggt ggctgtgatt tgcactccag 66120

gctaggttaa ggtacagata ggaagttttc cttgggaaag ttcagggagg gtaggttctt 66180

cgacatctgc agaggaagac tgtgatgttt tcagagccat gacagtgcat cagcagtagc 66240

agtagctaat agcgatattc agccttcagc ttggacaaaa caatcctgaa gtctagaatc 66300

tctgcaaaag caactgcaca gatgggtaat ggctaaatgg gcttgtatct agtcctcgta 66360

cattcagtgg tctagctagg tctcctcagc tccaagaaca gacctctgag ctctgctatg 66420

tccttgcagt cacaaacaac ttatgcacta cagggtgtct ctgttttggt cactgaatcc 66480

agaacccgaa tcagagtcta gtacacagtg ggattccacc catggcattc cccacaccat 66540

aacaacaccc cattatgtgc tagactgtac aagttctagt ttctagaata caggctctga 66600

atccagtgac cagaacagac actctgtagc aaataaactc cttctagtgt atttcaatag 66660

ccccatagag ctgcaatttc ggctacaaga agctgacata gattacttgc aagttcaagg 66720

cctgccagag ccctgtagtg agtttaaagc cagcctgggc aatttactga gcccttgact 66780

aaaaacttaa gtatttatat gggtggttaa ctcagtggta gagtgctttc ctagatatta 66840

agggcctgtc ttagttaggg ttttactgct gtgaacagac accatgacca aggcaagtct 66900

tataaaaaag caacatttaa ttggggctgg cttacaggtt cagaggttca gtccattatc 66960

atcaaggtgg gagcatggca gtatccaggc agtcatggct caggaggagc tgagagttct 67020

atgtcttcat ccaaaggctg ctagtggaag actgacttcc aggcaactag ggtgaggatc 67080

ttatacccac atccacagtg acacacccat tccaaccagg tcacacctat tccaacaaga 67140

ccacaccttc agatggtgcc actccctgga aggaaggaag gaaggaaggg agaaggagaa 67200

ggagagagac agagacagag ggccttttgt gagtggcgtt gaatgagcac gcagtgccat 67260

gaaggcgttg ggcacacttt gggagtacaa gtcggtgggg ttctgcttgt cagccctcaa 67320

atgccacaca tcaccactgt accaaatgca aacctttgca cccacccttg tgttggccaa 67380

attcccgctt tgtgtacttt gtgtcacagc tggagaagtc atctggggag attgtgtact 67440

gcgggcaggt gtttgaaaag tcacccctgc gtgtgaagaa ctttggcatc tggctgcgct 67500

ccgactccca cagtggcaca cacaacaagt acagagggta cctaccagga tctgaccacc 67560

gctggtgcag tcacacggtg ctaccgcgac atgggggccc ggataccatg cccgaactca 67620

ctccatccag atcataaagg tggaagagat tgcagttctc aagtatttcc actactccaa 67680

gagcaaattc ccattgcccc atgcagcgcc agcacaagcc acgcttcacc accaagaagc 67740

ccaacacctt cccaaaaaaa atgaggagaa ggaggaagag gaggaaggag aagggagagg 67800

aggaggagga ggaggaagag ggaggaggag gagggaggca gggagggaag tatttatgtc 67860

aaaaatcaaa aggaggcgag acagtcatat atctgtggaa gaatattctg gacagaacag 67920

cttgtgcaaa gaccctaagg tagaaacatg cttttcccgt tcaaagagca ttaagaaaac 67980

taaactagag tgactggaag gccacgaggt aacagagatc acagtaattt gcaaggacaa 68040

gataatttag gctgctttaa cctggatcta gaatgttctc cacagcctgt gtcccactga 68100

atcatcttgt cagcctcaaa gctatacttg tttgttcttt aaatatttgg cacagacagc 68160

tccactgcac tgggcaggtc cagcctgagg ctctgcagga gtggaaaata aggagtgaat 68220

tgaaaaaggc agaagagctg aggagagggc agctgagagg ggttggcagt agaagcgttt 68280

gctgtacaac ctgataagct gggtttgatc ccggctcaca cagaggaagg tgagaatgga 68340

ttcccacaag tctccacaca gataacatgg cacacagatg ccccatcttt cctcctcctc 68400

cttctacaca cacacacaca ggccatttaa agtgtttttt gtttgtttgt tttttcgaga 68460

cagggtttct ctgtatagcc ctggctgtcc tggaactcac tctgtagacc aggctggcct 68520

cgaactcaga aatccacctg cctctgcctc ccgagtgctg ggattaaagg cgtgcgccac 68580

cacgcccggc tcatttaaag tgttttaatt cctttgccat cttgagagcc caggctgtcc 68640

ctgggacatg cctgtttgca aacacattta tattttctgt tctttttctt ccagacagtt 68700

catccgttat taggctttta aggaatactt agtaagttag actgtgggca aacaaaaatc 68760

aataaaacgc tattcttgcc gaggcattaa gtcttgtggg agaaagaaac atttaaccag 68820

caaagacaaa aagtcaaatt cagcaggctc tgggagtctc ggagagctag cctctggctc 68880

cgttagggac agtggtcaca gatggattaa gacaagatcg gaagattaag atcggaagta 68940

aggttacgct gagaacatga ccagtaagac tcaattcaca gaggatagga ttagccacta 69000

ggcaaacgtg attaatttgt tgcctttaag tatgtaaaag cgctttttct ctccacaggc 69060

ataaattcaa acttgccgtg atctcccgtt attcccgcat agatttccag ccttgcctcg 69120

ggctagtggt tggttagctc tttaaagttt gagaatcacg tgggctaata tatgaatgat 69180

ttcagcagtc tccactagaa ccaattctag ccattcccct ccgttaaaga cgtcttacag 69240

gctggctccc tgcgagactc tgcctatagg gggcgctcga gggaggccga agaggtcagg 69300

gccgccgcag ttgcagagcg cagacttgtc ccacaaaccg cagccccctt tctagtaaca 69360

gtggagtccg gtgtgcagtt ttggcagcac tgggttcgcc gtgcccgatg cagagaacga 69420

gcatcagcag atttctaatt taagcaattc cggcctcttc tctttctcct ctccgagcgc 69480

tgtgggtggc agctgctccc caccttcgct catttgagtt aactttaagg cagggacgtt 69540

tgccaagagt tggagaggcg cagggcaggt gagggagcat tgctctcatc tgctgggtga 69600

gctgaaatgc tttgaaataa acaccctact atacacagga taggctccta caagggattc 69660

catccagtgt agcaccaata gcagggccgg gaaaccctgg tttacagtaa ccccttcagt 69720

tagagatgac atgtactaca aagccatcat tggtatacta ttaagactat gcagagacaa 69780

aacacctttg aaaaggaagg aaggaaggaa gaaaccactt gtggtggcgc acgcctttta 69840

tctcaacact cgggaggcag aggcaggagg atttctgtga gttccaggcc agcctggtct 69900

acatagagtt ccaggacagg cagagctgtt gaaaaaaaaa aaatctttgt attagatctt 69960

tctgtcaagt aatgttgtgg ttctgattaa atcctgctga ttcaactttt tcagagttac 70020

taaaagcagt taggctgggt gtgtgatgtt tttttattta ttttcaaggt taaaaaatat 70080

aaaatttata agattaaatg aggaattaac tagaggaaac ttaaaatttc atgttttaag 70140

tcaacgaatg aattgtaaag gatttatgat taactcgtca ttacaaaatc ttttttggtc 70200

tttaatccta gcacttggaa ggcagaggca gatggatctc tgtgagttca aggccagcct 70260

gggctacatg gtgacttcca ggactacata gaaagcccct gtctcaacac aaaaccaaag 70320

ccaaaaatca acacaacaca acacaacaca acacaacaca aacaccaaga ctggatacag 70380

tacataaata aggagtaatg gaattgttta tggcatcatg attgttggcg tagaacctaa 70440

gggggtgggg ggtttggagg actgctgtcc tcacagcaac ctcatcaacg ggattttata 70500

caatctatat tcatttatta tttaaaaata gcttattaaa aaatcctgct ctataatata 70560

ctgccccata aacgctgctc aacggaaacc tcaggctagc ggttggcagg tgcattctcg 70620

ttgccctttt ttgtttagcc aatgttgcag aaatgtacta cttgggggtg agggtgggga 70680

gagtcagctt tggtgaagac agaaccttgt ttgcatatgg tgctggcagc accgcccaag 70740

gacagcttga ggagacctag gctcccccct gtgccagagt ccctcggtct tgctactgag 70800

ttcgctccct taggcattcg cctcctggaa ctccagacga gttcttggtc atcttttgcc 70860

atgattttac caatcctcag agcagcaaag gtatttctta gaaagtccca ggtaacgggt 70920

gacctgaacc tggagccaat aacttgaaga ggttcctaat gtgtccagcc ctgttccagg 70980

caacctgcgt gtatttcttc atctaattcg tccaatagtg ccatgaagta ggtccttgct 71040

agtgttaatt cagcactgaa gaaaccaagc aaggctgggg tgaggggttg gggttggggt 71100

tgggggagct ggcagagtgc ttgctaaatt tgtgcaaagt ccacagctca attccctatt 71160

ctccagaaag caggtatggt cgcacgtgcc ggtaatccca gcacgcagga ggatccgaag 71220

atccaggcca tcctcagcta tatagaaagt ccaaggccag cctggggtac ttgagaccct 71280

ggctcaacac agatgagtaa gttaattaat aataaaaaag tttaaagaaa ataaaagaaa 71340

tcaaaatgca gaatgttcaa gtagcttgta tgtgctatca gacaagaatt tcaacaagct 71400

gtctaaatcg agggatcctt taaactaata tttaagaata gcccatacag tgtgttcaga 71460

cgcctcttat ctgtttaaag agccggctca aaccagcctg gagacagaat attttcaaac 71520

gtgaaccatc tggttgttac aagtcaaacc acaggaagcc tgcattagag ttggctctcc 71580

ggttgactgt gcccagcctc tctctctctg gtttccgctt ctctttgagt gttgacttca 71640

ttctctcaaa taatggcggt gcttcagact tgttttcagt gtgcttatta aagagtaccc 71700

tcaggatcag ctgcaagact gggcagaaga ggaagtcaag ccacgatgct ctcaaccagg 71760

gtcagctcag taagagcttc agaattctgc gctaacaaag agacagagta aataaaaatg 71820

acccgaaggc cttcccgtgg cccggaatct gacctcagca gtgacagtat atgactttgt 71880

tgattgtgct gacctctatc attgaaatgt cccgagggac tgaagagcac gcataccaat 71940

gagagaaaaa tgtcactgcg acgagggccg gaatcttgcg actccatcag ggagaattct 72000

ccctttcatg gtggctccca gatctcgttt agcctgtaag ctaacaagaa gaagaggcgg 72060

tcaacgcaca cgtggtggcc atcacgacaa acgggatgac ctaagagaca aacgattaga 72120

cggcagcgcc accgtgtggt agtccacgtc ctcaccgcaa gctgaggtcg gaagagggtg 72180

tctctgtgat caggaaacgt gcagtttttg tgacatacaa gcaaagggga agggcaggtc 72240

cgcgtcccca cactggattc atattttctg gatttgttga tgccctcccc cagttctgat 72300

cttgctggcc tctgtactgc ctgccttacc tatctgaact gaccgtgtac ctgtgagcat 72360

ccagtgtaat atgtttattc cacacagatt aatttcaagt cctcggggca gttttccgtc 72420

tgctctgtgc atagtgcaca aggctaatac agttacaaga gcccagaatg tggcgccggc 72480

ccagctctct ggtgatgctg ctgcacgcac acacgcggca ctatgctgac accttccttt 72540

tctagatctc ttgaataatc cacgttgttc tgctctgcat cggcctcctt tagatactcc 72600

aggaactgct aaacactcgc ttctctctga ggacacgtct agttcttgag ctccttgctt 72660

cagtttaaaa ttgaactacc tggctcaccc actctgcgtt ttcacaagat gcctggttac 72720

ctaccattta gtccaggttc tgccgttagg ggtcccacgc tctgtttagc ctttgagcgg 72780

caggtctaac actggatcaa atctccttgc tgtggctggc ctttggccat ctgtcccaga 72840

agggcaagcc ttgttctccc aaactccctc tgtcctctct ggattcttcc gtatcgccag 72900

catctcaagt agaagaaccc aaactcttgc tgaagcaaac ctccacccat tccagaacaa 72960

agccagggcg gtcctcaagc cagccaggtt tccccggcta tgggcatctg ttttccatgg 73020

catggtggct gcaccaagaa tgaggtcctc tctccgttaa agggcacacc cttcagagag 73080

gacctggagc acaggactct cctgtgtcaa cagcaagtgt ctgggatgtg atgcactcag 73140

tcagcacttt ggtccattgt tgagtagaca gagcacttcc ggggaacatc gtggagggat 73200

aggggacaat tacactgaga gcattaatgc attcaagttt tacagtaggg ttgttacttc 73260

tatccacagg tttctaagca tatctctctg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 73320

tgtgtgtgtg tgtgtgtgtg tgtgtaaatg tatgcattcc acggcaattg tctattgagt 73380

ttgctcctgg attacctgac tcctctcatc acctccaggt ctaattctta gcaacacgag 73440

gacgatattg cgccacctac tgtccaaatg tgtcctgccc tttctgcttc aaattctcct 73500

ctcattagac ctaaaatagc gccctcgtcc ccactcccag ccccctctgg ctgcctctca 73560

cggttgggtt tgtaatttcc acttggcctt tacatttcca tccagccccg tgggcagcca 73620

tctctaagcc tggtgtccag ctgagctggc ttttatgcgc tcacagcagc ccttccaggt 73680

gcccacggtg gtggtccaag gatcgcagca aaccggactg cttatgtaac tagagacata 73740

aattggtccc aggagtcctg cagcgagtta ctgaaataac attctcttta tcacaggccc 73800

gtttccacag ggccttttta cggggtgctt atgcaacagc cgccccgtca gcagaagtcc 73860

tggaagcgaa gagcagggtg atatttacag tgaataaatc atcgaattca ttacataaga 73920

acttaaaaat actagtgttt ctcctaagaa cttgggaagt tcagaaaaat ctatacccgg 73980

tcttagcaga aaagtgacgt ttcaaaaatt ccaaagcagt cggttaataa tttcaacaga 74040

agcgaaactc gagcaccgtg ggaaacgtct tcccagcctt gactggcatt gactttcggc 74100

tagtctcttc aatgttaagc aatgtcagcc agttaaataa atgtccacaa acgaagtctt 74160

tgttaaattg aaatttgttt tgctccaaaa tgactgttta cacaagacag agacctatgc 74220

agtggaaata taaaggcata aatgaggaag gaggctacac aggagatttc aactctcttc 74280

gaatagtagg tatatcagag ggcttcatgt tactgtaacg aaatgcctga gacaatcgac 74340

ttacaaagag gaaaggttta tttttgctta tagctctaca agtggtaggt agcctagaac 74400

cagatggcct taatctttgc ggctctgaaa agatgtggca aggccatcct cctacatctg 74460

aaagacactg agatattggg gtcccagagt ccccttcaaa gacatgtttc tagtgaccca 74520

aggaccgaag gttttatgaa acctcacctc ctcacagttt ccccacttct caataatact 74580

ttcctggagt ctaaaccttt aacacagtgg tctttggaaa ctactaccca ttttcaagcc 74640

atagtatgtg tgtgtgtatg cacatatatg catgttggtt tacctttgct aaggcttgaa 74700

cccagggcct tatacatacc agactctgcc accatgctac atcagcagcc tataatttgt 74760

tttattattg ttattatttg cgtatgtatg cttatgttca tgtttacatg agggtacagt 74820

tgtgcatgtg actgcatata ttcctgtgtg tctgtgtctg tgtgtgtctg tgtgtgtgtc 74880

tgtgtgtatc tgtgtgtgta tgaatgtgta tgtctgtgtc tgtgtgtgtg tgtgtatctg 74940

tgtgtctgtg tgtgtatgta tctgtgtgtc tgtgtctgtc tgtgtgtgta tctgtgtgtc 75000

tgtgtgtgta tgtatctgtg tgtctgtctg catgtgtatc tgtgtatctg tgtgtgtgtc 75060

tgtgtgtgtg tgtgtatgtg tgtatgtatg tgtgtatgta tgtgtgtgtc tgtgtctgtc 75120

tgcatgtgta tctgtgtatc tgtgtgtgtg tatgtgtgtc tgtgtgtgta tctgttcatg 75180

tgtctatgtg tgtgtgtctg tctgtctgtg tgtgtatgta tgtgtgtgtc tgtctgcatg 75240

tgtatctgtg tatctgtgtg tgtgtatgtg tgtctgtctg tgtgtgtatc tgttcatgtg 75300

tctatgtgtg tgtgtctgtg tgcatctgtg tgcgtctgtg tgtgtgtagg acagagggca 75360

aacctacttg tcattcctca ggaactatgt tttgagaagg ctctctctct tgaccagaac 75420

acacccatta agctaggttg gttggccagc aagccatggg atctacttgt gtctggttcc 75480

ccagctctgg gattacaaac ctgccaccaa gtctgccttt ttagataggt tcaggcattg 75540

aactaggatc cccgtgctcg tataaccagc attgtacctg ctgagccatc tctccaatcc 75600

tatattttta aaatggtaag tagatatact gggactcatt gtgtttattt attagttatt 75660

tattgattta gtttgagaca aagtcttgcc ctatagttca ggctttccag aaactccata 75720

tttagcccag attggcctca aattacaatc cctttacttc agatttctga atgttggggt 75780

aacaggcagg ttaccaccat cctgccttta tatttttctg tcaaaaagta aattcaaaaa 75840

gaaagcatac ttttggaggg gtagggggct ggggagatgg ctcagtggtt cagaactctg 75900

gctgctcttc caaaggattc aggttcaatt cccagcactc ataggtcacc tcacaactgc 75960

ctgtagttcc agatccaggg gatctgacag cttgacacag atacacatac aggtaaagca 76020

ccaaaatcaa tcaatcaatc aatcaatcaa tcaatctagt gtctctgctc acctaaagta 76080

aatatgaggc catggaagct gctagcctca gggacatata ccttgttcct ccttccattc 76140

aacatccatc acactcgcct gcattttaat tccctgcatg tctttctgtc tctctcagtg 76200

aactatgaaa cttcttcact ttcttcactt tctttctttc tttctttctt tctttctttc 76260

tttctttctt tctttcttct ttctttcttt ctttctttct ttctttcttt ctttctttct 76320

ttcttccttt ctttctttct ttctttcttt ctttctttct ttctttctat gtatatgtgc 76380

atgtttgtat gtgttcatgt gcatcacgtg tgtgcagtac ccgaggaggc cagaagaggg 76440

catcatatcc tcttgaaatg gagttcctat gattggaagt tgtcacatgg gtgctaagac 76500

ccaagcctgg gtcctctgca aaacagcaag tgctcttaac ccctgagccc tctctacagt 76560

cctgggctgt gcactcttgg ggtgggggtg ggagagtctg attatttttc actgtacttt 76620

ccctatcacc caacctaatt ctaaaataaa tatgtgctct gtagacattt gttctgtggg 76680

cagctattga aagcagggcc ccaaggcagc tggccccagc accgtgaaaa tgtcagccta 76740

atgttttctc cttgacttct cttatttcct tcataaaatg aactgcaagg aagtggctca 76800

gttagtagaa ggctgttgtg tcttttgcaa agcactgggt ctcatcccca gagctgcata 76860

aagccgtgca cggaggaggt acacacatgg aaccccagca tttggaaagt gaggtcagga 76920

gggccaggag gtcatggtct tcccagatgc acagtgaggt gggtctgggc tgtatgaaaa 76980

cctgtctcca aaaaaaaccc caaaattctc aagaggctgt ttaagctaga catcatatag 77040

ggaccatggc aatgggtcaa ctgcatctct aattgtctct taggagcttc ttagagacac 77100

actcaggaca agctgcccgg agtctttgtg aagaccatgt gtgaggatca tgctagaaat 77160

gtctcaccct cactgagccc ttaccacagg ccaccaggta ggtattccga tctccactca 77220

ctgtggtgaa ggaaactagg ccccagagag ccagcccata gtctgtgacc gtctccttgc 77280

agtctgagcg ggatgcatcc agtgagactc ctctgtctga ctctgaagct caggtctgtc 77340

cttcttccta cctgctccac accagggcac atcgttttga cctgaaggga ctttgcggtt 77400

gttcatgcaa tctatatttt gttacttcag gactttgcca agagaaacaa agaatgctcg 77460

attatcctct aagtctataa ttgattgaat aaatcttaaa attagccaga atccttttgg 77520

gccatattgg gtgaatgggg cctgctgagc cattctgcct gtccggggca gacactttgg 77580

cttggattca ctctgtatgg gatatttttc cccattcatc tctaagccta gctcattggt 77640

ctgtttcacc cctacctccg ctgccagggc agcccactct gacatttatt atcctcagtg 77700

tctgctggga tcagggcttg ggtgtatagg agttgtctca gctcatcctg agatccaatg 77760

agaaaagagg tgtctttcct cccatttcac ttttaagaaa taagtaactt ctcacggtgg 77820

accccacgtt ggggtccaga aatccacagg aggcgttccc ccctaccccc cacccccacc 77880

ccgcggtccc ataacgtgat gcaatgcttc gcacgctgcg ctcacagtgt gggcttcgcc 77940

ccctgccgct cagccttttc ctggtggtct tcggggcaag cggtggtctg agtttgtcat 78000

tgtttcagaa acagagcctg gctttaagga cctgatcagg cacaactcca gcatctaaat 78060

ccccttccta agctgaaggg atttcctttg tctggagttt taaaaagacc ctttccttaa 78120

aaaaaaaaaa aaaaacctct ttgtggagcg aagcaaggga ttctgttttc aacaactggc 78180

ttgagaacag gcgcacacat cctctactgt tcatcgtcgc tgttactggt aggtttgcag 78240

cttaaaggga gtgggcgaaa cgcgctcaag gctactgctt gctcgttgga gatctcagtg 78300

aacggaatta atcaggtgac atttgcatgg tgcctagaac tttgccacca cagactttgt 78360

ttttttcatt aagaggattt atttcttagg taacacaaat ataaacagtc agtaaataga 78420

tccccaggag caaaatcgga gaagcagtga aaatcctgga gacggacgaa ggagaaaatt 78480

ctggaagctc tgtttatgat cgcctccttt tgggaattga agaaaggaaa gcagatgtca 78540

gacagtggaa ggttttacag agaccggttg atggttccgt catcttgaag acaaggtgaa 78600

gtaaaacgga gatgtaatgt ttagcttgga acgtaaggtg aaccggaagt gatttggtat 78660

tgttgaccaa gacctcacca tgcagattgc cgaatccata cctaacccat accaattaaa 78720

ggataggaca gtgagaaact gtctctgcac ttacgagtgt gcgcatgtgc atgtgaaaat 78780

gacattttat gtgtatgagt gttttgcctg catgtatgtc tatgtaccat gtgtggctgg 78840

ctcccaaaga ggccacaaca tgtcatatcc cctagaactg gagtcacaaa tgatgatgag 78900

ccaccatgtg ggtgctgggc attgaaccca tgtcctctgg atgaacagct agcggtgcta 78960

accactgagc catctctcca gtcccaacta ttagggttct taatgataaa tgtgtattag 79020

ttcctttttt attgtgataa ataccatgac cagggcaagt tatagaagga agaaggaaga 79080

gtttatttgg ggatagggtt ccaggaggag agtctataat gagagggaga cactgtggcc 79140

cagaactgag agacctcatc ttcaaatgaa aacacggagc aggatgaact ggagtggggc 79200

cagactgcaa agttcaaaag tctgcaccca gtgatgtata cttcctccag caggctttgc 79260

ctcatgaaga aaggttccac ttcctcccaa aagagccacc agccaggagc tgaccaagtg 79320

tttaaataag tgaccctgtg cagcaggtgg aggggacagt tctcagtcaa accaccacta 79380

tgtgtgaaag catctctctt tcgatatctt tgggttaagt ccagtctcca gagagagcca 79440

tttatctaca gtcacaaagt ctgtgtgaga caccctgtgt taggagcacc cactttgggc 79500

atactgagga cccactatgg gcagatgtca gagactcaca ataaacaatc tttagcagtt 79560

gttgtatgga atgcagtttt ccaaaaattt ctatagttct atgctaacct attcttgtta 79620

gcattctatc tggactctgc ccccacagtt acctggcaac aaccaggtag gctcctcccc 79680

acagttacct ggcaacagcc aggttgcctg gcccaaccgc tgcttgcccc ctcctctctt 79740

tcttactcct acttctctcc cttgcctctt gctccccctc tccccattcc cttcccactc 79800

tctccaagtg gtcatggtca tggctagcct ctatactctc tctctctctc tctctctctc 79860

tctctctctc tctcctccta ctactactac cctttaaact cccttcccca tgcccttaat 79920

aaactctatt ctatactata ccattgtgtg gctggtccct cagggggaag gaatgccttg 79980

gcatgggtcc gctgaggcac tcctttcccc cacacctaca tagaacatat ctctcctctc 80040

tttatctttt tatagtcaca acaatcctgg gataggattg ccagaaaggg agacaaaacc 80100

tgcccaggcc tgccacaagg gccctactca tagaatgtaa ggagtgcagg ggagagctgc 80160

tgaggtcggt cttctacaga caggaggtct gggctccttg ggagtcagtt ctcagccagg 80220

tcttcctgac tcagtttgtg agtagggatc ccttgttcag catccatagt gttagaccta 80280

gtggggaaac ccctacccaa ttcccaattc ggcgtgcacc caaaaaaata cagccctgtc 80340

tgttctgctt tgttctcggc cccaggccgc gggctcatag acaactcttt tacatgaatc 80400

acaagttaca aaatctcatt ttctttcaat aggaaatggc aagctacagt aaaagtagtg 80460

tagcttcact ccgacctcct acaaccttcg aaagttatgc aattatggct cagttggtaa 80520

aatggctgcc atggatgtag caagcacata aaaagctatg cctggccggg cgtggtgatg 80580

cacaccttta atcccagcac tcaggagaaa gaggcaagct gatttctgag tttgaggcca 80640

gcctggtcta caaagtgagt tccaggacag ccagggctat aaagagaaac cctgtttcaa 80700

acaaacaaaa caacaacaac aaaaaagcta tgcctggcac cgcagtgcct tggtggtaga 80760

caggaagggt catgaccttc ctggcctgcc gattttgcca aatcagtatc ctccaggttc 80820

tgtgagagac cttgtcttaa aaaaaattaa gtgagtacca ggcatggtga tgggtggtac 80880

acaccttcaa tcttagcaca tagacagagg caggcagata cacacacaca cacacacaca 80940

cacagacacc acagagattt ttttattggt aaaaaaagat tctactagaa tcaagtgtct 81000

aaataatagg atttaaccaa ggggatcaaa agggattaga gtgaaacacc aaacccacga 81060

agacaaagaa aatcgaaacc agaaaccact ctctccccac ttcctgctgg ctcatttcca 81120

ggtgcctttc tgtctggtcc cattgtctta cttacttgat aatctccctg ggccaggagg 81180

gtgttgattt agagcacctc ctactccgac tgataacagc ctccaggcct tccatgctct 81240

gtggggtctt cttcagcctc ctaatggccc acccgctgtg ctttctgtta caaatgtgaa 81300

aaccttcatc cccgctaacc taccaagtgg taaggtgtta tttattgaac agataccagg 81360

agcacatgaa gggccggggg cacagctcta aatggttggg gaacctcaga gagccaaaca 81420

gataagaaac ctgctcttag ctgcttgtat cttcctgagg tgggagaggg tgcccagaga 81480

atactcagaa acacaacaag caagtgtaag gtgtacttgg caggtgggct atgggaaagg 81540

tcacagtaca gggatggagg agggcacctg attctcccct gttatcagtg ggaggctgca 81600

gtggagcaaa acacccccag tccagatctg cccagcttct gccctaattt ccagtttgat 81660

ctatctggga ccctttcctt gatctccact gaaaacagga acgatgttaa gtgcagctag 81720

gcacgcatat cctaaagccg gactatttga aacagatttc cagctcagca aaatgctccc 81780

cttggaccag taagctgctc tctgctaata aaatttacct gtgtcacagg ttatggagag 81840

ggtgatgtca cccacggaga gagtgatgtc acccatggag agggtgatgt cacctatgga 81900

gagggtgaag tatgtttagc ataagatgag cagagctgag cgtggcacag cggggcagat 81960

gtcacattaa agacagaatg cccgaactgt ggggtggagg acagcgtctg ctcagaaaac 82020

gatggcaggg tgaggttgtg cgacccattt ctacccttca acagctgtac agcctgctgt 82080

gtgggccccc ctgctcaaag cccgaggcta tgccaacatg aacacaagca tgcctcttct 82140

tgtatccatt ggtgtgtttc cgaaaaccgc tcttggtttt ctgtagtgag gagggtgggg 82200

ggttgtttgt ttaaaacttt ataaacagaa gaatttttta aaaatcttat agtcactgta 82260

atttacatgg tacagggctg gcactgacag gtcccttcaa tgacaagtcc gttcaatgac 82320

agagagtttg tctgaagccc aggcttgggt ctccagaatc tctgcaccaa accaattgtg 82380

cagtacattg atcactactg atggagaaat aggagttacc tttctgtttt caagatctat 82440

ctatctatcc gtctatctat ctatctatct atctatctat ctatctatct atccatgaga 82500

tcttagagtt gaacaacagg aaactatacc agtgttccaa accacctggc aactgtcaga 82560

tgttgccagt cacaaaatcc acttccaata agatgccaga cctgtcagca gccttggttt 82620

gggaggttct tagggacact tgctactggc aatatgctgg caagcctacc atgttcacag 82680

cttgtgtgaa aaatatcacc caggcacttc atgtcctgtt gcccgacgac ctccaactgc 82740

tgtcctgcct ccctcagggg gatggggtta gcagaggata tccactcaca atgagatgtc 82800

tcactttctg cgttcagatg agacaattct cactgttttc tttagtaaga aaatgtttcc 82860

cgagagaatt cttcttctta ccacatcatt gagctctaag agaagttagt atctctcaat 82920

gatggagaca cactcttcaa gatctaggct ggcctggtct acatagtgag ttccctgtca 82980

cccagggtgt tacagtgaga ccctgtcctt taccgaaagc agccttcatt ttaatttggt 83040

gttgcttggc ctgtactgtg gggctgttaa aagccacacc catagcctct gtagaagaaa 83100

tagatggcaa aaacttcagt agaacatcga gggtggggct ggagagatgg ctcagcaggt 83160

aaaagcaatg actgctcttc cagaggtcct gagttcaaat cccagcaacc acatggtggc 83220

tcacaaccac ccgtactgag atctgaagaa cattgagggt ggatgagatt gaccagcttg 83280

tcttaggaag gaaagaaggc tcaggatttt gccaatgacc aagggtttct gccagtctca 83340

tcacctcgga agctgagaat gaaggcatgg aagagaaaat tctgcagggc tgagttaggg 83400

ttccagtgat gtggcacatt acctgagatg cacatattca ataacttttc tctttcacta 83460

cctctgctgg gtggtggact agaggagagg gtgaaccttt ttaaaagtag gctggaaaaa 83520

aaatgtgtgc ctccagggcc tcttcaaggg cacaccccaa tgtcctgtgt tcctttatcc 83580

cccttcctaa ccgttctcaa ccttcctaac acttccactc tttgagacag ctcctcatgc 83640

tagggtgacc cccaaccata aaagtatttt cattgctact tcataactgt aaatttgcta 83700

ctgttatgaa tcatgttgta aatatctgat atgcagatgg tctaaggcga tcctctgaaa 83760

gggtcgtttg acccaggtgg gttgcgagct acaggttgag aacagcagct gtagacacta 83820

cggtcctcca aagggttaac tttggctctc agtttgggtt cagagtccat catagttgga 83880

aggtccagtg cagacgcaag aggcttccgg tcacatttca tccacaggca ggaagtagag 83940

ggccttgaat gctgtcacgt gacttaatcc ttttcgttta gtcaagggtc ccagcccata 84000

gcctacatta ggacgggtct tctcacctca gcccaactta gaaatccctc acaaacatgc 84060

tataggtttg ttttttaggt gattctagtt cctgccaatc tgatgatcga agagggtgtt 84120

tttgtttttt ttttagattt ttatttattt attttaattt tatgtatgag tgggttttgc 84180

ctgttatagc tgcaccacat gcatgtagtg cctctagaat ccagaagagg gcgccagaaa 84240

ccaatgtaac tggaattagg actgagccat cttatggatg ctctgacccc aacctggggc 84300

ctctgcagga acagtaagta ctcttaactg atgagccatc tctccagccc ttttaatgat 84360

cagtattaac cttgacattt tttaaaacat tccatcatct cccaagaaca ccacaggtga 84420

gcgagcaaac ctttagcaaa cggactctta ggggacactt aaggtccaaa tcataacaag 84480

tggctagaga tgattaaaca agaatactgg caaactcaac aagtctgggg agacatagtt 84540

gcaattaccc tgtttgaagc gctcctgttt ctgtgcccag gaacggcttg aaacctggag 84600

gagaaacact ttctcaccag ggggaataag gagctggaag tttcgggacc atcttgcttg 84660

cacacctgct gctcagaatc tgggggagag gcctttgcta agccaagtcg ccaacacaaa 84720

gcttctccaa aggccaacag gaatagtggc attgcaagaa gatgtgtgcc atggtaaact 84780

ctgagtgtgg ccaccccaga gggtcgtgag gcagggccag cttttagtgt attcgttgct 84840

ggagttacag aaaggaaggc ttctaaaggc aaaaatgaga aagatcacac aacctctttt 84900

gttgcaacaa tcctttggcc aggcagacta gtggacaagg gcgttcaaag gtcagaggtc 84960

agtccaaatt taaacagaag acggcggatc aaatgtcctt gcacaatcgt ggtgtagtgt 85020

gtaaggttgt agatgccctt gttcgaggct gtgattctgt cagtctttgg agggcagtcc 85080

tttttatcca ggacttggtg acagtcgcta ctccagggct ttccagcttg acctaagtga 85140

ctctgttttc tctctgacaa cctttacact ttccaacccc accctgcgaa ggcctatacc 85200

tgatacaaga tcccctaagc tgttaccgtt cgttcctacg tcaggacttg ctcttaccct 85260

gttctctctg acctacaatg tgccatctca ggtgaccttc tgtgtcccca tggcctggag 85320

acaacgcatt gtcctgggct cccctgttct acctcctttt ccgcccctgc cactctggca 85380

aattctactg tttattaaag ttcccaccct ggcctgtgaa ctggaagtgg ctgtagattg 85440

tggaaagaaa atgtggtctt gtgtgggagg ttccggcacg gtctagaatt tcagcactct 85500

cagtaattct caccctgagt tgacacaaag actaggtgag tgaagatgcc tagattctac 85560

tgtagctgaa atctgggggg gaagggagga ggcatgaaag gtgggagcac tcctaacagg 85620

gatctgccaa gtaaatgcca ggtgtctgaa cctgtactgt ccagggagcc tgtgtgtctc 85680

ctctggacca ggaactggtc tatctttgag gccactgggg ttccggaatt cagagccttc 85740

tcagaagccc ctatcccacc ccaccccgcc ctccgcgtgt cccggaggca gctcagccac 85800

tttgccggac tccctgccca gctgaacctc aacattcctg atgagaccga ggaaggcgct 85860

aattaaacta atcacgggag agagctgctt cccgctcgtg atccgtttgc gagcacctcc 85920

ataacgaggt tattattatg tgtaagggga gtttggctct aaattttaat ttcctgggac 85980

accagagcca atgccaagga ggagtttatg gaaatgcagc ctgatgccaa aaacaaatta 86040

gatttgttct gccacaggat attttgtgaa gttgtgaagg attggccaca tggggagaga 86100

cttaccttga ctgcttcatc tccctgtcgg tctatgccca ggccttagaa tattctcgat 86160

gtaataaagt cccttaaaat gcagactgat tcctttttta aatccacata cttaatttat 86220

aggctcttgt tccctgccct tctccacagg agaaccttga ttgtcttttt taagctgggt 86280

tttacactgt tttcttctcc tgtgtggttt gggaactatc gaagagaaga acaatgccta 86340

acaaggaaaa acagaaatac tcaattggtg tctgactaat caggttacga ctcagaccag 86400

gctggcctcg aactcagaga cttgtctgtc tctgcctcct gagttagcca ccagtgcgca 86460

ggcccagttt ctttcttcgc tgtccctctc catgctttaa acccctggtg tatatttggg 86520

caacttcaag ataactcata ggtgagttaa aggtcccagt acctttgtgt acgatgttca 86580

caacagtacc ctgtgattgg ccctctgtta gttcagtggt ttaagtcaac cttggaacac 86640

acagagttca ctctgcttct gagagtttgt acaataaaca tgttgtatct ggacacaaaa 86700

ggacaaaaga tgaacaataa gccagggaga agaattccat gctgtgccca caggcaaggt 86760

taaatgttgt tattacacac aacgctctcc tgaggatctc tccttatctc accagcccac 86820

cttcgggagt ttgcttattc aatgagaatc tgtgtaataa accacattgt agttgggcat 86880

gagaagataa aagatgaaca gtaaattggg ggcagcggtc acatgccgag ctcacaggca 86940

aaggcttagt gttacacaca aagctcccct taatatcttc ccttagggtt ccctttacgc 87000

cataaaaacc acccaggagg ctagagaggt ggctcagctc ttccgaacac ttgttgctct 87060

tagagagaac ccagcttcaa tgtccagctt ccacatggtg gctcacagcc atctatgact 87120

ctgtcacaag agatctgttg cccctttcat agtgggtcag gcctacctgg ctgttgccag 87180

gtaactgtgg ggaggagcat acctggctgt tgccaggtaa ctgtggggtg gaatttagac 87240

agaatgctaa cagtggagtc ttggtattct tagtgggaca tggctgagac caggagggag 87300

gatgtcgtgc tgaaggttgg cagacagaga gcataggaaa tagtcatctc tggctggatg 87360

tgggggagtg gataggatag gaatatggga taagcttgct ttgtgggctg aaggcttctt 87420

gaacgttgat attctaaatc aattatactt atctatctac ctatctacct acctacctat 87480

ccatctatcc atcaactatc tatctaccta cctacctatc catctatcca tccatcatct 87540

atctatctat ctatccatcc atcatctatc tacctaccta tccatctatc catccatcat 87600

ctatctatct atccatctat ccatccatca tctatctatc cagctattca tccatcatct 87660

gtctatccat ctatctatat acctacctac ctacctacct acctgtctgt ctgtctatct 87720

atctatctat ctatctatca tctatctatc tatttgtgta tatttgtgtt gtgtcttgtg 87780

tgtgcttggg catgtatgta cacacatgtg aaggtcacag gacaggagtt gattctcttc 87840

ttctaccagg tagaaccagg ggtggggggc tattctcaag tatgggcctt ggtaacaaga 87900

gctttaacct gctcagctat ttattccacc ggtccagaga tcctaagttt aaggtgacta 87960

cttggtatgg gcgaggcttt tcttgagtct gtctggttgc tgggacacag ccgaatcagg 88020

gcatcatatg tcggattgag aagtgtgtcc tttcaaagag tcctctgttg aagccctaac 88080

cacagatatc atcacactgt ttgaagatgg agcctagcag gaggaaacaa aagttgaatg 88140

gtactggaaa gtgcggcctt aatcctggtg tcctcgtaag aaggaacaca agttgtccct 88200

ttctacgcat gtgcagaagg cggtgtgagg gaccagtgag aaactggctg tgtgccaagc 88260

acaagaggaa cctcctgggc gaccatccct cccagtacaa ccttaggctt tctgcctcaa 88320

gaactggtag gccatgaatt tctgttctca aagtcaccta cctgacctgt ggcatttcgt 88380

catgacaacc ctggtggctg aagatcctgg ggaagagttt ttcagggcaa aagtcaccga 88440

agatgcagca gggagtgaca caacacagga tcacacaagg gaagaccaga agatggcagt 88500

cacttagcca aggacagctg ctggtagctg cttccagaga gccagttctg ctgcagtgcc 88560

catgtgtcag ggacagtgct gggatctcag tgttttgcag tagatgtagg ccctcaagga 88620

tctcacggtg ggtaaaggac acacaaacac agtgccacta caacatggtc aacggtcagt 88680

gctgctttaa gaaacaaggc gagttgtcat gtagaggatc tgtctcaggg gaggtggtgt 88740

ttgagatgaa catcaaatct gagacggggt ttgccgggag aagagaactg agagtgtcag 88800

gctggagaga cagctcagcg ggtaagagca ctgactgttc ttccataagt cctgagttca 88860

attcccagca atcacaatga gatctgacgc cctcttctgg tccatctgaa gacagctaca 88920

gtgtacttag atataataat taatacatct ttaagagaga gagagaggga gagagagaga 88980

gagagaaagg aagaaagaga gaaagaactg agggtgtaca caccaagggc ccgtggcagt 89040

tgaggcagga ggacagccac atagcaaatt ccaaactaga gaagactgca ggtcaaaaca 89100

ctgggaagcc caagcgagtg caggtcaggt tgggggtggg gcagagtaga ggaagcaaat 89160

tgtgatcaga atgtgaaatg cctgtggggg accatgcaga atggagctgc tacataatct 89220

ctagacggaa gagaaaatcc aggtagtact gcagagtatg agctaagaca ggaaagccac 89280

ttctaggaag tatatgtggg agagaactca agttgatggc tgagatgaag gggatatttg 89340

ggtgaggtga gacctaacaa tgacttctaa tcttctgctt ggatgcctga gatcggtaca 89400

acacttgtca gcattaggga ctgataggtg agaaacagag actgttggct ggggggaaaa 89460

tgggacacag tggcccaagg ggtgttgatg aattccggtt tagacatgcc cactttgagg 89520

ggcccgtggg gaatgtcagc aggcagcttg aacaccgatt tggagttctg ggagaatgtg 89580

ggtaaagttg aacggtacgt acggtaacgg agaatgtggg taaagttgaa cggttaccgt 89640

acggtaactg aagtcaggca ataaacagag ggagcaaaga gccatcagct atcggctggt 89700

tgtcctatgt ccagttgtat gttcctgtgt gttatccacg gccagcaatg ttgtttaggg 89760

gaaactattg ccaccgtctt tttccaatga atactcggag gcacagagag atattaagag 89820

agtttcccaa gcttacataa ttgacaggtg aatttaaacc tgtgtgatct gtccctcaga 89880

catatttatg ttcaaggggt aaggaaagaa gagcccgagg agaaaccatt tgacctcgga 89940

ccaacaacaa agccaaaggg attcttagaa gccagatgga gggcatcaaa tgctcaaagg 90000

cctgcaaaga taaggggagg ccattaggaa gaagttctgg agacaagctg tggtcagaca 90060

gctaggagac cgcagaggca cacggcatta gcagacaggg agcaaaggca gaaagcaaat 90120

gttaagatga ggctgtggtt tgatataaac catgcaacaa ggttagatag gccccgaggg 90180

aggaaggacg ggtggtgggc tggtactgta gcaagcatat gcatgatgtc atgttggcaa 90240

gtttatctcc agtcgacacc ctcctctcca gtgttgaagg cccagaacca agcttgccag 90300

agcaagggtc tgcaggctcc tgtgagctta tgtttaaaaa accagaatgt tgattaatta 90360

attgtttctg agtaatatac tacaaaggga gccagatgtt cgtggtttgt taatgagaac 90420

attatagtca gtaggcagtg cctcatgata attaaaatgt tgccagggag tgtgagctcc 90480

attattagtt atatttgctg actatctcag gtgtccatga tccatagcca tctttccgtg 90540

ggcagacatg aagtccagtg acagattttc tttcttttct tttcttctct tttctttcct 90600

ttccttttct tctctctctc ttctctttct tttttcttcc ttcctttctt tctttttctt 90660

tttaaagatt tctttatatg taagtacacc atagttgtct tcagacaccc cagaaaaggg 90720

catcagacct cattaaggat ggttgtgagc caccttgtgg ttgctgggat ttgaactcag 90780

gacccagcca ccatgtgctt gctgggattt gaactcagga tccaagggag ggcagtcagt 90840

gctcttaact gctgagccat ctctccagcc ccagattgga ttgtgtttgt atgtatgtgt 90900

tcctgtggat gtgtgagtgc ttaggggtgt gtgtgcacac atgccattgt gcgtgggggt 90960

cagagatcag catcagggat cttctaccaa tcccactcct ccttgctttg agacatggtc 91020

ccttactgac ctcagtgctc acagattggc tagaatggct ttccaacaag ctctagggac 91080

acactgtctc tgtctctcta gggacgggat tcccaggcag gccaccatgc cagggttctt 91140

tcatgggtgc tgcggggatc gaactcacat ccttaggctt ctgcagcgag tgccttgctg 91200

gctgagccat ctcttcagct cacatttgga ttgttctctg gcactttgat gtagtgcttc 91260

tcacacggga tcaacatttt atctatatgc aacttgctat gcccattcac cacttccctt 91320

aggtttcctt tttttttttt cccgttaact aaaaattcac agtaaaatat ttcactataa 91380

atatttcagt aaaatattta ctaatgtata ttttaaacgt aaataaatat tttacaaaga 91440

ttatattatt ttaaaaggaa aaatctgtgc aagaaatgtg aatggaggaa tgggctgata 91500

acttgatatt aatcccagtg gtccgggggt agagacggga ttcccagggc aacctggcta 91560

gttacgatag ccaaattgat gagctgcagg ttcacaggca ccactgcctt actgaggaaa 91620

atggagagcg atcgagaaag tgacccaaaa gtccactgct ggcctccaca catgggcaca 91680

cacatatatg cacacacatg ctgtcacata gcacatggga aaatgggaaa atctgactgt 91740

gtgatatgct cctaaatcat tgtgacatct agcagtgacg tagccgagct cctgagaatg 91800

cctttctcaa acaccatgca atccctccat gccatcagag cctgccactc acagcactgc 91860

tccacaccgg acacaggcag atgtataggg attccttcct tccttccttc cttcctttcc 91920

acctcctctt ctttctttct ttcccctctt ccccatctcc ttcctttcca tgttcccact 91980

aggaaagctt gaatatttat gagttagagg aacagatgaa gcccaacgtt tggtaaaaga 92040

ggcaacatca gatagcgaag gagcggcagt aagactcagc ccagaaagga gggagaatcg 92100

ctctgtgatg gaggacttcg ccatgttgat aaaagaattc tcattttaca tcaaaggtga 92160

aagaaaagaa gtttatttag tgcaccaaaa ggagcccccg agtatgaaag tgacagaatc 92220

accagtgcag ttttggttag gggaaactac ttcaagggca atttcccaga atgcaattcc 92280

cagtatgcaa atgagggtgc ttctgtggta tcagggtgtg gctaattctg tttgctctcg 92340

ttattatgca ttgtgctatt tgggttgaac ggagccatga tttttctgtt ttggagatgg 92400

cttatggacc aggcttgcac tgggaaccca tttccttttg ggcctctcaa tgatgagctt 92460

tttttcctac cccacccccc acaatgcagc aacctcccaa agagaaccat atgcaggcac 92520

tgattggatg cgctatttgt attgcgtgtc cggagaggaa gcaccatctg gagatagaac 92580

tgttgaaccc cagaagaaat aaatttgaca agaaggttaa aaatgccgag gtcaaacgca 92640

agaaagaaat gctgataaat ggtcccagaa caacacagaa gccgtacagg cacagccatg 92700

accagacatt tttttctgat tccaccaatc ttccttgttg ctgaaaatca ctgacatttc 92760

gggggttttg atgcgtttgt cccgattcct tttacttcct tcaacatggc acaaggaccg 92820

tctccaacac tggtcagtaa tccagggcct ggggactttt aagtataaca gctgtgagca 92880

aattcaagtc ttcttattcc tcttttactt ctctgcattc agaggaaaca ctgaccagga 92940

gcaatgtatc acaggctcac agatttgtct atcccaagta ggcttttggg gtcccctttg 93000

aagagaaagt gtggatcctt aactggcaca tcactgacag ggcgaacaca tatttgtacc 93060

taagatactt tctgagaaac aaattattta ttctggatag agggacgtgg taatttcctc 93120

aagtttgata gccattgggg tagtctcatg cagaagacac aactggggga aatgagagag 93180

agagagagag agagagagag agagagagag agagagagag agtaaaggag ccaaaggggc 93240

ttttcatggc ttttcacgta tatccagaag agtaaactct aaggaacctc ttactgacca 93300

ggccaataaa tgtccctgtc ccaaacaaaa ggtgaaagct agctgagaaa tgataccaga 93360

ggttgtacat acacatacat acatatacac acatacacac atacatacat gcacacacat 93420

gcatgcatgc acacacacac acacatacat gcacacacac acacacacac acacacacac 93480

acacacacac acacacacac acacatatgc acacacacag aatcacagga ggccagaccc 93540

tctcctctga ggcctgcaag gttctctgtg agtaatgaag tactctgggc atgctccatg 93600

cctacgtggc atttcagatt ctcctgtggg ccctgctttc tttttggatt cagaggaaac 93660

actgtggtaa gacagaggac tgaggcatgt gaggaggtcg ctcactggag ccctgttagg 93720

cagctttgat gaagcttgta gggtgtcagg ctgtgcagct gagagagtcc gatccttctg 93780

aaactctcag gttctggttc ctccaggacc caggtcaact gaaggaggag aaggaggaag 93840

tcaaagagga aggaaggact gctagtaaat ctgagttgct ggagggattc accacagagc 93900

agggtctttg tccattaggg tttgaggact gaagttctca gtgagttcaa agagaatgtt 93960

tagtgggaag aacacttacg aaagaagagg caggggcttg agcaggagtt ctgaaacaga 94020

gctgaggagg taccagtggt ggtaaggcct gtgtgacacc ctgagagaga cttcagtgta 94080

gaagaaacca ggagagatta agaacccaag aaaaagatca ggctatgtcc atactctgag 94140

tgacttcacg ttgtataaaa caatggattg gtttggtttt ggcttaaggt tttggtttaa 94200

ggttgactcc atcttgagtg tggggaataa tctcactcct ggggggacaa acaccacaat 94260

ctctctggca ctacacatgt gcccagggtg atacatgact tattccccag agtggaaaag 94320

gtgccactct gtgacttaat ggggcaaagt gagattgtag gccaataagg gtgcattaaa 94380

aaaaatcata ttggaaaaac cacactgaag aacttagtgg acaggccaga ccaaggaaag 94440

gtgtaacttc ctcatctggc cccccctaaa atgatggaca cctaacatag caataataat 94500

ggacacacac tgacctatta tccaatacac agtgtgtatg tacaccaagg agggaaagac 94560

agaactctga actgtccctg ggacttcacc ttagctcatc ccccattgct gaccgggcct 94620

attcaacgtt catctgccca tccacctgtt cgcccatctg gacctgcttc tgtttttacc 94680

tagaagctgg actcaattct tcagcatcac tgtgaaccct gactccccca cagcctgtag 94740

caccttcttc cctgcagatg gtttctggtt tgttactgta tcaatataga ccctgcttaa 94800

tttccaccag cctatttcaa aaacctcttg accgctgctg gattctcttg taggggaatg 94860

ggtctttcat atgtggtctg gtctgttttg aaaacttctc agtgccagtc acaagagata 94920

gaaatcagaa caagcagtgt aaagggaccc tttcctgtct cacatcctct ctgtttctgg 94980

gcacactaga gaaccaggaa tgatggactt tctggagaca agagtgttat agctagagaa 95040

agaaagatga attgtccatt tttttaaaac cccaggacat atgtattaat gtctttggtc 95100

tcatagatat gactaaggac ttccatatga gaaggctgtg ttggattatc tgggtttgag 95160

gtcaccatga tcatatgggt tcctagacac ttggaaagaa aagttgtctt agttagggtt 95220

ttactgctgt gaacaggcac catgaccaag gcaactctta taaaggacta catttaattg 95280

gggatggctt acaggttcag tggttcagtc cataatcatc aaggtgggaa catggcagca 95340

tccaggcagg catgatgcag gaagagctga gagttctacg tcttcatcca caggaagcca 95400

ggggcagact ctcctcgggc agctaggaag agggtctcaa agcccacccc cacagtgaca 95460

cacttcctcc aacaaggcca caactacacc aacagggcca cacctcctaa cagtgccact 95520

ccctgggtca agtgtattca aaccaacaca aaattcaaga gcagagaaaa aagaaatatg 95580

aagataaagg gaccagaggt tggagttatg ggattgctgg ctctgaagac agaggaagaa 95640

atctcaaact aggatacatg gcagcttcta gcagttaaga aagtaagtta aatttatcaa 95700

ccaattctct ttggggcctc cagaaagagc tgagttctgg tgacaccttg atttttatct 95760

cagggaagcc tacttggaac ttccagcctc caagaatgta agataataaa attgtagtct 95820

ttaaaacctg tttggtttgg gtgggttgtt tttgttttgt tttgatttgt ttttccagca 95880

aaatcaacta atacagagag aaactgtttg ataagatctc ccacccccac ccccaaggat 95940

ggcattttca aagagtgtga aagagcagtg cattgtgggc agtaaggtac agacacaaga 96000

cctacctctg gccatctgga tatgtatgtg tgcaagacga gcttttccac ttcagagggc 96060

tggattgcaa gggacagaag cctctggggg aatacaaatg tcagctaaag ccaggaagta 96120

gaggaggtga ctcaaggaaa ggacacaggc ctcgagaact attttaattt ttggaaggac 96180

aacaagagtt tcaagagttt caggaataca atgtcaataa gaagacccta ggctgctgga 96240

ccaaatggag taggagagga atagaacatg aagtgtaacc cgatctggaa taacaggcta 96300

cacacattct atgggtctct tactgagaac cctggtgggg gggggggggg ggaaggaggg 96360

tgggtggacg gacaggaagg tcagagatgg ggctaactcg gcttagcagc tttaatcaaa 96420

tgccctttag cttcttcttt gattttaccc atgaattatg atgcatgaga ctcatttctc 96480

ctggccctgg ccaaatttca gtcataaaac ttgttttgtt tttgaaagga ctgagacaaa 96540

tttttatgca gaacagcatg tcaaagtaaa gctaacgagt gagtaattgt caaaacaagt 96600

ctaaaagaat tcacttcctc cctgttataa tacaaaatta tttctggtta atcgcgtctc 96660

actctgaagc ccaagttctc taaaggagtt gctttttttt tttttttggt acagagccga 96720

tgggaagttt gacctcagct gagcaaaaca gccagccggt ccagagatac aatgttgcag 96780

atcaagagtc aaacaatctg gggttatctc actcctttta acagccattt tcttttgacc 96840

tttgtgtctg acccaacatc ctgcgaatgc ggggtggggg tggggtggga aactcttggg 96900

aagtatttct aactttaaca gagatctatg gtctctgcta acccccaaag ctaggaatgt 96960

tacaagatag tatctgaatc ctatagaaac aatctccttg cttagtgtca ggacacctgc 97020

cggatgctcc cgccaacatg cctgctacat gtgttggttt atgaagcttt tgttctgtgg 97080

ctatgacagt tcatgcttct tttcccgtgg tgggagtgta tgtgagggag caggtcattc 97140

cgggtaactt ggacccctgt tcccagccca tgctcactcc tctttagctc agaataaacg 97200

gtttccttat atcctttaaa gcagagccat tattacatgc tgccggcatt actgggctcc 97260

taagtgccat caggtttgag ttacaagaag ctatagagtc caagactttc cctgaggctc 97320

tgatgtatat agaaaaggga cacttccccc ctccccccaa aaaccaggat cttacgtagc 97380

agagctggaa atagcctggg gaacatttcc ttgaggccct gctgtatgga gaaggaacat 97440

agtttctttt ctaaaaccag gaatatgctc ggtagagcca gtaatggcct ggactcagag 97500

tcttgaggga gcaatggctt ttgacactga gaacccagct aaggggatat ggttttcagt 97560

cccaaggctg ttgtgtgttt aatctgtgac cactcttgag atactcacat tccctttgtg 97620

caacattgct tgatgaactt cctgctgact ctgtttcatt gtatggtagc cttctgctaa 97680

ccctgcctca tgtctttcct ggaaatcgta tataagatgc catgcttctt agtaagcttg 97740

cttggcataa gacacggcgt gtgcaagacc ttctgatccc aaacttccct ttcctcttat 97800

cttttggtcc aggaccccat cactaaagaa ccagttgctg gtataattca gggacgcttt 97860

atagcttcat acaagaccat ggtgcttgct ttatttacaa gatggaaaga gggacattct 97920

atatagcatt ctcaatctat gggttgagac tcaattaggg cctgcccact ccaggaaagc 97980

cagtgtttcc aaaacactgg gttctatcct cagacaagaa aacacagatg cttacatgat 98040

aattcataac agtagcaaaa ttacagttat gaagtagcaa cgggaatagt tttatggttg 98100

gggtcactac aacatgagga attatattaa cgggtcgcag cactaggaag gttgagaacc 98160

actactctat agcaatatca gattcaaggt cacaaggcct caagctcttt agccctacta 98220

gatgaggtca gccttgtagg tttgtcaaga ccctcctttg gcattcctaa cacgagacct 98280

gtgacatgag cagtcttggt actttcctac tacactgagc tcctacatca cagacattag 98340

ttagatcttc tggatttcag gtactaggta acaaaatggg ccaaaccaac gggaatgagg 98400

taagagtatt tctgaaggaa aactggagga gacctggcct ttgctatcca gaatttggcc 98460

ttatctagat agatgtctct ccaaagcaaa ggatcgcctg acgtctctta ctgcattaac 98520

tgaacagata agagaaggat ccagggaagg caacacagga agaggtaggt agagtagaaa 98580

tttctagttt ctttggggac tcagttaggt tataatgatg tttttggctg gggcaaacag 98640

gtgaaaggat gttttgttac agcagacatg tgaaaggata cgtgatgttt agaaggaata 98700

taaagatgac cccacagaca gtggaagcta gagcttcact ttgctcccca ctcaatagtc 98760

ttcacacatc cacatgtatt ggttcgcctt acgttgtgtt gctgagcttc actgtggtga 98820

ctttggagag ggagatactc accaaagagc ttcccataag attcctgtgg cttcttgcca 98880

cttctgcttc cgtggacttg aggtgagcct cgtggtttct tctggattga actgatgttg 98940

ctgattcgtg tgtggtgtct gccaagtgga ctggttggca gctgctgctt cgtgtttggt 99000

gtttgctagt agactggaca gagaacaaag aagattggaa tcgtcccaaa gaactcttgc 99060

taaacaggtc ttcttccccc aagtcctaat aacctttctt ttccactgcc tctggtggat 99120

ggtgggctag agaggaggtt gaacctttat taaagtaggt agagaaaagt atatgcctgt 99180

aggtagaaat gcaaacagat cagtcactga caagtcagtg cttttagtct ctttgttagt 99240

tatacccaag ttccatcctt ggtctgggct tgatagcaca cacctttagt cctataactc 99300

aggaaataga ggaaagctct atgagtttga gcctagcgtg gtctacatac tgaattttag 99360

gataaccagg gcgacagagt aagacttggc ctttaaaaga aaagttccat cctgtgctgg 99420

ctagttttat gtcactttga cacaagctag aatcatcaga gaataggaag tttcaactga 99480

gaaaatgctt tcctaagatc aggctgcagg caagcctgca gggaatttca ttaattggtg 99540

atggatgcgg aagggccttg ctcattgtgc atggtaccat ccctgagctg gtgtcctggg 99600

ctctataaga aatctgtctg agaaagtcat gaggagcaaa ccagtaatta gcatcccatg 99660

catcagttcc tgcctcctgg tccctgcctc caggtttctg ccttgcttaa attcctgtct 99720

tgactccctt cagtgatgga gtgtaatgag gaagcataag ccaaataaac cctttccttc 99780

ccatgttctt tttagttaca gtgtttcatt aaagcaataa caaccctaac taagatatac 99840

aactttatat tgtgatacaa ttatgtcctt gatggtcaaa catttcctct cgtaaggtgg 99900

aacaggaggt gggagatact gagacgagtc tccttgtaaa ccagattcca aacttgcaaa 99960

ctaaaagtta ttgctgcaca ctttcgttca ttttgtgtat tccctccatt ggtccctccc 100020

tgagtacaaa gctaattgct tctttaatta agccctcttc cacttttact aaagctgttc 100080

acatattcca catcactttc cgctcttggt ttcccactgg accgaggcgg gcatttccag 100140

gacccagctg gcactgttta tctgcagtgg gtaccaggtg ctgggaggat aaattactgt 100200

ttttcaactc tgtttcttct ccttccagtg gatgcatcta aagtggaacc cagggagaac 100260

ccagggagcc tggctgcaga caccgtggag cagtgagtct caccaaagaa agatctcagc 100320

tggatgacag tctttagagc caaacttcaa attagccgtc ttggaaacgc ctttggttac 100380

tccaaaggga cgtaggtgct aatgggcagg catacacacg agcacaccag tatccctctg 100440

tattgtcacc gtgaggctca gtctcataat aattttgtct ccttgtgcag taacagcgag 100500

gaggttattc tgatttacct gagtgtcttt tttcataaat actgaggtgt attcccaggg 100560

ctggtctatt atctgtctgg atagtggatg ttccaacagg gccagcttca tgagcaacac 100620

atagcactct acacttcaaa gggcccaggc ttgttttaat gtactgacac catcatctcg 100680

aaattattat tttttttgta tttcatatac tgtttgtttg tttggttggt ttggtttggt 100740

tttttgtttt ttttttttgt tgttgttttt tttttgtttt gttttttttt tttttttttt 100800

ttcgagacag ggtttctctg tatagccctg gctgtcctgg aattcacttt gtagaccagg 100860

ctggcctcga acttagaaat ccgcctgcct ctgcctccca agtgctggga ttaaaggctt 100920

gtgccaccac cgctcagccc aaggcaactc ttataaggac agcatttaat tggtgctggc 100980

ttacaggttc agagattcag gctttatcat ccaggcagga gcatggtagc agctaagcag 101040

gcatggttca ggaggagctg agagttctac atcttcatct gaaggctgct agcagaatac 101100

tggcttccag gcagctagga gaagggtctt aaagaccaca cccacagtga cacacctact 101160

ccaacaacac cacacctcct aatagtgcca ctccctgggc caagcatata gaaaccatca 101220

cactgcgtaa acatattttt gtcttaatct ttatatacaa atccttattt attaaaattt 101280

ttatctatca tttttcatgt tttaatatta tgagacataa taatcacgta gatagatctt 101340

ccactgtttt aaaaaataaa attgattggg atggcctgca tagcttttta attgtaaaaa 101400

aatattaata tttaactatt attttgtgat tttaggccag tacagctgga cagaggaggg 101460

tggcaattac tttagaagat agggcagaag ccctgtattt gggtccttaa aaacacattt 101520

tagggggctg attttagtat ttgtttgtgt ttaaagaggt caaacatatc ttgaaaatcc 101580

tttaagtttg tgtgtgtgtg cgtgtgcgtg tgcgtgtgtg tgtgtgtgtg tgtgtgtgtg 101640

tagaaaggca tatacataag taacttttat ctaaagggga aagcctgcct ttaggcagta 101700

aaactcaaca gaacccacct gaacagctcc cttgtggcag cagctgtaat tgccctttag 101760

ttcttgatgt tgttattgtt gaaatctgtc aaccttaaag tgttagcctt ttctgttagt 101820

atgctagtgt tgtatatcat acaaacttta ttaaaattaa gacaaaacaa accattaaaa 101880

tcataacata aacatcattg tagctccagg aagttgagag aagagataaa ctagaggaac 101940

gctttaagtc tcactaagga atgtcagcac cttgatttta gtttttacct tttgtattta 102000

tatctcaggc tgtgttgtcc tccttcttgt cacacagagg gtccttttga cccaggacag 102060

ggattaagga catg

Claims

1. Inhibition of long non-coding RNAslincRADExpressed small interfering RNA molecule, the sequence of the small interfering RNA molecule is shown as SEQ ID No 1-12.

2. Inhibition of long non-coding RNAslincRADExpressed ribonucleic acid molecules with small hairpin structures, and the sequences of the ribonucleic acid molecules with the small hairpin structures are shown as SEQ ID No 21-24.

3. Use of the ribonucleic acid molecule of claim 1 or 2 for the preparation of an agent or medicament for inhibiting adipocyte differentiation.

4. The ribonucleic acid molecule of claim 1 or 2, in its preparationslincRADUse in low expression model animals, characterized in that the model animals are adipose tissueslincRADReduced expression level to wild type animal adipose tissueslincRAD60% or less of the expression level.

5. Use according to claim 4, characterized in that the model animal is selected from the group consisting of mouse, rat or rhesus monkey.

6. Preparation methodslincRADA method of underexpressing a model animal comprising the steps of:

1) obtaining a codeslincRAD-a DNA fragment of the shRNA expression cassette;

2) introducing the DNA fragment into pronucleus of animal oosperm by microinjection, and then transplanting the oosperm into the pseudopregnant congeneric animal body to make it develop into adult animal;

3) identification of the resulting integration in the genomeslincRAD-transgenic animals for shRNA;

4) mating the transgenic animal with the same wild animal to obtain a progeny animal;

5) identification of the obtained GeneGroup integrationslincRADThe progeny of the shRNA isslincRADKnock down transgenic animals.

7. The transgenic animal model with the expression of slincRAD knocked down is applied to screening of drugs for fat metabolism-related diseases.

8. Use according to claim 7, characterized in that the disorders related to fat metabolism are selected from the group consisting of metabolic syndrome, obesity, hyperglycemia, hyperlipidemia, diabetes.