CN111321171A

CN111321171A - Method for preparing gene targeting animal model by applying CRISPR/Cas9 mediated ES targeting technology

Info

Publication number: CN111321171A
Application number: CN201811535721.7A
Authority: CN
Inventors: 琚存祥; 赵静; 张明坤; 吴丹; 侯欢欢; 高翔
Original assignee: Gempharmatech Co ltd
Current assignee: Gempharmatech Co ltd
Priority date: 2018-12-14
Filing date: 2018-12-14
Publication date: 2020-06-23

Abstract

The invention discloses a method for targeting by applying a CRISPR/Cas9 system gene, which comprises the steps of selecting a mutant of Cas9 with DNA single-strand cutting capacity, using a tracrRNA/crRNA binary complex or an sgRNA aiming at each targeting site, and identifying conserved interval adjacent motifs near the targeting site by the mutant of Cas9 to generate DNA single-strand cuts without generating DNA double-strand breaks and inducing the homologous recombination and repair of the single-strand cuts. The invention also provides a method for preparing a gene targeting animal model by utilizing the method-mediated ES targeting technology. The invention reduces the insertion deletion mutation caused by HMEJ repair in the gene targeting process, and obtains high-efficiency targeting positive clone. The invention can be used as a platform technology to be widely used for preparing gene targeting animal models.

Description

Method for preparing gene targeting animal model by applying CRISPR/Cas9 mediated ES targeting technology

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to a method for preparing a gene targeting animal model by applying a CRISPR/Cas9 mediated ES targeting technology.

Background of the study

Animal models are indispensable tools for studying human medicine and health, and the physiological and pathological processes of the animal models in life activities have many similarities with human beings and can be used as references for human research. Through different gene editing methods, animal models of gene modification can be obtained, so that the occurrence, treatment and the like of related diseases can be researched.

Gene editing is realized by replacing, cutting off or adding or inserting a foreign DNA sequence into a nucleotide sequence of a target gene in a cell genome to generate heritable changes. Unlike random mutation of DNA caused by ray or chemical mutagen, the gene editing technology is directed to change the composition and structure of gene and has the features of high efficiency, controllability and directional operation. Therefore, the gene editing technology is evaluated as ten-major breakthrough science technology in 2014 by the science and technology review of majors science and technology, wherein CRISPR gains the breakthrough prize of life science in 2015.

After a single-strand break or deletion of a DNA molecule in a cell, it is easily repaired by various repair mechanisms in the cell without any change, but the result of the DNA double-strand break is very different. There are two major modes of repair of DNA double strand breaks in cells, Homologous recombination repair (HDR) and Non-Homologous End-link repair (Non-homologus End Join, NHEJ). After double-strand break of DNA, if there are sequences homologous to both ends of the break in the cell, then homologous recombination repair is carried out, and exogenous DNA segment can be inserted into the broken sequence to knock out the original gene or insert exogenous gene into genome DNA to form so-called gene knock-in; double-stranded broken DNA molecules are joined by NHEJ if no homologous sequence is present in the cell, and such "hard" joining is likely to result in deletion, addition or alteration of bases to cause mutations due to the lack of available template. DNA double strand breaks are a serious injury to the genome, but such changes are needed to study gene function.

The traditional ES cell (embryonic stem cell) targeting is a molecular biology technology based on homologous recombination and embryonic stem cell technology. Although homologous recombination-mediated gene targeting can change the homologous sequence with high accuracy, the recombination efficiency of the target gene is very low, and great challenges are brought to large-scale application of gene targeting experiments.

In recent years, a new idea is provided for optimizing a gene targeting technology for the research and application of a CRISPR/Cas9 technology. CRISPR/Cas9 is an acquired immune defense mechanism that has evolved from bacteria and archaea to cope with constant attacks by viruses and plasmids. In this system, crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form double-stranded RNA, and this tracrRNA/crRNA binary complex directs Cas9 protein to cleave double-stranded DNA at the targeted site of the crRNA guide sequence. In the process of genome editing, tracrRNA and crRNA can be fused into 1 single guide nucleotide (sg RNA) for expression, and can also play a role in targeted shearing, and cells repair broken double strands, so that accurate genome editing is realized. However, the double-strand break can introduce non-negligible off-target mutation at the relevant site, which brings risks to clinical application. Cas9 is a nuclease Cas9 nicke (Cas9n) that creates a single-strand nick by creating a double-strand break through the two nuclease domains RuvC and HNH, converting one of the two key structures to alanine (D10A or H840A). Cas9n requires a pair of sgrnas to guide to achieve targeted double strand cleavage, reducing off-target effects.

However, when the Cas9 protein-mediated ES targeting technology is used for gene modification, both the WT Cas9 and the Cas9n protein introduce double-strand breaks, which can cause HMEJ repair of the genome, lead to the occurrence of non-target bands during genotype identification, interfere with positive band judgment, and are not favorable for selecting positive clones for injection inheritance.

Furthermore, whether homologous recombination can occur efficiently is influenced by a number of factors, Thomas et al (Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse-derived stem cells. cell,1987,51(3): 503-12) found that the efficiency of homologous recombination can be increased by a factor of 10 when the length of the homologous sequence is increased from 4kb to 9 kb. However, the identification difficulty of the PCR method is increased by overlong homologous sequences, the targeting efficiency and the identification difficulty of the PCR are balanced, and the existing overlong homologous sequences have no good method for solving the problem of the PCR identification.

Disclosure of Invention

Aiming at the defects of the prior art, the invention utilizes the characteristic that the Cas9 mutant generates DNA single-strand break, uses a tracrRNA/crRNA binary complex or a gRNA to generate DNA single-strand nick aiming at each targeting site, does not generate DNA double-strand break, induces single-strand nick homologous recombination repair, and reduces insertion deletion mutation brought by HMEJ repair. Furthermore, the CRISPR/Cas9 gene editing technology is combined with the traditional ES targeting technology, and the ES cells (embryonic stem cells) are used for testing the sgRNA cutting efficiency to screen out the high-efficiency sgRNA. And then Cas9 mutant protein and the screened high-efficiency sgRNA of the targeted recombination site are added in the ES targeting process, so that the targeting efficiency can be obviously improved.

The specific technical scheme of the invention is as follows:

a method for gene targeting by using a CRISPR/Cas9 system is characterized in that a mutant of Cas9 with DNA single-strand cutting capacity is selected based on the CRISPR/Cas9 system, a tracrRNA/crRNA binary complex or an sgRNA is used for each targeting site, and the mutant of Cas9 recognizes conserved spacer adjacent motifs near the targeting site to generate DNA single-strand cuts without generating DNA double-strand breaks and induce the homologous recombination repair of the single-strand cuts.

The types of gene targeting in the method of the invention include gene knockout and gene knock-in.

The mutant of Cas9 in the method of the invention is preferably Cas9n D10A and/or H840A.

Furthermore, the method of the invention can design at least 2 targeting sites aiming at each target gene to improve the targeting efficiency or simultaneously carry out multiple gene targeting, the interval of each targeting site is not less than 100bp, and the adjacent DNA single-strand nicks are ensured to be separated by enough distance without generating double-strand break.

Another object of the present invention is to provide a method for preparing gene targeting ES cells using CRISPR/Cas9 system, comprising the steps of:

(1) selecting a proper targeting site according to the type of gene targeting and a target gene, and designing a homologous DNA donor and an identification scheme;

(2) the method for targeting by using the CRISPR/Cas9 system gene is adopted to prepare a mutant of Cas9 or an expression vector thereof, and an sgRNA or a tracrRNA/crRNA binary complex or respective expression vectors are designed and prepared according to a target gene and a targeting site;

alternatively, a mutant of Cas9, sgRNA, or a co-expression vector of crRNA and tracrRNA is prepared;

(3) transfecting the mutant of Cas9, sgRNA or tracrRNA/crRNA binary complex prepared in the step (1), or respective expression vectors or co-expression vectors into ES cells, and selecting a non-homologous DNA template according to the type of gene targeting to enable a targeting site to have base insertion or deletion, or simultaneously transfecting homologous donor DNA to insert exogenous DNA into a genome;

(4) extracting ES cell DNA to perform PCR identification and/or Southern Blot detection and karyotype identification, and identifying the clone which passes the identification to be the gene targeting positive ES cell.

The invention can directly prepare a mutant of Cas9, sgRNA or a tracrRNA/crRNA binary complex to transfect into ES cells, can also prepare respective expression vectors or co-expression vectors to infect into ES cells, preferably directly prepare the mutant of Cas9, sgRNA or the tracrRNA/crRNA binary complex to transfect into the ES cells, and can avoid the problem of expression efficiency after the expression vectors or the co-expression vectors are infected into the ES cells.

In the above method, preferably, the step (2) further comprises detecting the cleavage efficiency and off-target effect of the sgRNA or the binary complex of crRNA and tracrRNA. The detection comprises an endonuclease detection method, an SSA report carrier detection method, a Sanger sequencing method and a Digenome-Seq technology. Preferably, the method for detecting an endonuclease is an enzyme selected from the group consisting of SURVEYOR enzyme, T7EN1 enzyme.

In a specific embodiment of the invention, a wild Cas9, sgRNA or tracrRNA/crRNA binary complex, or respective expression vectors or co-expression vectors are transfected into an ES cell, a cell genome is extracted, a target cutting region is amplified by using a specific primer, the obtained PCR product is subjected to T7EN1 enzyme cutting, electrophoresis detection is carried out after enzyme cutting, and the detection result shows that one or more bands are added as effective cutting compared with a control group; or performing Sanger sequencing on the obtained PCR product, and comparing sequencing results to show that double peaks in the sgRNA area are regarded as effective cutting; and screening high-efficiency sgRNA or crRNA and tracrRNA.

The method for transfecting the ES cells comprises viral transfection, lipofection and electrotransfection.

The invention also aims to provide a method for preparing a gene targeting animal model by using CRISPR/Cas9 mediated ES targeting technology, which comprises the steps of injecting the gene targeting positive ES cells prepared by using the method for preparing the gene targeting ES cells by using the CRISPR/Cas9 system into animal blastocysts, respectively transplanting the blastocysts into surrogate mothers, feeding, and obtaining offspring which is the F0 generation gene targeting animal model. Preferably, the gene targeting animal model of the present invention is a mammal, preferably a mouse or a rat. Preferably, the gene targeting animal model is a KO, cKO, KI, KO first and TM mouse model.

The method of the invention is suitable for knocking-in or knocking-out all genes. The animal models specifically listed in the invention are PRKAA1-KI, CD27-KI and ANXA8-CKO mouse models.

The invention also aims to provide a method for improving screening efficiency of the target-targeting ES positive clone mediated based on the CRISPR/Cas9 system, a CRISPR/Cas9 system is utilized to select a mutant of Cas9 with DNA single-strand cutting capacity, a tracrRNA/crRNA binary complex or an sgRNA is used for each target-targeting site, and the mutant of Cas9 recognizes conserved spacer adjacent motifs near the target-targeting site to generate DNA single-strand cuts without generating double-strand breaks and induce homologous recombination repair of the single-strand cuts.

The wild-type Cas9 and the mutant Cas9n or expression vectors thereof can be prepared by referring to the prior art (Cong, Le, et al, "Multiplex genome engineering using CRISPR/Cas systems." Science (2013): 1231143.) and can also be prepared by entrusted commercial companies. sgRNA or tracrRNA/crRNA design and preparation sgRNA for engineered sites sgRNA can be designed from targeted genes and targeted sites using the Cas9sgRNA design website, e.g., using the website http:// cruispr. Sgrnas with scores of 60 or more were selected, with higher scores indicating higher sgRNA specificity and smaller corresponding off-target effects.

The sgRNA transcription vector can be constructed by reference to the prior art or prepared by entrusted commercial companies.

The specific operation is as follows:

1) designing an sgRNA primer;

the sgRNA sequence was ordered as a forward primer and the reverse complement of the sgRNA sequence was ordered as a reverse primer.

2) Constructing an sgRNA transcription vector;

the forward and reverse primers were annealed to form a double strand, and ligated to BsaI single-digested pUC57-T7 vector to construct a transcription vector containing the sgRNA sequence.

3) Preparing an in vitro transcription template by PCR amplification;

PCR was performed using PrimerStar Max system, sgRNA-F, sgRNA-R as primers, and pUC57-sgRNA plasmid with correct sequence as template, at 95 ℃ for 5min, 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 40s, 72 ℃ for 5min, 30 cycles. And (4) after the program is finished, carrying out agarose gel electrophoresis, cutting a target band, carrying out gel recovery, and recovering the obtained final product as a transcription template.

4) Transcription in vitro using transcription templates and purification.

Reagent I: HiScribe^TMT7 Quick High Yield RNA Synthesis Kit(NEB#E2050S)；

And (2) reagent II: ambion Megaclear kit (Ambion AM 1908);

press HiScribe^TMThe RNA was transcribed in vitro in the manual of the T7 Quick High Yield RNA Synthesis Kit (NEB # E2050S), and RNA was purified according to the manual of the Ambion megaclean Kit (Ambion AM 1908). the concentration of the recovered RNA was measured by UV spectrophotometry, and 9. mu.l of 1 × Loadingbuffer was added to 1. mu.l of the RNA sample, and agarose gel electrophoresis was performed. The remaining RNA samples were stored at-80 ℃.

The invention has the advantages that:

(1) according to the invention, by utilizing the characteristic that the Cas9 mutant generates DNA single-strand break, a tracrRNA/crRNA binary complex or an sgRNA is used for generating DNA single-strand nick aiming at each targeting site, and DNA double-strand break is not generated, so that single-strand nick homologous recombination repair is induced, and insertion deletion mutation brought by HMEJ repair is reduced.

(2) The invention combines CRISPR/Cas9 gene editing technology with traditional ES targeting, firstly uses ES cells (embryonic stem cells) to test the sgRNA cutting efficiency, and screens out the high-efficiency sgRNA. And then Cas9 mutant protein and the screened high-efficiency sgRNA of the targeted recombination site are added in the ES targeting process, so that the targeting efficiency can be obviously improved.

(3) The invention utilizes CRIPSR/Cas9n mediated ES targeting gene editing technology, combines protein obtained by in vitro expression and efficient sgRNA obtained by ES cell screening to realize genome multi-site targeting, cuts a plurality of sites on a target gene, improves targeting efficiency while not increasing the length of a homologous arm, realizes the purpose of genome modification, and can be widely used for preparing gene targeting animal models as a platform technology.

Drawings

FIG. 1 alignment of sgRNA cleavage T7EN1 in example 1.

FIG. 2. example 1WT Cas9 mediated ES targeting PCR assay target clone map.

FIG. 3. example 1Cas9n D10A mediated ES targeting PCR assay target clone map.

FIG. 4 target clone map in example 1WT or D10A Cas9 mediated ES targeting Southern assay.

Fig. 5 alignment chart for sgRNA cleavage sequencing in example 2.

FIG. 6 is a graph of target clones in PCR assay of example 2.

FIG. 7 is a map of target clones in Southern assay of example 2.

FIG. 8 is a genotype test chart for the mouse of example 2.

Fig. 9 alignment chart for sgRNA cleavage sequencing in example 3.

FIG. 10 is a graph of target clones in PCR assay of example 3.

FIG. 11 is a map of target clones in Southern assay of example 3.

FIG. 12 genotype test for mice of example 3.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The present invention is further illustrated by the following specific examples.

Example 1 preparation of Prkaa1-KI ES cells

The WT Cas9 and Cas9n D10A used in the present invention, which were purchased from Qiang Biotechnology, Inc., Suzhou, were tested for nuclease activity and used in subsequent experiments.

Design and preparation of sgRNA and detection of cleavage activity

1. Design and preparation of sgRNA for target site

1) Design of sgrnas for sites of interest using Cas9sgRNA design website

sgRNA specificity was analyzed using the website http:// criprp. mit. edu/. Sgrnas with scores of 60 or more were selected, with higher scores indicating higher sgRNA specificity and smaller corresponding off-target effects.

Because the two ends of the homologous DNA donor fragment are respectively provided with homologous arms with the size of about 5kb, the integral skewness is longer, and 1 cleavable sgRNA is respectively designed and screened within 100bp of 2 homologous arms.

The sgRNA sequence is as follows:

sgRNA name	Sequence of	PAM
			Prkaa1-5S1	gaacacuagugcacuuaucc(SEQ ID NO:1)	CGG
Prkaa1-3S2	uccugaaaugacuucuggug(SEQ ID NO:2)	CGG

2) Designing an sgRNA primer;

3) Constructing an sgRNA transcription vector;

and annealing the forward primer and the reverse primer to form a double strand, and connecting the double strand with a BsaI single-enzyme digested pUC57-T7 vector to construct a transcription vector containing the sgRNA sequence.

4) Preparing an in vitro transcription template by PCR amplification;

PCR was performed using PrimerStar Max system, sgRNA-F, sgRNA-R as primers, and pUC57-sgRNA plasmid with correct sequence as template, at 95 ℃ for 5min, 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 40s, 72 ℃ for 5min, 30 cycles.

And (4) after the program is finished, carrying out agarose gel electrophoresis, cutting a target band, carrying out gel recovery, and recovering the obtained final product as a transcription template.

2. In vitro transcription using transcription templates and purification

Reagent I: HiScribe^TMT7 Quick High Yield RNA Synthesis Kit(NEB#E2050S)

And (2) reagent II: ambion MEGAclear kit (Ambion AM1908)

Press HiScribe^TMThe manual of T7 Quick High Yield RNA Synthesis Kit (NEB # E2050S) was used for RNA in vitro transcription and RNA purification according to the manual of Ambion MEGAclear Kit (Ambion AM 1908).

The concentration of the recovered RNA was measured by UV spectrophotometry, and 9. mu.l of 1 × Loading buffer was added to 1. mu.l of RNA sample, followed by agarose gel electrophoresis, and the remaining RNA samples were stored at-80 ℃.

3. The sgRNA and the WT Cas9 protein are transferred into mouse ES cells by electrotransformation

1) Preparation of ES cells were thawed from frozen ES cells in liquid nitrogen, expanded by passage, and counted one day before electroporation to inoculate 5 × 106 cells per 10cm dish.

2) Preparing an electrotransformation sample: cas9 was mixed with sgrnas as electrotransfer samples at 14:00 pm on the day of electrotransfer under RNA-free conditions, according to the electrotransfer requirements.

3) Cell electric transfer, after the cell digestion inoculated in the previous day is stopped, 1 × 106 cells are taken, centrifuged for 5min at 1000rpm, the supernatant is sucked off at negative pressure, 100 mul of electric transfer buffer is used for re-suspending, the mixed electric transfer sample is added, the mixture is transferred into an electric cup after being mixed evenly, after the electric transfer, 500 mu lES-DMEM is added into the electric cup, and the mixture is transferred into a 6-hole plate paved with gelatin.

4) And after 48h, collecting cells, and improving the DNA identification and cutting effect.

4. And (4) carrying out gene identification to obtain the sgRNA with high cutting efficiency.

The PCR system was loaded as in Table 1, the PCR primers are shown in Table 2, and the PCR program is shown in Table 3. The PCR product was digested with T7EN1, and the digestion results are shown in FIG. 1. In fig. 1, the left graph is a graph comparing the cleavage of sgrnas detected in the 5-end homology arm, and the right graph is a graph comparing the cleavage of sgrnas detected in the 3-end homology arm, which shows that 2 bands appear after T7EN1 enzyme digestion, indicating that the sgrnas detected in the 5-end homology arm and the sgrnas detected in the 3-end homology arm can cleave the target genome.

Table 1:

table 2:

note: the 1 and 2 primers are amplification primers containing the positions of 5-end sgRNA and 3-end sgRNA, respectively.

Table 3:

secondly, positive ES clones are obtained by screening through a Cas9D10A mediated ES targeting technology (meanwhile, WT Cas9 mediated ES targeting is used as a control)

i. Feeder cells were plated in 6cm dish one day before electroporation.

And ii, placing the required ES-DMEM, ES-0 and pancreatin in a 37-degree water bath kettle for preheating on the day of the experiment. The electrotransfer instrument is turned on and the parameters are set according to the cell line.

Digesting the cells with pancreatin, neutralizing with ES-0, centrifuging at 1000rpm for 5min, connecting the sterilized suction tube with the soft tube part of an electric suction apparatus (the suction tube is put into a clean bench in advance), and sucking the supernatant under negative pressure.

Using Ca²⁺/Mg²⁺free PBS 20ml resuspension, counting, 2 × 106 cells, centrifugation 1000rpm5min, negative pressure suction supernatant.

v. after resuspension with 100. mu.l electrotransfer buffer, the mixed Cas9D10A protein and sgRNA and homologous DNA donor (sequence shown in SEQ ID NO:7, homologous DNA donor contains knock-in site homology arm, replacement CDS, resistance selection element (G418), mixing and transferring into an electric cuvette.

After electroporation 500. mu. lES-DMEM was added to the cuvette and transferred to 6cm dish plated with feeder cells.

After vii.48h, it was replaced with fresh ES-M + G418 containing 200ug/ml G418. Placing into carbon dioxide incubator at 37 deg.C and 5% CO₂Culture and screening for 7 days.

After 7 days, single clones were picked into feeder-plated 96-well plates and when ES cells grew to 80% confluency, the cells were plated at a rate of 1: passage 3, one of the plates was used for DNA extraction and the other two plates were used in a freezer frozen to-80 ℃.

ix.96 well plate DNA was identified by PCR.

x.96 well plate cell expansion: after the PCR positive clone is confirmed, the 96-well plate is taken out from a refrigerator with the temperature of-80 ℃, the surface of the 96-well plate is sprayed with alcohol, the 96-well plate is wiped clean, the 96-well plate is placed in an incubator for about 30min, the placing position of the 96-well plate is changed (the 96-well plate which is not completely melted is changed for 2-3 times, the temperature of the bottom of the 96-well plate is lowered after the 96-well plate is placed in one position for 10 min, the bottom of the 96-well plate can be maintained at 37 ℃ again by moving the position so as to be rapidly melted), after all the ice crystals are melted, all the cells are sucked into a 24-well plate, each. Each clone was amplified to 2 10cm dis by changing the medium, passaging, etc., one dish for cryopreservation and one dish for DNA extraction.

Southern identification and karyotype detection: the amplified cell DNA is used for southern detection, and the clone passing the identification is the targeting positive clone.

PCR detection system and results:

PCR system as in Table 4, PCR primers as in Table 5, and PCR program as in Table 6.

Table 4:

table 5:

note: the 5 'LR-PCR primer is an amplification primer spanning 5-terminal homologous sequences, and the 3' LR-PCR primer is an amplification primer spanning 3-terminal homologous sequences.

Table 6:

the results of target cloning in the WT Cas 9-mediated ES targeting PCR assay are shown in fig. 2, where: b6 is a negative control, which is B6 genomic DNA; n is blank control, no template control; p is a positive control; TRANS 2K band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp; A1-H6 are samples of different clone numbers. The positive clone amplified by the 5 'LR-PCR is matched with the positive clone amplified by the 3' LR-PCR in number, and the positive clone is correctly recombined. Positive clones were obtained from 48 clones numbered A1-H6: e1, H2, E3, H3, H4, E6; the positive rate is as follows: 6/48% the remainder was wild type or negative.

The results of target cloning in Cas9n D10A-mediated ES targeting PCR assay are shown in fig. 3, where: b6 is a negative control, which is B6 genomic DNA; n is blank control, no template control; p is a positive control; TRANS 2K band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp; A1-G4 are samples of different clone numbers. The positive clone amplified by the 5 'LR-PCR is matched with the positive clone amplified by the 3' LR-PCR in number, and the positive clone is correctly recombined. Positive clones were obtained from 40 clones numbered a 1-G6: a1, A3, a5, a6, B1, B3, B4, B5, B6, C3, C5, C6, E1, E3, F2, F4, F5, F6, G1, G2, G3; the positive rate is as follows: 21/40% 52.5% remaining wild type or negative.

And (4) conclusion: comparing the PCR identification results of FIG. 2 and FIG. 3, it was shown that CIRSRD10A mediated PRKAA1 gene recombination more efficiently than WT protein.

Southern detection and results:

the cleavage protocol is shown in Table 7, the probe primer details are shown in Table 8, and the template was a mouse BAC: RP23-240A 23. Table 7:

note: the difference between the target genotype and the wild type is distinguished by means of enzyme digestion to produce different fragments.

Table 8:

note:

primers

1 and 2 are amplification primers of 5-end and 3-end probes respectively, are placed on the outer sides of respective homologous arms, detect a target band, and eliminate random insertion interference.

The results of target cloning in WT or D10A Cas9 mediated ES targeting Southern assay are shown in FIG. 4, in which: B6N is a negative control, and is B6 genomic DNA; marker strip: 15000bp \10000bp \7500bp \5000bp \2500bp \1000bp \250 bp; : a non-destination strip. The results show that: by using the Cas9n D10A protein, the genome is not fractured, and the detection band only comprises a Target band and a WT band; using the Cas9WT protein, DSBs appeared in the genome, with non-Target bands in addition to Target and WT bands.

Example 2Cas9n D10A mediated ES targeting technology CD27-KI mouse model

Design and preparation of sgRNA and detection of cleavage activity

Sgrnas were designed, prepared, and cleavage activity was examined with reference to the method of example 2.

The sgRNA sequence is as follows:

sgRNA name	Sequence of	PAM
			CD27-5S1	uggcauggccaccucccuac(SEQ ID NO:16)	TGG
CD27-3S2	acgggcaagagcacaccg(SEQ ID NO:17)	AGG

The PCR system was loaded as in Table 9, the PCR primers are shown in Table 10, and the PCR program is shown in Table 11.

Table 9:

table 10:

Table 11:

the sgRNA cleavage sequencing alignment results are shown in fig. 5, where: the left figure is a graph comparing the cutting of sgRNA measured in a 3-end homologous arm, the right figure is a graph comparing the cutting of sgRNA measured in a 5-end homologous arm, and the black area is a sgRNA sequence. Sequencing alignment shows that double peaks appear at the sgRNA position, which indicates that the position is cut, so that base insertion and deletion are caused, and the sgRNA can cut a target genome.

Screening and obtaining positive ES clone by using Cas9n D10A mediated ES targeting technology

Referring to the method in example 2, positive ES clones were obtained by screening using Cas9n D10A mediated ES targeting technology. The homologous DNA donor sequence is shown in SEQ ID NO. 22.

PCR detection system and results: PCR system as in Table 12, PCR primers as in Table 13, and PCR program as in Table 14.

Table 12:

table 13:

Table 14:

the results of the target clones in the PCR assay are shown in FIG. 6, where: 129 and B6 are negative controls, are 129 and B6 genomic DNA; n is blank control, no template control; p is a positive control; TRANS 2K band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp; A1-G12 are samples of different clone numbers; the positive clone amplified by the 5 'LR-PCR is matched with the positive clone amplified by the 3' LR-PCR in number, and the positive clone is correctly recombined. Positive clones were obtained from 96 clones numbered A1-P6: a3, F3, G3, E5, C6, E6, F10, G11, positive rate: 8/96% 8.3%, the remainder being wild type or negative.

Southern detection and results:

the cleavage protocol is shown in Table 15, the probe primer details are shown in Table 16, and the template is a mouse BAC: RP23-394N 13.

Table 15:

Table 16:

note:

primers

1 and 2 are amplification primers of 5-terminal and 3-terminal probes respectively, are placed on the outer sides of respective homologous arms, detect a target-in-band, exclude random insertion interference, and 3 is an inner primer, and verify the integrity of a target-in-band region.

The results of the target clone in the Southern assay are shown in FIG. 7, in which: B6N is a negative control, and is B6 genomic DNA; marker strip: 15000bp \10000bp \7500bp \5000bp \2500bp \1000bp \250 bp; the LR-PCR positive cloned DNA uses the enzyme cutting scheme and the corresponding probe in the table above, the detected band is referred to the negative control and the Marker band, and the positive clone with the recombination target at the two ends and the integrity passing is judged.

The Southern identification results show that: a3, C6, E6, F3, G3, G11: positive clones passed for targeting and integrity, but random insertions were present in E5, the target band in A3 was weak, and injection was not recommended.

Thirdly, carrying out positive ES clone injection and transplantation

i. Preparation of 2.5 day recipient mice: and (3) selecting ICR female mice with qualified body weight in week age, combining the ligated male mice and the female mice at a ratio of 1:2 at 3:30 in the afternoon, starting to detect embolisms of the receptor at 8:00 in the morning on the next day, marking the embolisms as 0.5 day, and taking the mice with embolisms for 2.5 days as the receptor of the embryo transplantation experiment on the same day.

Preparation of 3.5 day embryos: selecting suitable week-old female mice, performing superovulation (injecting PMSG and HCG), closing ligated male mice and female mice at a ratio of 1:2 at 3:30 in the afternoon, detecting embolus in recipients at 8:00 in the morning on the next day, marking the infected mice as 0.5 day, killing donor mice with embolus for 2.5 days after cervical vertebra is removed, cutting uterus and oviduct, flushing oviduct and uterus from the orifice of the oviduct with an injection needle with D-PBS, selecting embryos at 8 cell stage, cleaning, transferring to a culture dish with M16 liquid drop prepared 1 hour in advance, placing into a culture dish with 37 ℃ and 5% CO, and placing₂The incubator was incubated overnight.

injection cell preparation, providing positive ES cells to the injecting person 10:00 a.m. the day of injection, according to the injection requirements.

injection: preparing proper fixed tubes, injection needles and operating dishes, selecting 10-15 small and bright ES cells with smooth edges by using the injection needles, fixing the embryos by using the fixed tubes, slightly rotating the embryos by using the injection needles, searching the intercellular spaces, inserting the injection needles, and slightly blowing the ES cells into the cystic cavities after the injection needles enter the cystic cavities. The blastocysts injected with ES cells were transferred to ES-medium and cultured for 3-4 hours for recovery.

v. embryo transfer: after the blastocyst is fully recovered, the embryo is transferred into E2.5 day recipient uterus. Mice that had undergone the transplantation procedure were placed in clean mouse cages and incubated on a 37 ℃ hot table until the mice were awake. The mice after revival are transferred to the corresponding animal feeding rooms to wait for the birth of a newborn animal.

Breeding the mice: the mouse born by the transplant recipient is marked as a chimeric mouse, male mice with the fur color chimerism rate of more than 50 percent are bred, and the mouse of the offspring is marked as F1. And (3) making a breeding plan according to the breeding target, breeding 3-5F 1 with the genotype meeting the requirement according to the plan, and establishing a line.

And (3) identification: performing tail shearing within 1 week after birth of an F1 mouse, identifying the genotype, and detecting mutation; all mice generated in the process of establishing the strain are subjected to genotype identification so as to ensure the genetic stability of the mouse genes.

PCR system and procedure are shown in Table 17, PCR primers are shown in Table 18, and mouse rat tail DNA is used as a template for identification.

Table 17:

table 18:

note: identifying KI and KIN as target genotypes; WT was identified as wild type.

The results of mouse genotype detection are shown in FIG. 8, where: the number is the mouse tail number, P is the positive control, WT is the C57BL/6J wild type, N is the negative blank control, M is the DNA Marker band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp. Mice # 107, # 108, # 110, # 112 were KI positive F1 mice.

Example 3Cas9n D10A mediated ES targeting technology preparation of Anxa8-CKO mouse model

Design and preparation of sgRNA and detection of cleavage activity

The sgRNA sequence is as follows:

sgRNA name	Sequence of	PAM
			Anxa8-5S1	caaguguguaagacga(SEQ ID NO:39)	GGG
Anxa8-3S1	ucuggucagucagcgug(SEQ ID NO:40)	GGG

The PCR system was loaded as in Table 19, the PCR primers are shown in Table 20, and the PCR program is shown in Table 21.

Table 19:

reagent	Volume (μ l)	Specification of
			10X Buffer	2.5
ddH₂O	17.75
			primer	0.5	10uM
primer	0.5	10uM
			Mg
²⁺	2	25mM
			dNTPs	0.5	10mM each
Taq	0.25	5U/μl
			Template
	1

Table 20:

Table 21:

the sgRNA cleavage sequencing alignment results are shown in fig. 9, where: the left figure is a graph comparing the cutting of sgRNA measured in a 3-end homologous arm, the right figure is a graph comparing the cutting of sgRNA measured in a 5-end homologous arm, and the black area is a sgRNA sequence. Sequencing alignment shows that double peaks appear at the sgRNA position, which indicates that the position is cut, so that base insertion and deletion are caused, and the sgRNA can cut a target genome.

Secondly, screening and obtaining positive ES clones by using Cas9D10A mediated ES targeting technology

Referring to the method in example 2, positive ES clones were obtained by screening using Cas9n D10A mediated ES targeting technology.

The homologous DNA donor sequence is shown in SEQ ID NO: 45.

PCR detection system and results:

PCR system as in Table 22, PCR primers as in Table 23, and PCR program as in Table 24.

Table 22:

table 23:

table 24:

note: the 5 'LR-PCR primer is an amplification primer spanning 5-terminal homologous sequences, and the 3' PCR primer is an amplification primer spanning 3-terminal homologous sequences.

The results of the target clones in the PCR assay are shown in FIG. 10, in which B6 is a negative control and is B6 genomic DNA; n is blank control, no template control; TRANS 2K band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp; A1-H12 are samples of different clone numbers; the positive clone amplified by the 5 'LR-PCR is matched with the positive clone amplified by the 3' LR-PCR in number, and the positive clone is correctly recombined. PCR positive clone (3-terminal homology arm internal short primer): b1, C3, F3, D4, a5, E5, F9, B10, D10, E10, E11, C12, positive rate: 12/96% 12.5%, the remainder being wild type or negative.

Southern detection and results:

the cleavage protocol is shown in Table 25, the probe primer details are shown in Table 26, and the template is a mouse BAC: RP23-20D 5.

Table 25:

Table 26:

note:

primers

1 and 2 are amplification primers of probes at the 3-terminal and 5-terminal respectively, are placed on the outer sides of respective homologous arms to eliminate random insertion interference, and 4 is an internal primer to verify the integrity of a target region.

The results of the target clone in the Southern assay are shown in FIG. 11, in which: B6N is a negative control, and is B6 genomic DNA; marker strip: 15000bp \10000bp \7500bp \5000bp \2500bp \1000bp \250 bp; the LR-PCR positive cloned DNA uses the enzyme cutting scheme and the corresponding probe in the table above, the detected band is referred to the negative control and the Marker band, and the positive clone with the recombination target at the two ends and the integrity passing is judged.

The Southern identification results show that: a5, B10, C12, D10, E5, E10, E11: positive clones for hit and integrity pass.

Thirdly, injecting and transplanting positive ES clones;

with reference to the method of example 2, injection and transplantation of positive ES clones, breeding and identification of mice were carried out.

The PCR system and procedure are shown in Table 27, and the PCR primers are shown in Table 28.

Table 27:

table 28:

note: fln and Fl are used for identifying the target genotype; WT was identified as wild type.

The results of mouse genotype detection are shown in FIG. 12, where: the numbers are rat tail numbers, P is a positive control, WT is C57BL/6J wild type, N is negative blank control, M is a DNAmarker Marker band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp. 56#, 59#, 62#, 65#, 66#, 72#, 73#, 77#, 80#, 84#, 85#, 87#, 89#, 91#, 92#, 97#, 98#, 102#, 109#, 119#, 120#, 122#, 128 #, 134 #: the mice were positive F1 generation mice.

Sequence listing

<110> Jiangsu Jiejiaokang Biotech limited

<120> method for preparing gene targeting animal model by applying CRISPR/Cas9 mediated ES targeting technology

<160>61

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gaacacuagu gcacuuaucc 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

uccugaaaug acuucuggug 20

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

acttttacct gctgagccta 20

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gtgtatgtgc taagctagtg gt 22

<210>5

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gaactcttga ctggtcactt tttg 24

<210>6

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggccaatgtg ttacttttat gtc 23

<210>7

<211>18829

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ctcctggctt gttttgctct taccttacca taagtgtgcc aagcaggctc ccaactgggc 60

cctggccctg gctgttcctt ccccctgccc acttcacctt cctcctggcc aaggtagtct 120

gctcctccct tccttggggc tctgctctgg tgtactctgt gaccttcctg gcatgctctg 180

cctcttactt aattactgtc tgtgtcctgg cccctgcatc aagaacataa caaacttgct 240

tactgtctgt ctcctggcac ctgcatcagg accataacaa aaacaggaga ttggctttgc 300

tcccccacag acttctagca gctagcacag tacctggtat cttataggtg cttagtaaat 360

ctttactgaa tggatgaata cacgtgtttt aatattagta gataatgttt tctctcacca 420

ggtaccaggt catcagtaca ccatctgata ttttcatggt gatggaatat gtctctggag 480

gagagctatt tgattatatc tgtaaaaatg gaagggtaag cagttctcat ttaattctgt 540

acacagtttg gtaactctct gtccccctta ttgcattata gtgtcagcaa gcaattttca 600

gaggtctgtt tagtaataag tccccttagc atgaatttgt gtagcatcat aggctaatga 660

gaaggaaagg acctcaaaag tgatccctga tcccatttaa ttgacatgga tataaagact720

acttgttgag tatttgttca aggtcacgtg atttactagt gatttacaga gttcacgtaa 780

gtccctaaaa tttattatcc tcagatttaa ggtttttagc aaaacaaact tttatttatt 840

tttttggaga cagggtcaca ctatgccact gtgactgcca tgtagatcag gctggcctcc 900

agtgcatagc catgcccctg tctctgtctc ccaagtgctg ggattgaagg tgtgcctggt 960

ctgtgctcac ccttaatgtg cttcctaaac tttcctaaca cgctttccta tgtgggcctc 1020

ccaaaatacg tcagtaagta cttaaacgat gtaggccttg ttcttcaacc ctggccacat 1080

ttcagttgtg ctcgtgtgtt ggtttctgat gtgcctgcag tagaaaggaa tagggatttt 1140

gaaggagcta ggaagtagac ttacagggct tagtgtttga aggaaccttg aagacgttta 1200

gttctaagca caaattctga gcaccgtccc ctgcccatac gcactctcac gttatctgac 1260

tggcacagcg ccctgctgaa aggccccaca cctaggagag aagagagcct ggctcccctt 1320

cagtcggaag ctatttgcat tcttcattct ttcatgttac gcactgtctc tctttaatca 1380

ggatcagact cattaagcac gctagtctgt tttttctcaa tgtggttaat ttctacctaa 1440

agttttccct ttctcatcct tcaaggccct attcctgtcc actggaggaa ttcttccaaa 1500

ttgatcaatg cctgcatttc tcatctctct ctttttttcc tcccatttgt catttctagt 1560

cattttgcta tacattgtgg ggacgtcttt agtgttacct tttgaatgcc aaccttccag 1620

ctgtcacatg tttgtatttc agatttctgc cttattttct gaaattccct ttggatagct 1680

gcctgtttgg ccttacctgt ttgagaatga cattttggtt ttaaatgtct tttggccact 1740

gcaccgctcc gttttccgtg ctttccgctc gcctcctctt ttttttcccc ccttctgttc 1800

attttgtctc cccccccccc aaccctccca tactcttagt cagatatctg gccattctta 1860

gctgtccact taaatttcag aacaaagtca caccacatgc caagccagtg gatgtgtgct 1920

gtttatggtt ttccctcagg ttgcattgtt ggagacatta attttggtag ggtaaattct 1980

ttgctcacaa gcacaagtgt ttttcttcgt ttgatttggt atctgctttt tcctattttc 2040

ttgaaggtat tggtttagct gttggaactg agtcgggaga tggggtcagg tctttcctgt 2100

tagtatgcac ataatctaca tcttccttaa cggcctggcc aacccgagga gctacaggtc 2160

ttactccccc ttggtggtac ctacagcttt ctgcacaggc ctgctgtctt gtttccgtcc 2220

gcccctcatt gcttcagaaa gtcatggtgc acccactgct cagcgttccc agggttccgc 2280

tgtgcacttg cgttcatttt ctcctctagg atttggtttt gcttttctct cttttgctga 2340

gttttcatct gctgtccata cattcctctt gatggccgct gtctctccat ctttttgttt 2400

tatgggcctt gtgtgttgct gacagcaccc tggggcgaca caacagggag tgacacaaaa 2460

gactcagtgt gggatgttta tcagcttgga gtgttctttt acatggctca tcttccttta 2520

gtcgctgcca ttctttgtac tgtattttta taaataaaat tcttttcacc tttgcagcct 2580

ttctgttact tatcatttgt taagttttct cttcctcttg cataattttt ttttaaataa 2640

acatctactt gaacaaaggt gtgagattgc aaaagtatta atgtcttttt cttaagggag 2700

aggaccttac atttcagggt aagggtatgc tacacttcag tagcattttt aattttgtat 2760

tcaaagcaat ggctggtttc tctttgtccc acgttatcca tgcggtgaag gtgtggggtt 2820

ctggatttag aatgctcact cctgacctgc caaggacctc atgtgagcat ccttgcctgg 2880

gccagtagct cacttatcga atcccctgtc ctggaacccg ggcaaagcag tgctgcttcc 2940

cctgctgagc aaacgccaga aaatgcagag actggaagat gatttttaaa agattgtact 3000

taaaaattgt aattgtagtg taagtgtttt ccttcaggaa agatgcaaag ggtaaatgtg 3060

ctaaggattt tcttttggtg aagtgaaaac atcttagacc tgaagagatg gcacccatgt 3120

caggtggctc tcgattgtaa ctccgggtcc agaggaccca acattcttag cattcttgtc 3180

tctgtaggca cctgcaggca gatgcacaca cacacacaca cacacacaca cacacacaca 3240

cacacacaca tatacacaca cacacacata tacacacaca cacacacaca cacatacaca 3300

cacacacata cacacacata cacacacata cacacacaca cacacataca cacacacaca 3360

cacacacaca cacacacaca cacacacact taaaaatagc ttaaatttca gagtacaggc 3420

tatctttgcg ttgtggctca attccaatac ttacctgtca cttctttgtg gtctgtaatc 3480

gtcccccatg caagcatcac ttggtatatt ctctttttct ttcccttttc tccgattaga 3540

tttgctcttt gacagttgcg tccatgtgtt ctgcgccctc tgtgtacgct cccaccctga 3600

cccccttctc ttccttcccc tcctccctgc aggtccctgg tttacactca tgactttgtt 3660

ctgttctggg aaccactgag tttgactaga agcatctgtt tccccctggg tttggatctg 3720

ctcattggaa cgctttctgt atcactctgt gacccttttt tcccactgtt acgctcatgt 3780

ttggtttggt tttggcctta ttttgtctac tagagtaggg ccttcctctc actcatctct 3840

gtgctatgga tatctaacca gtacagcata ttgaatatct aatactatgg atatctaacc 3900

agtacagcat attgaatgtg ctcagtaaat atttgttgaa ggagggaagg attaaacacc 3960

actaattgga aaacattccc agtttcaggt agaagttgaa agtactagaa atagcccatg 4020

agctccagaa gaagatgtgt ttttaagtgt agtttatata tgtgataggg tcaaagagtt 4080

gagttgctga tttggtcccc gtaagccctt gcttggcatt tcagtgtagg gctgcaggcc 4140

ttacttgccc atttcaaagt acctcatggg tgaagttcgc ctggaagctt tcaggcgttg 4200

acagtccaca ggctggagaa ttataagaat gttttcattg agagacacag cttaaaatgc 4260

ctttcttctt ttcctagttg gacgaaaagg aaagccgccg tctgttccag cagatccttt 4320

ccggtgtgga ttattgtcac aggcatatgg tggtccacag agatttgaaa cctgagaacg 4380

tcctgcttga tgcacacatg aatgcaaaga tagccgactt tggtaaggat ggttgtgatg 4440

tttaatgagc ataagtatca tcttctggta gtgtttatgg ggtgcactag aaccccactc 4500

tgcaagtgaa cattcagaag ggaaaacagt aaggtacgcg cgcgcgcgcg cgcgcacaca 4560

cacacacaca cacacacaca cacacacaca cacacacaca cgccacagga tagctttggg 4620

ggatgtttct attcttccag cacgtgggtc ttagagggtg gatttaggtt gtcaggcatg 4680

gtggtaagca cttttacctg ctgagcctac ctgccagccc tgaaattcag ttttgtgatc 4740

ttaaatattt caacttctta acattagaga gatcatttga aaagaaaaga ctattatttg 4800

tcaagaaggt agcaacttga tttctaatat ttctttttgg tggagtatgg agaaaatgga 4860

aagtgggatg gaattattga cataacattg gaatctagaa agatgaatta tatgaggaaa 4920

ctttttttat aaaattaata tttaaatgct gtaaagttgg ttttcagatg tagtggtagg 4980

tgccttcagt ctaagaattc cagagatgga ggccgataac ttcgtatagc atacattata 5040

cgaagttatg tagagatctt tctgttcagc tgggtctatt ttgcctagca gtatccatat 5100

taaaaggtag acatttcaag gatcttctag tattcaaaac caccactagc ttagcacata 5160

cacattaaac tctagatgtt gaaattcatt gtacattttg tgtataaggt cagaatattt 5220

aagaaataaa gactataatc aagttaaaag tttttgtttt tttgtttttt ttttctgttt 5280

ttattttatt ttattttatt acatattttc ctcaattaca tttccaatgc tatcctaaaa 5340

gtcccccata ccctcccccc ccacttccct acccactcat tcccattttt tttggccctg 5400

gcgttcccct gtactggggc atataaagtt tgtgtgtcca atgggcctct ctttccagtg 5460

atggccgact agaccatcgt ttgatacata tgcagctaga gtcaagagct ccaggatact 5520

ggttagttca taatgttgtt ccacctatag ggttgcagac ccctttagct ccttgggtac 5580

tttctctagc tcctccattg ggagtttttt tttctgtttt acttgctttt tgtccttaag 5640

aactcttgac tggtcacttt ttgtcatttc aggtctttca aacatgatgt cagatggtga 5700

atttttaaga acaagctgtg gctcacccaa ttatgcagct ccagaagtca tttcaggaag 5760

attgtacgca ggccccgagg tggacatctg gagcagcggg gtcattctct atgctttgct 5820

gtgtggaacc ctcccttttg atgatgacca tgtgccaact ctttttaaga agatatgtga 5880

tgggatcttt tatacccctc agtacttaaa cccttcagta atcagccttt tgaaacatat 5940

gctgcaggtg gatcccatga agagggccgc aataaaagat atcagggaac acgagtggtt 6000

taaacaggac cttccgaagt atctctttcc tgaggaccca tcttatagtt caaccatgat 6060

cgatgacgaa gccttgaaag aagtgtgtga gaagttcgag tgttcggagg aggaggtcct 6120

cagctgcctg tacaacagaa accaccagga cccactagcc gtcgcctacc acctcatcat 6180

agacaacagg agaataatga atgaagccaa agatttctac ctagcaacca gcccacctga 6240

ctctttcctg gacgaccacc atttaactcg gcctcaccct gaaagagtac cgttcttggt 6300

tgccgaaaca ccacgggccc ggcacaccct ggatgaatta aacccacaga aatccaaaca 6360

ccaaggtgta cggaaggcaa aatggcattt gggaattcga agtcaaagcc gacccaatga 6420

tatcatggca gaagtttgta gagcaatcaa gcagttggat tatgaatgga aggttgtaaa 6480

cccctattat ttgcgtgtac gaaggaagaa tcctgtgaca agcacatttt ccaaaatgag 6540

tctacagcta taccaagtgg atagtaggac ttacttgttg gatttccgta gtattgatga 6600

tgagattaca gaagccaaat cagggactgc tactccacag agatcgggat ccatcagcaa 6660

ctatcgatct tgccaaagga gtgactctga tgccgaagct caaggaaagc cctcagacgt 6720

ctcccttacc tcatctgtca cctccctcga ctcctcccct gtcgacgtag ctccaagacc 6780

aggaagtcat acaatagaat tttttgaaat gtgtgcaaat ctaattaaaa ttcttgcaca 6840

gtaactgtaa gtctgcagaa attgatgatc tattaaacaa taaagatgtc cactaaaatg 6900

gaagtttttc ctgtcatact ttgttaagaa gggtgagaac agagtaccta cattttgaat 6960

ggaaggattg gagctacggg ggtgggggtg gggtgggatt agataaatgc ctgctcttta 7020

ctgaaggctc tttactattg ctttatgata atgtttcata gttggatatc ataatttaaa 7080

caagcaaaac caaattaagg gccagctcat tcctcccact catgatctat agatctatag 7140

atctctcgtg ggatcattgt ttttctcttg attcccactt tgtggttcta agtactgtgg 7200

tttccaaatg tgtcagtttc atagcctgaa gaacgagatc agcagcctct gttccacata 7260

cacttcattc tcagtattgt tttgccaagt tctaattcca tcagaagctt gcagatctgc 7320

gactctagag gatcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 7380

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 7440

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 7500

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 7560

gctgcgactc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt 7620

aaaaaacgtt taaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 7680

gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 7740

aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 7800

aatgtatctt atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat 7860

ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata 7920

aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa 7980

gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 8040

tgtccaaact catcaatgta tcttatcatg tctggatctg cgactctaga ggatcataat 8100

cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct 8160

gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa 8220

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 8280

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggatccccatcaa 8340

gctgataaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc 8400

tgtccccagt gcaagtgcag gtgccagaac atttctctaa ggcgcataac gataccacga 8460

tatcaacaag tttgtacaaa aaagcaggct ggcgccggaa cgcatgcaga tctacgcgtg 8520

attaacttta aataattggc attatttaaa gttattaatt aaccccccca aaaaaaaggt 8580

gcctatcacc cttctgccta cctgtgtatc ctcaggacat ggtgggcctc tctggttggc 8640

agaaagcaca acaaagcctc ttcatcctat actacttctc ttgaccagat gccaagtcta 8700

atatgaactg caatcctcta tacaccaaaa gttcatgggg gcaccgtgag gtcccactcc 8760

acctcagcca attccttgtt gctgccccac tcacctcctg agcctccctc tgtttcctgt 8820

ccacctttca gcttccctct caggctggga gcaggggcca gtagcagcac ccacgtccac 8880

cttctgtcta gtaatgtcca acacctccct cagtccaaac actgctctgc atccatgtgg 8940

ctcccattta tacctgaagc acttgatggg gcctcaatgt tttactagag cccacccccc 9000

tgcaactctg agaccctctg gatttgtctg tcagtgcctc actggggcgt tggataattt 9060

cttaaaaggt caagttccct cagcagcatt ctctgagcag tctgaagatg tgtgcttttc 9120

acagttcaaa tccatgtggc tgtttcaccc acctgcctgg ccttgggtta tctatcagga 9180

cctagcctag aagcaggtgt gtggcactta acacctaagc tgagtgacta actgaacact 9240

caagtggatg ccatctttgt cacttcttga ctgtgacaca agcaactcct gatgccaaag 9300

ccctgcccac ccctctcatg cccatatttg gacatggtac aggtcctcac tggccatggt 9360

ctgtgaggtc ctggtcctct ttgacttcat aattcctagg ggccactagt atctataaga 9420

ggaagagggt gctggctccc aggccacagc ccacaaaatt ccacctgctc acaggttggc 9480

tggctcgacc caggtggtgt cccctgctct gagccagctc ccggccaagc cagcggcgcg 9540

ccgccaccat gggtgctagc gagctgatca tctctggctc ctctggagga ttcctgagga 9600

acatcggcaa ggagtaccag gaggctgctg agaacttcat gagattcatg aatgaccagg 9660

gagcctacgc ccctaacacc ctgagagacc tgaggctggt gttccactcc tgggctagat 9720

ggtgccacgc tagacagctg gcctggttcc ctatctctcc tgagatggct agggagtact 9780

tccttcagct gcacgatgct gacctggcct ctaccaccat cgacaagcac tacgccatgc 9840

tgaacatgct gctgtcccac tgtggcctgc ctcctctgtc tgatgacaag tctgtgagcc 9900

tggccatgag gagaatccgg agagaggctg ccaccgagaa gggagagaga accggccagg 9960

ccatccctct gagatgggat gacctgaagc tgctggatgt gctgctgtct agatctgaga 10020

gactggtgga cctgaggaat agggccttcc tgtttgtggc ctacaacacc ctgatgagga 10080

tgtctgagat ctctaggatc agagtgggag acctggacca gaccggagac accgtgaccc 10140

tgcacatctc ccacaccaag accatcacca ccgctgctgg cctggacaaa gtgctgtcta 10200

ggaggaccac cgctgtgctg aatgactggc tggatgtgtc tggcctgaga gagcaccctg 10260

acgctgtgct gttccctcct atccaccgga gcaacaaggc taggatcacc accacccctc 10320

tgaccgcccc tgccatggag aagattttta gcgatgcctg ggtgctgctg aacaagaggg 10380

atgccacccc taacaagggc cgctaccgga cctggaccgg ccactctgct agagtgggag 10440

ctgccatcga catggctgag aagcaagtgt ccatggtgga gatcatgcag gagggcacct 10500

ggaaaaagcc tgagacactg atgagatacc tgaggagggg aggagtgtct gtgggagcca 10560

actctaggct gatggactcc gctagcggcg ccggtcctaa gaagaagagg aaagtgtgag 10620

gccacgtagg cccgcgatga ataaatgaaa gcttgcagat ctgcgactct agaggatctg 10680

cgactctaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa 10740

aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac 10800

ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 10860

aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 10920

catgtctgga tctgcgactc tagaggatca taatcagcca taccacattt gtagaggttt 10980

tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 11040

ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 11100

caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 11160

tcaatgtatc ttatcatgtc tggatctgcg actctagagg atcataatca gccataccac 11220

atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 11280

taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 11340

aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 11400

tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cccatcaagc tgatccggaa 11460

ccgcttggct gcaggtcgtc gaaattctac cgggtagggg aggcgctttt cccaaggcag 11520

tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg 11580

cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc 11640

ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg 11700

tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc 11760

accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc 11820

ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg 11880

ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa 11940

aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc 12000

ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga 12060

actaaaccat gggatcggcc attgaacaag atggattgca cgcaggttct ccggccgctt 12120

gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 12180

ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 12240

gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 12300

ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 12360

gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 12420

tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 12480

accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 12540

aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 12600

aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga 12660

atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 12720

cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 12780

aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 12840

ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 12900

ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag 12960

gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 13020

catgctggag ttcttcgccc accccccgga tctaagctct agataagtaa tgatcataat 13080

cagccatatc acatctgtag aggttttact tgctttaaaa aacctcccac acctccccct 13140

gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa 13200

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 13260

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccgggcggc 13320

cgctaacttt aaataattgg cattatttaa agttacaccc agctttcttg tacaaagtgg 13380

ttgatatctc tatagtcgca gtaggcgggg taccgctagc ataacttcgt atagcataca 13440

ttatacgaag ttatgtagag atctttctgt tcagctgggt ctattttgcc tagcagtatc 13500

catattaaaa ggtagacatt tcaaggatct tctagtattc aaaaccacca ctagcttagc 13560

acatacacat taaactctag atgttgaaat tcattgtaca ttttgtgtat aaggtcagaa 13620

tatttaagaa ataaagacta taatcaagtt aaaagttttt gtttttttgt tttttttttc 13680

tgtttttatt ttattttatt ttattacata ttttcctcaa ttacatttcc aatgctatcc 13740

taaaagtccc ccataccctc cccccccact tccctaccca ctcattccca ttttttttgg 13800

ccctggcgtt cccctgtact ggggcatata aagtttgtgt gtccaatggg cctctctttc 13860

cagtgatggc cgactagacc atcgtttgat acatatgcag ctagagtcaa gagctccagg 13920

atactggtta gttcataatg ttgttccacc tatagggttg cagacccctt tagctccttg 13980

ggtactttct ctagctcctc cattgggagt ttttttttct gttttacttg ctttttgtcc 14040

ttaagaactc ttgactggtc actttttgtc atttcaggtc tttcaaacat gatgtcagat 14100

ggtgaatttt taagagacag ctgtggctca cccaattatg cagctccaga agtcatttca 14160

ggaaggtatt gctgccatta ggctactttt gatgtgcctg ttgttgctta tttctcttaa 14220

gtttgttagt ctgtactgtc gccttgagga tgtgatgtca ctaacgagga cataaaagta 14280

acacattggc ctgtggtaac tgtcatctcc gaggctcgct gcctcagtct gctaacgtag 14340

gtctaggcct ggaagcttct agcttgtgta caatctaatc taggcctaga aagacttcag 14400

ctgctgaggc ttgctgctgg gtaagcgcgc cccttccttg ttctttctga tctatggctg 14460

attcaactca gatgttttgg ctctcaaact cctctccacg ataactgatt caatctggct 14520

tctctgggct tctcctgaat ttgccctgct tgacctccaa ctctgtcatc gttctaatct 14580

ggctccttct cagtctctgc ttgttctgtc ttcacctgtg tctggcttgt ccttcctctg 14640

caagctgtct ctgtacatct gtcctggtac agtggcctcc tctccctgca ctgaccctca 14700

agtacttcct ctctcttccc gtgagaggtt gggcatatcc tattctgtca ggtctttttc 14760

tgatttgtca tttgtctgcc actcaattag aagttacttt caaacatggg tgctttcttc 14820

tatgaactaa ctttgccaca gggattaaaa gtgtgtgctg aggccgaggc acatcacaac 14880

tagaagcagc tttttcagta aataacacaa tcttgaggtt ttacaatatg ataagatatc 14940

ctgcaacaga aagaagggaa tatatttctg tacttgcaga ttttctattt tatgaataat 15000

cacaaggagc tggagagatg gcttattggt taagaggact tgctgctctt ctagaggact 15060

ggaatttggg acctggcaac cacaatctgg gtggctcaca gttgtctata actttgcctc 15120

caggagaccc aacacactct tggtttttgc aagcagttac atacatggat gcacctcaac 15180

acacacacac acacacacac acacacacac acactaataa taataataat aataataata 15240

ataataataa tagaaataaa taaaaaaaac aagtggaaaa acaagtccat ctttggagga 15300

gagtgttggt ttgcaagtca cagtaaagta gttagaatga agtgctttca tttttgtctc 15360

tggtgggcga attgaaagcc aaccagctgt aaaggtggat aagccgctga gaacctgcct 15420

actgcagtac tcactcccga ggaatgcttt acttaggcga acattaaaaa atgtaacatg 15480

atgggcaatc cttgctggta attctacacc ttctcatgac accatctcaa gaaaaccaaa 15540

ccaaactaga ccgcacagag agcagaacag agttacaagt gtaacgtcac gtgtagttgt 15600

ataagtattc aaatagctgg cattcatgta gtagcttcat gttttatgag gaaggaaact 15660

aaagaagtgg tcctttaaaa aaacagacca gctgaatgta ttgcatgcct atagcctgag 15720

tatcgcacat ggatgccaag acagggctat ataggcctgg tatcaaggaa aatcgaggtg 15780

aggtgagggg caagagggtt ataaaaaggg agaaggaagg agagagactc atttatgttc 15840

agtcttgatc tagatctgct agagattgaa gtgtaagact gctgaactctgaactactat 15900

ctagcactaa aggacggatg tattttcatt agttactttt taaatagcgt gaaaacaaaa 15960

tttaatttgt gttttatttc cttgttttgt tcaaggttca agttcctcct cctcctcctc 16020

tccttcctct ccctcctcct ctccttcctc tccctcctcc tctccttcct ctccctcctc 16080

ctcctcttcc tcctctgtga tagagacagt tcattaacaa agagaaggca cgcctaaatt 16140

tggaagtggg gtagtggtct gggaaaagat atttgcttaa agttgtaaaa tttaatttaa 16200

aagacagact ttattttaaa ccatatatgt atgtgtgttt gtgtgagggt atgtgtacat 16260

tagtgtaggt gctgacagag gtcagagccc cccaacccca cccccccaat cctcaccctt 16320

cagctattgc tacgggcagt tgtaagctgc cttgtgtgtg tgtgtgtgtg gggggggtga 16380

gaactaaacc agggacctct ataaaaacag cgcaggttct caatggctga gacatccctc 16440

cagctcagag aatttattat ttttttccat tttttaaaat taggtattta cttcatttac 16500

atttcaaatg atatcccgaa agtctccaat accctccccc aacctgctcc cctacccacc 16560

cactccccct ttttggccct ggtgttcccc tgtactgggg catataaagt ttgcaagacc 16620

aatgggcctc tcttcccaat gatggccgac taggccatct tctgatacat atgcagctag 16680

aaacacgagc tccggggtac tggttagttc ataatgttgt tccacctata gggttgcaca 16740

ccccttcagc tccttgggta ctttcactgg ctcctctatt gggggccctg tgatccatcc 16800

catagctgac tgtgagcatc cacttctgtg tttgctaggc acccgcatag cctcacaaga 16860

gacagctata tcagggtcct ttcagcaaaa tcttgctggc gtagagaatt tatttttaag 16920

atcaacgctt tggctaggct tggtggtgca ccttacccag gaggttagac agcagtttgt 16980

ctccagctca ggctccttag tgagacaggg ccagcttggg aataaataaa atgctcttgc 17040

aaaaacaagc aagcaaagaa agaaataaaa aacaagtaaa agtcaaagca aacaaaaaca 17100

cattgcttcc tcattgctgc tgaaacacta atgaggtagg ggaggcctgt tgctcgccag 17160

tgcccacaat cccgtccacc tcaaccattt ccttgtttaa atgcttcaaa tggtgatgga 17220

cctaatttaa aaaaaaatcc ctgtgctttc tatgttcatg tgcttatttt tttttgactg 17280

ctcaaagaaa aggtaatgaa gtgaattaaa aagattcatt tgtgttttct atcttaattc 17340

ctttgatttg agaaaaattc aaaatctaga tgctacaggg aatataacta aagaaaaaag 17400

gcatgcgtca tgttcaagca tgcataaaat gggctcttgg ggaaatgtac agtttggatg 17460

tctttccttg aggtctgtgt gaataggagc actcattttg agaacagtgt ttactgctgt 17520

ctataggaag gaagcactct cgtcactctg agggttcgtt ttccccctga aggcagcctc 17580

ggatgattca gctggtttgc agctagttag aatccgtgtc tccagtgctt ccttcacgta 17640

gacgcactca cgcacgaaga gggcgagtgt tctgtgagct ccactgccct tggcttgctt 17700

ctgcagctcc agcttcctga actgtgatgt gagggtttta ggagaaatgg gttcagagtc 17760

tgtcactaca gacttgaatc atgggcccag cgagttggct cagctggtaa aacatttgct 17820

gccaagtgcc tagagtctga cccctgaatc ctgggtaaag gtaggagaga actgattcca 17880

cagagctgtc atctcagctc cacacccact gtggcatgtg cacaccatac acacgcacat 17940

gcatgtgcac acacacacac acacacacac acacacacac actaaaagtt acttaaaggt 18000

gagggtgggg gaacaaaaaa ggtacttaaa aaaaactatc cctgtggctc aaatcaacat 18060

tgatcatgat ggctcaaggg ttgaccttat tggtaggaag tttgagggtc atagtaaggt 18120

agtacaattt gacccatgga tcacaagtta caaaatagct tatttttaaa caggtaatac 18180

tgataagact agtatcttta tgtgtgatgt gcaacaacca ctatagatac catattctga 18240

ccactgattt ctttaaaaat tattttctta tatttttcat aaagtatttg tatttgaaga 18300

gtgttgaccg atgctttttg atattgtgat taatacagtt agcattctct atgcagattg 18360

tacgcaggcc ccgaggtgga catctggagc agcggggtca ttctctatgc tttgctgtgt 18420

ggaaccctcc cttttgatga tgaccatgtg ccaactcttt ttaagaagat atgtgatggg 18480

atcttttata cccctcagta cttaaaccct tcagtaatca gccttttgaa acatatgctg 18540

caggtggatc ccatgaagag ggccgcaata aaagatatca ggtaactcac tttatctgcc 18600

tacttgacca accagccaac aaaccatctg tctgtctgtc tgtctgtctg tctggactgt 18660

gtagtcttgg ctatcctgga actcactcta aagacaaggc tggccctgcc tccctctgcc 18720

acccgagtgc agtgctagga ttaaaggcgc gtggtattac cattgcacag caatattagg 18780

tgatttttaa accaatgtat ttttctacct tttcgagtct ctagatacc 18829

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ggtcttcata ccgccactgc tt 22

<210>9

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ggcctgcgta caatcttcct g 21

<210>10

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

cacccagctt tcttgtacaa agtgg 25

<210>11

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gcacagctgc cactatcagt catgc 25

<210>12

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ccttcggtgt taccttgtag gct 23

<210>13

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tctggaggaa gagatggagt g 21

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

cccttcttcc accattgctg 20

<210>15

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ccattttgcc ttccgtacac c 21

<210>16

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

uggcauggcc accucccuac 20

<210>17

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

acgggcaaga gcacaccg 18

<210>18

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

catgtctgtg aagctcagca ga 22

<210>19

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

gaatgtacct gagagcagag g 21

<210>20

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

tctgtagccc tcacattcag ag 22

<210>21

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

tctgtgcaaa cacaatcggt cag 23

<210>22

<211>21864

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

agcatgttca acgcagtgag ccacctctgc tgcctccatc caccgatgca atgcatcggt 60

ccagcgccca ggtaaactgc tgccccttca tctccactcc ttgcgacttg cagactcaag 120

taccagtgaa ggggaaaagg cacaagtatc ctctgagccc ctcaacaaag tattttagtt 180

agggttttac tgctgtgaaa agtcaccatg accccagcaa ctcgtttttg gttggttggt 240

tggtttggtt ggttggttgg ttggtttggt tggttggttg gttggtttgg ttggttggtt 300

ggttggttgg tttggttggt tggttggctg gttggtttgg ttggttggtt ggttggttgg 360

tttggttggt tggttgtttg gtcggttggt tggttggtta gtttggttgg ttggttagtt 420

tggttggttg gtttggttgg ttgtttggtt ggttggtttg gttggttgat ttggttggtt 480

ggttggttgg tttggttggt tggttggttggtttggttgg ttgattggtt ggttggtttg 540

gttggttggt tggttggttg gttgtttatt gctttcccag acaatggtcc cctccctggc 600

atctttgagg atttgcttta ctaggctgat cttcccagac caaaaccctc aggacgcagc 660

attaaagcgg cacacaccaa ttcacgtgac atctgctttc ctaaatcagc ctcacttcct 720

gtcaggattg cctccctaca ccgtgggcac atgcaaccct gagtgttagc aggccctacg 780

acaaaaacag agagagaaag gaaaccacaa aggaggcacc cggaacctgt gacctcagct 840

cccacaacaa acacctgaaa cactgcacca cgctttcaca ttagggttca gctcaccaga 900

aagaaatggg caaaagggtg gacatgtggc tcacttagta gattgttctc tcaaatagac 960

tcagtgctaa gggcgggggg ggggttattt ttgggttttt ttgagacagg atttctctgt 1020

gtagccctgg ctgttctctc tgtagaccag actggtgtca aagtcagaga tctgcctgcc 1080

tctgccttcc aaatgctggg attaaaggtg tgagccacct ctgcctgact gatgttaggt 1140

ttaacattta gcactgcata aactaggtgt ggtggtgcat ggctctcatc cttatcatac 1200

atatatacat acacacacat acacacacat atatatatga catatatatt atgtatatat 1260

catatatatg ataaagatga gggacatata tatatatgta tgtatatgaa tgataagaat 1320

gtatatgtgt gtgtgtgtgt atcactaaca tcagaaagga atgtcaagta ccatggctgg 1380

ggacagcact gaatccagag gcagctgcca aggcgctgtc agcaagcact gtccagagct 1440

tcgcttaccc agagtctgag aaagaaaacc gttagagcta ccagacagga cactggacaa 1500

taaatcctcc taaataaagt agaagcctta agggaatcta aacaaagagc cggaacctac 1560

tcaacaattc agaagctcag ccatttgtct gctccaaaaa atgtcttcccagagggcctc 1620

aaaacagcac ctgctgtagc cttgctcctg ctgtcaccac agtcagatgt gaagacccta 1680

ttgctgaaaa cgtaacacag tgtgggcagt gtggtcgcta gagagatagt tctgggttat 1740

atagaaagca ggttgagcaa gccagtaagt gctgtccccc tgtgatctct gcttcagttc 1800

ctgcctccag gttctgtaag tcagaagtct aaaggaggcc tcagccaggg atgcagctca 1860

cggcctggcc tcagccccca actcattgcc ctcatcttcc ttccgtcttg caaatcagca 1920

atgagtcctc atatgactgc ttgtttgttc ttctgcttcc acttgagacc acttaatatg 1980

cccagcagag ctgtgcacct ctttaatccc aagcattcag gtggcagagg caggtggatc 2040

tcagagtttg agaccagcat ggtctacaga tgtaaacaac accgagagac agggtcctcc 2100

tggctggcac aggcttcggc cttttctttc tcccagcatg agattcctgg ggaaattcca 2160

gtttcttggg ggaccatttt tgcagtcatt tctgtagcca aaggatatct tcattcctac 2220

cagggcagga gaataaaaac attcaaacac ataaaataaa caaacccttt gaaattattt 2280

taaaatattt tcaaaaacat atttctaata agagattagt atctgaaaca gtaactaaaa 2340

tcaagatgat tagaacagaa gcaaaagacc taataaacat ttcttcagag aagaggtaca 2400

attctcgata agcatgtgaa aggctaagga gaggagaatt gggatctcac ggtttagcag 2460

gtatggagtt tggggttata ttgcttgttc atgtatgagg gggagggcgt tggtctcact 2520

gcttctggcc aggatggtct cctggactta aatgacccct cccactcaag ctcccgagga 2580

ccaagaacct cagacaggca catgtcatca tgtccagctt aagagttcca attttatagt 2640

ataaagagaa tatacagatg aaaggtgatg cgttggctgt catggttaca gcgaaactac 2700

ttcagaagac aacttggaag gagaaaggag ttgacttcgg ttcacagtgt cagagatgtc 2760

aacctctgac tgactagtgc cattgcttct gggcctgtgg tgaagcagaa ctttatggtg 2820

aaaagggtgt ggcagagtcc tctcacttca cagcatctag aaagcagaga caccgaggga 2880

gacggagaga ggaaggaagg ccatgaggat aagacacacc attcaaaggc ttgattctag 2940

tgagccacta attctaactt ggccccactg cctatttagc catgtagtca tcaagggagt 3000

acttcgtgga tgaagcctgc ttacctcccc atggcactgc tgctccagcc atgagccctc 3060

tgcggtacac ttcatatcta ccttgttagg gtttccattg ctgtgaagag acaccatggc 3120

cagggcaact cttataaagg aaagcatttc attggggctg gcttacagtt tcagaggttt 3180

agtccattat catcatggca ggaagcatgg cagtgtgaag aaggcacggt gcgggaggag 3240

atgagagatc tacatcttga tcctaaggca gccaggagac tttcatccac actgggtaga 3300

gcctgagcat tggaggaaac cttaaagcca caactataca cttcttccag caaagccata 3360

ctctaaccag gccacacctc ctaaaaatgc cactccctgg gccaagcatt caagcacatg 3420

agtctatggg gcccaaatct attcaaacca ccacattagg caaacctaac accagtgtta 3480

cttcaggaga gccaggcagg ccctgtccct gtcctgagcc tgtggaggaa gatagacttt 3540

gaacctagag gtggaggctt tagatgagga cacaactcat ggtctggcca agatgttaag 3600

ttctctgtgc actcaaagcc tctttttagc aactttcttt ctacagttct tcaacgccag 3660

gtcctattca ttcctacagg gtcagccgtc agctccccgc acccatgaca tctgtcttca 3720

gatttatgac taacggtgaa gatgttggag cctgggaatg aggaaggaaa ttccttccgt 3780

tgtaaagttg tcgcggcctc tgctgggcat gtggtggagg gtggtttcct ttaaacagaa 3840

catgctgcag ctcctggctc gagtggaccc ttgaagagct gtagtggggg ctggggaaag 3900

ggaggagggt ggaaaaaatg aagagacagg aagctgaagc atgagataga aagaacagat 3960

ctaacatccc ccaggtgcga ccacagggct gtgacatttc accaacacta tggaaacaac 4020

tgtgaagccc ccaaatgaga catatccttg agataagtca gcaggggaaa gagtggaggg 4080

ggttcaatgg cccgtgcttc tccagtgtag caactccagc atcctcaaat cagaattccc 4140

agaggagcat gaggcttaag atgatggccc acgtaggtgc ccaccttcaa tcccatgtgc 4200

ccagggccat gaagagagat ggctcaggta cctgaagata ctagtgacca agccagccag 4260

aagcaggaag gataaaaatg ggattaggtc aagaaggaag tttggcaaaa gttcccccag 4320

gcagtgggtc acatctgtgt cacaggctgg cattaagaag aaataaaacc aggctggagt 4380

ggtagctcag tcaggaaagt acaagggcct gagtctgatc gccaaaaccc agtttttgag 4440

ttcatttctt tgtttgtttg ttttgctaaa ggatcccagg gtttgatggt catccagcct 4500

cctctactag gcaagttgca ggccagtgag aaataataat ctccatcttc tttatcatca 4560

ccaccaccat catcatcatc atcgatgtcc ctgaggaatg aaaccaaggt tgtcccctga 4620

cctccatacg catgaacacc tacacacaca cacacacaca cacacacaca cacacacaca 4680

cacacacaca catgtgcaca caaggattaa agggacttga tatcacagaa cagaaaaaga 4740

cacagggaac acagggctaa ctcttgccct caattgtatg caatgttcaa gagcttgtat 4800

agccagaaac tcaaacctca gttatataag gctggttgtc atgactcctc ccctttatac 4860

atgcaatagc tttgctgcgt gtgctgtgtc caagctggga atatagctca gacagcagtg 4920

tttgcctagg atgcaaaaag ccctgggttt catccccagt accacataaa cgggatgcgg 4980

cagcacagcg ctatggcacc agcagcagtg gcttggtagc aaaccctgca acaccaggaa 5040

cactcaggag agggaggcag aaagatcaga agtcccaagt catcctcagc taccaagcaa 5100

ccttgaggct accttggcat atataagacc gtcttccaaa ctgttgtgct tagccgtgtc 5160

accccacctc taatggggtg ttttgtggta ctagttttag gcaaaatctg aaaatgtcat 5220

gattttaaaa ctaacctgcc cctaggagct tcagaaataa tctgtaatct gctcctatct 5280

ggtcacctct ggaagggctg ggctatgaga gaggacactt gccagggaca gatgagggca 5340

cccagcgtcc atccctatca acaggtccgg cggctgtggg tattcattac tgagagaggt 5400

gcccaagggg ggcacatgtc tgtgaagctc agcagagctg ggtttacccc ttcatagtct 5460

ctccggagtg cggaaaagag aagggggtgg aggctgctga gagagagaca aagaaaacca 5520

aaacagccac aactgccttc aaaggtcggt ttaccaccca agtggagtcg cagtggctgc 5580

tgcccagagc acataggagc caccagtgac tcggtacaag cagttggggc tcagaaaaga 5640

tctccctggg caggagctat ggcacggcca catccctggt ggctgtgcgt tctggggacc 5700

ctggtggggc tctcagctac tccagccccc aagagctgcc cagagaggca ctactgggct 5760

cagggaaagc tgtgctgcca gatgtgtgag ccaggtaaga gggggccttg gtaagggcca 5820

ggtgagtggc gaaagagaga ggactggggt taatacagta aataggcgca gggtgagact 5880

gagctcaagc aaggagggaa atcctgcagc tgtggggagg caccaccttg aagagggcag 5940

agaaccagcc cttctcaggc cttgatccct taccctctcc tcccaggaac attcctcgtg 6000

aaggactgtg accagcatag aaaggctgct cagtgtgatc cttgcatacc gggggtctcc 6060

ttctctcctg accaccacac ccggccccac tgtgagagct gtcggcactg taactctggt 6120

gaggtgggca agggtgtgta ggtggggacg atggacaagc atctggggga gcaaggctgg 6180

tgacgggttt gggggtgcaa ggaggatgac ggggccaaag ctttggcctt cttcaaggct 6240

cacagcaagt ggagccaatg ctgggaaatg cggcacccta ggtggggcat gaattaacgt 6300

gggcagacat ctagtattcc aggaaaggga taaatagaat ttgagggatt ggtgagaacg 6360

ggtctatgga taggatcaag acaataaaat gagagaagtg gctctatgac cccagatttc 6420

gggcaagcaa cccccaacca ataaactgac tgtgttccca gagtgcacat gtgcaggttg 6480

gccattggga atttggaata catctttcta aggaaataac aaagcaaccc aataggtaac 6540

aacctttttt tgcacaaaag ccaatttgag aaaatgagta tgtatgtagc ctgtgttttt 6600

attttatagt tccaaaaaaa aaggtttttt ttaaactaag aagtataata tctgcagttg 6660

tgcttatgaa tgctgttatt atttctccat cttcgctaca atttcagaaa ctacggccag 6720

tatacactag aatagcaggc tttaaagtgg cccctttgaa caaatgttct ctagcaagac 6780

actttttaga accaggtaac tgtaactatt aaactaccta cttgcaaaac gtgtttggtg 6840

tcactgggtc tcttggtact gggttttgtt ttgtttagtt tagagacaag gtctagctct 6900

gtcacccagg ctggagtgca gtggtgcaat catagctccc cacagccttg aactcctggg 6960

ctcaagcaat cttctagtct cagcctctga aagtgccagg attacaggtg tgagctgtag 7020

tgcctggtcccgtaaaaaca aaaacaaaaa ccaacaccag atgtggtggc tcatgcctat 7080

aatcccagca cttgggaggc caaggcagaa gggtatttgc atattttgta gagacttgag 7140

cccaggagct tgagaccagc tgggcaacat agtgagacct tgtctctaaa aaaattggct 7200

ggtcatggta gtgcacctgc agtcccagct acttgggagg ctgagatggg aagatcactt 7260

gagcccaaga ggtccccaag aggtccaggc tgctcacatc actgcactcc agcctgggta 7320

acagagcaaa accctttttc acacacacac acacacacac acaaaaagcc agagcagtgg 7380

ctcacacttg taatcccagc actttaggag gccaaggcag gcggatccct tgagcccagg 7440

agtttaagaa caccctgggc aacatggcaa aaccctgtct ctactaaaaa tacaaaaagt 7500

tagccaggcg tggtggcacg aacctgtagt cccagctact cagaaggctg aagtgagaag 7560

atcacttgag tccaggaagt ggaggctgca gtgagccgtg actgtaccac tgcactccag 7620

cttgggagac agagtgagca gagtgagacc ctgtttccaa aaaaaaaaag aaaacaaaag 7680

cttttccatt gcaaatcaac caaaaaggaa cgtctttaat acacaagaaa tattcttcag 7740

tctacttaac ttaaaaatgt aactgtaata gatgcttttc tcttgacagc ggccccggca 7800

gggttggaac ttttcctagt caaaaccccc ctactcactc tgcagaccct tcctgaaggt 7860

cttcgcccca tactgagccc atcttcagtc ccttcacttg ttccaaacta gcccaatcca 7920

tgacccttct ctgggagctc agccaataga tcccaaagta ccacagcagc acctggctgc 7980

agaaggaccc ccgggaaatg cagtcagggg tgagaagatg ggtgggctga atgagctcgg 8040

ccctctctgg gccttagtgc tggcatctat gagatgagga catgcctgaa gtcagcctgg 8100

aattttgagc ccaggttaaa ggcgcataac gataccacga tatcaacaag tttgtacaaa 8160

aaagcaggct ggcgccggaa cgcatgcaga tctacgcgtg attaacttta aataattggc 8220

attatttaaa gttattaatt aaccccccca aaaaaaaggt gcctatcacc cttctgccta 8280

cctgtgtatc ctcaggacat ggtgggcctc tctggttggc agaaagcaca acaaagcctc 8340

ttcatcctat actacttctc ttgaccagat gccaagtcta atatgaactg caatcctcta 8400

tacaccaaaa gttcatgggg gcaccgtgag gtcccactcc acctcagcca attccttgtt 8460

gctgccccac tcacctcctg agcctccctc tgtttcctgt ccacctttca gcttccctct 8520

caggctggga gcaggggcca gtagcagcac ccacgtccac cttctgtcta gtaatgtcca 8580

acacctccct cagtccaaac actgctctgc atccatgtgg ctcccattta tacctgaagc 8640

acttgatggg gcctcaatgt tttactagag cccacccccc tgcaactctg agaccctctg 8700

gatttgtctg tcagtgcctc actggggcgt tggataattt cttaaaaggt caagttccct 8760

cagcagcatt ctctgagcag tctgaagatg tgtgcttttc acagttcaaa tccatgtggc 8820

tgtttcaccc acctgcctgg ccttgggtta tctatcagga cctagcctag aagcaggtgt 8880

gtggcactta acacctaagc tgagtgacta actgaacact caagtggatg ccatctttgt 8940

cacttcttga ctgtgacaca agcaactcct gatgccaaag ccctgcccac ccctctcatg 9000

cccatatttg gacatggtac aggtcctcac tggccatggt ctgtgaggtc ctggtcctct 9060

ttgacttcat aattcctagg ggccactagt atctataaga ggaagagggt gctggctccc 9120

aggccacagc ccacaaaatt ccacctgctc acaggttggc tggctcgacc caggtggtgt 9180

cccctgctct gagccagctc ccggccaagc cagcggcgcg ccgccaccat gggtgctagc 9240

gagctgatca tctctggctc ctctggagga ttcctgagga acatcggcaa ggagtaccag 9300

gaggctgctg agaacttcat gagattcatg aatgaccagg gagcctacgc ccctaacacc 9360

ctgagagacc tgaggctggt gttccactcc tgggctagat ggtgccacgc tagacagctg 9420

gcctggttcc ctatctctcc tgagatggct agggagtact tccttcagct gcacgatgct 9480

gacctggcct ctaccaccat cgacaagcac tacgccatgc tgaacatgct gctgtcccac 9540

tgtggcctgc ctcctctgtc tgatgacaag tctgtgagcc tggccatgag gagaatccgg 9600

agagaggctg ccaccgagaa gggagagaga accggccagg ccatccctct gagatgggat 9660

gacctgaagc tgctggatgt gctgctgtct agatctgaga gactggtgga cctgaggaat 9720

agggccttcc tgtttgtggc ctacaacacc ctgatgagga tgtctgagat ctctaggatc 9780

agagtgggag acctggacca gaccggagac accgtgaccc tgcacatctc ccacaccaag 9840

accatcacca ccgctgctgg cctggacaaa gtgctgtcta ggaggaccac cgctgtgctg 9900

aatgactggc tggatgtgtc tggcctgaga gagcaccctg acgctgtgct gttccctcct 9960

atccaccgga gcaacaaggc taggatcacc accacccctc tgaccgcccc tgccatggag 10020

aagattttta gcgatgcctg ggtgctgctg aacaagaggg atgccacccc taacaagggc 10080

cgctaccgga cctggaccgg ccactctgct agagtgggag ctgccatcga catggctgag 10140

aagcaagtgt ccatggtgga gatcatgcag gagggcacct ggaaaaagcc tgagacactg 10200

atgagatacc tgaggagggg aggagtgtct gtgggagcca actctaggct gatggactcc 10260

gctagcggcg ccggtcctaa gaagaagagg aaagtgtgag gccacgtagg cccgcgatga 10320

ataaatgaaa gcttgcagat ctgcgactct agaggatctg cgactctaga ggatcataat 10380

cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct 10440

gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa 10500

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 10560

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgcgactc 10620

tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc 10680

ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt 10740

attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 10800

tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 10860

tggatctgcg actctagagg atcataatca gccataccac atttgtagag gttttacttg 10920

ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 10980

ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 11040

tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 11100

tatcttatca tgtctggatc cccatcaagc tgatccggaa ccgcttggct gcaggtcgtc 11160

gaaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc 11220

agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc 11280

caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc 11340

tcccctagtc aggaagttcc cccccgcccc gcagctcgcgtcgtgcagga cgtgacaaat 11400

ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg 11460

ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag 11520

gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc 11580

ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct 11640

gttctcctct tcctcatctc cgggcctttc gacctgcagc ctgttgacaa ttaatcatcg 11700

gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat gggatcggcc 11760

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 11820

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 11880

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 11940

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 12000

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 12060

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 12120

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 12180

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 12240

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 12300

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 12360

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 12420

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 12480

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 12540

gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 12600

catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 12660

tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 12720

accccccgga tctaagctct agataagtaa tgatcataat cagccatatc acatctgtag 12780

aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga 12840

atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata 12900

gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 12960

aactcatcaa tgtatcttat catgtctgga tccgggcggc cgctaacttt aaataattgg 13020

cattatttaa agttacaccc agctttcttg tacaaagtgg ttgatatctc tatagtcgca 13080

gtaggcggta cctggtaaat agaactccca ggtctccgga tactcaccac taaaacaata 13140

gcacctcatt gtttttgttt tgttttgttt tgttttttaa ttatacttta agttctaggg 13200

tacatgtaca caatgtgcag gtttgttaca taggtataca tgtgccatgt tggtgtgctg 13260

cacccattaa ctcatcattt acattaggta tatctcctaa tgctatccct cctccctccc 13320

cacaccccac gacaggcccc agtgtgtgat gttccccttc ctgtgtccaa gtgttctcat 13380

tgttcaattc ccacctacga gtgagaacac attgttttta accctcccca gcagcttctt 13440

taacccctcc atttcccttt tggcggagac ccttgtcatc actcctctct accaccagtg 13500

ctctcccctc ctgcactgtc ccaggcttcc tcattcttcc cttgcaggtc cttatccaac 13560

atggctcaca attcctggga agccacaagg aaaccaaatt ctagtagcaa ctcagagtca 13620

gcattcatta gcaggtcaca gggccataag gatgccaaga aggaatttgt ccctctcctt 13680

gtcctgacaa aaggcataag tagaagaaaa ttgttaggtt tggttatgac ctttgacatt 13740

ctctctttga catcctccct ttctgcaccc ctcttccacc tgtcagggca cctagggctc 13800

cccgggtccc cacaagtgtc tgccctaagg cccaccaggc tttcttgcag cctgtcccac 13860

atctgggtgt cactgccttc ctctgaatca ccctcctctc aaaccagccg ccagcgagcc 13920

cagcccagcc cagctagtgc cctaccacac ccgcccccag gataattaac acccagccac 13980

ccagcatcgg acctctacac ttgagcaaaa tgtatatttt aaaggcaaat tctcacgcct 14040

gtaatcccag cactttggga ggccaaggcg ggcggatcac gaggtcagga gattgagacc 14100

atcctggcta acacggtgaa accccgtctc tactaaaaat acaaaaaatt agccaggtgt 14160

ggtggcacac acctgtaatc ccagctacta gggaggctga ggcaggagaa tcacttgaac 14220

tcaggaggcg gaggttgcac tgagccgaga tcgtgccact gcactccaac ctgggcgaca 14280

aagcaagact ctgtcaaaaa acaaacaaac aaaataaagg ctaagtaact gtaatcaagt 14340

aactatttat tatttattta ttttagtgta tccacaggtt gtgcagccat caacactatc 14400

taattctata acattttcgt taccccagag aaacttgtac cccttagcaa tgactcccca 14460

tttcttcccc ccaaccaacc ccagccccag ccccagcccc tgggaatccc taatatactt 14520

tgttttgttt tttgttggtg gtgtttgaga caggatctca ctctgtcacc caggctggag 14580

cgcagtatca tgatctcagc tcactgtaac ctccacctcc cgggctcaag cgatcctccc 14640

acctcagcct ccccagtagc ttgggcctac aggtgtgcac caccacaccc agctaatttt 14700

tgtatttttt gtagagacgg ggtttcacca tgttggccag gctggtctca aactcctgag 14760

ctcaagcaat ccacccacct caacctccca aagtgctggg attacaggcg taagccactg 14820

cacctggcta ttttctttct ttcttttctt tttttttttt ttttgagacg gagtttcact 14880

ctgttgccca ggctggagtg cagtggcgtg atctcggctc actgcaactt ccacctcccg 14940

ggttcaagca attccctgcc tcacccggct attttatatt tgttgatctg ctgctggtta 15000

tgcctgtgag ccaaatttgt acaaattctt aagctttaca atcatgcgtg cctctctgta 15060

cgtatattat actttaatta aaagttatta aagtattggg gccgagcaca gtggctcacg 15120

cctgtaatcc caacactttg ggaggctgag gctggtgcat cacctgaggt caggagttta 15180

ataccagcct ggccaataca gtaaaacccc gtctctacta aaaatacaaa aaattagccg 15240

ggtgtggtgg cacacacctg taatcccagc tacttgggag gctgaggcag gagaattgct 15300

tgaacccggg aggcagaggt tgcaacgagc caaggtcacg ccattgcact ccagcctcgg 15360

caacaagagc gagactccat ctcaaaaaaa taaataaaaa ataaaaaatg tatcagggag 15420

tacaagagaa agtcatataa gctctttcta cttttcatgg tttctatgtc tgaacccatg 15480

aatcttcctt ctgaaaggtc caactttctt ctttaataac aataataatg gcaaaggcat 15540

tgggggaccg tgagcaaagg gcaggccttt gcaggggtgg gaatggaaag ggaagcacgt 15600

ccctagaggt gggcctggga tgggggttgg gggatgaagc aagtggacct tgaaggtctc 15660

cacaggtctg agtgtcctat gctccctggg cctctcttcc cccaggtctt ctcgttcgca 15720

actgcaccat cactgccaat gctgagtgtg cctgtcgcaa tggctggcag tgcagggaca 15780

aggagtgcac cgagtgtgat cctcttccaa acccttcgct gaccgctcgg tcgtctcagg 15840

ccctgagccc acaccctcag cccacccact taccttatgt cagtggtaag ttccaggcaa 15900

ctctctgtgc catcacgtgg ggtagcggtg ataccccaac cagtactccc cactcctacc 15960

cctagataag gtcagcctgt ttctgccttc ccatcccatc cagcacctct caggccttca 16020

gatgtgccct atggggtccc ctgctgctac tcattctgtc tctgtttttc cagagatgct 16080

ggaggccagg acagctgggc acatgcagac tctggctgac ttcaggcagc tgcctgcccg 16140

gactctctct acccactggc cacgtgagtt ttctccttaa tccccaccgc tagagagaat 16200

gcatacacga ggggccagga gggaagccag acagaaagct cctaggatta gggataagag 16260

gaggggaaaa agcagagtcc actgtttagg agaggagttg gccaacggtg gcgggtggga 16320

tagaataagg tgggggaaag gggagaggca aggtgacagg agggctgggc tgagggagcc 16380

aagggctaga ccctccccta acccctgtgt gtcccctcct attacagccc aaagatccct 16440

gtgcagctcc gattttattc gcatctttgt gaccttctcc agcatgtttc ttatcttcgt 16500

cctgggtgca atcttgttct tccatcaaag aagaaaccac gggccaagta agacacaggc 16560

cttccctcct gtctctcagc tgtgctaccc acacctaaag cccccacacc tactccaccc 16620

acctactctg gctcctcaag tgtccttccc ccagactccc caagcccccc agctctcagc 16680

cctccacttg ggcttttctc ctcttttctc cccttcacct ttcttctctc cactgctgac 16740

cgattgtgtt tgcacagatg aagaccggca ggcagtgcct gaagagcctt gtccttacag 16800

ctgccccagg gaagaggagg gcagtgctat ccctatccag gaggactacc ggaaacccga 16860

gcctgctttc tacccttgac cgggtgctgg tggggccctt tctgacgagg ggccatccac 16920

agagacctca atggtggcct gctcccctgt catggtcatc agaacccttt cctgtgaata 16980

ctcaacaaac tgcccttctg agaccaacag ggacaagagc aggatcccag tgtgtggccc 17040

agcaagctgg ggagactcag gctctagcta taaagcacac ttactctaag tgaaaccgtc 17100

cagctgacaa aactacatgc caggggtgga aatggctcac caagggctcc agagggggct 17160

tggcagtgag gagcacgaag tgttcctcca gaggaccaga gtttgattcc cagcactgtc 17220

aaatggatcg aaaccagctg taactctact ttcagagggt ctgacacctt tgacctcctc 17280

tggcatctac aaacacagga cggacacaca cacacacaca cacacacaca cacacacaca 17340

cacgattaaa aataataatt ttttttaaaa aaatggaatg agccaccggc aggtgccctg 17400

gtaccagaag gactgactgc cgtgagagac actatctaca gacacacaga cttcctggcc 17460

ttggttctgt cttctctgta agttgtgaca gtgctgtggg aaagctgagg ctgagacttt 17520

ctctcagttg agctaaaact ataaataaat tcatttgtct acttctatac acattgtccc 17580

ttctctctgt gcaggcaaac actggaaccc ggagccaagc cttacggccc agggactcta 17640

gtaacaagcc agctatggac actctccatg tgctattccc cttcctacag aatgagggta 17700

ggggaacaag aggaggagga ggaagaagaa aaaaaagaag ggacagtgaa ctaggaagaa 17760

ggatagtgta aatgctaact tcaaatacgc acacctacaa ctgaaacaaa caaacaaaca 17820

atccctcagt gtggccctga gcttgtaatt caaggggtta agtggtgtga caagggcggg 17880

gcttggagta gagacagcag gagatcactg ctgtgcttgt tggctaccaa acaattccag 17940

gttcagaaag agaccctgtc tctgaagaat aaggtggggt aatagagcaa gacacccagt 18000

gccctcaccg agcctccaca cacaggcagg gctgggcata ctcacccaca caagtgtata 18060

caccgcatat acacaaatac aagttaaaag tgagacttca catagctgtc cagcctccta 18120

aaattccaat cctagaacta ggccacaccc catctcgtct gttcgatcac agatctgaac 18180

agggagttgt caccagggca gacccaaagg atttaacatg gactgaaagg aagaagggtc 18240

cagagtaccg gggaatctat cttctggctc caccccctct ccagccctct catagttggg 18300

gctgatggga aagctgatgt ggtgatacca ggttttgaaa tcacctgggg atttcttggg 18360

tgcggggtgg accttaggga gaacagaaag cagagctggc tgcagccatt actggcctcg 18420

ggcgggcggc cacagaggca gttgaagtga aagtgaaaga gaaacgataa gagaacggag 18480

accacaggtg ctaagtgagg gtgctcacag aaccccctct tcagccagag atcactagca 18540

ggggaactgt ggagaaggca gccagcaagg aagagcctga gagtagcctc catgggcttg 18600

gagcccagct ggtatctgct gctctgtttg gctgtctctg gggcagcagg gactggtgag 18660

tgcggagacc aggctggggc ctgggggact gggggggagg aagcgccaag ctaaaggagc 18720

cccaggaagc caccttctca gaacagcacc agcttgtctg ctaagcccag caaatgagtg 18780

cggcactcac aacatgcgga accagtttgg agaggggtac ggcagagaac tcacagaaaa 18840

ggggggcttc ctcgatggga aggcagggtg ttagcattct gtgggacagc tcactgccct 18900

gagttgaatc cttagaagag gaaggggctc cccaggctaa gaaatcattg ggttggtgac 18960

tcctggtggg cgtggctatg gcagaaagca gaggcctctg ggaaagttgg gtgatgagaa 19020

aaaggttgtc aatgtcccta ggaacagcag tgtcttggct cctctcccct gtggcagtat 19080

ttcttattcc tttttttttt tttttttttt aacctgagaa ttcctttcca gaaattccct 19140

tgcatcccac agctgagact cagaaataca aagcataaac ccctctggct ttgatcagga 19200

cagatctagg acttgaaata actcacagag ccgctgcctg agtagcaggt gcctcagcag 19260

aagctgggtt tatagatgac accaggacag cggggctcag aaggcacctt tacctgctgc 19320

tcagcccggg gcttggtgaa ggggtgggaa gagtgtggag gacgagggtg cttgttccac 19380

tttccctgca gaccctccca cagcgcccac cacagcagaa agacagcggc agcccacgga 19440

catcatctta gactgcttct tggtgacaga agacaggcac cgcggggctt ttgccagcag 19500

tggggacagg gagagggcct tgcttgtgct gaagcaggta ccagtgctgg atgatggctc 19560

cctggaaggc atcacagatt tccaggggag cactgagacc aaacaggatt cacctgttat 19620

ctttgaggcc tcaggtaaaa tctccccatc ctaggcttcc tccacaggaa cagccccgtt 19680

cccagttcta tctacccacg cctgcccccc cccccccccg gtctggcgtt gtccatccaa 19740

cattacttct gtatgctgcc cttctcccca gtggacttgg tacagattcc ccaggcagag 19800

gcgttgctcc atgctgactg cagcgggaag gcagtgacct gcgagatctc caagtatttc 19860

ctccaggcca gacaagaggc cacttttgag aaagcacatt ggttcatcag caacatgcag 19920

gtttctagag gtggccccag tgtctccatg gtgatgaaga ctctaagaga tgctgaagtt 19980

ggagctgtccggcaccctac actgaaccta cctctgagtg cccagggcac agtgaagact 20040

caaggtgaga aaacaaaatg cagtggcaca ggaacatttt gtattattgt gtgtgtgtgt 20100

gtgtgtgcgc gtccatgcgc acatacgtgt gcacatgcca cagcacatag gtagagatca 20160

gaggacagct tatagaacct accatgtggt ttccagggat tgaactagtt cactatgctt 20220

ggcagcaagc actttatgca ctatgccatc tcaatagccc caagatagtt ttttggtttt 20280

ttggttttgg tttttttttt tttttggggg gggggttgtt gtttttttaa tttgtttttt 20340

gtttgtttgg ttggtttgtt ggttggtatt ttgttttctt ttgtggttag agatgggata 20400

gaaggaagaa aacaaatatc ccacaggtcc tcctgaggta cccatagagc ttcatctttt 20460

ctttctaaaa tatcctgagt caagaagagg ggatgctagg agatccttat tcgagcaagc 20520

atcaaaacat aataatcatc aggtattttc tccaaagtgg ggaagcaaca catgaggaca 20580

ctgtagctac acatgtcact acactcttat ttagccaaac tcatgtttcc tcccagggag 20640

agggagtctg gtctggtgct tcatggcaat tacagcagag cacagggaga aagaagtgct 20700

cagtagctct tactagtgag cccattcctc attctctccc acttagtacc tcctaccagg 20760

ttcaaaacca agaggaaaca tattgacagg cacctggaga gtccagttca ttattttgcc 20820

tccaaaaagg agaaaatcaa gtccacagaa gatgacgatg agggggcccc tctcctatcc 20880

ctgcaaaccc ctaaaacatt agcctttagt attatgttgt cccatactga ggatagaacc 20940

tagagccttg tgctaaacac gagctctact actaaactac agcttcaatt cctttttcag 21000

ttttattgtg aaacaggggt ctcacccaga tgccctggca ggcctggaaa cttgtgattc 21060

tgtcatgcct ccttagtagc tgatggtaca ggcatatact accacaccct ccggctctac 21120

agtcctttct gaatgtgcct tgccacatca gaaaagatgt tcagcatctc ctatccttgg 21180

ccatgggtag tttagtgctt ctcccttttt gaacttattc agatctacaa ataaatgaaa 21240

caacataccc ccacccagcc gtgacttatt tctgacctat ttcgtcagct gtcatagttc 21300

ctctctaccc gactaacata gatatcctcc cctggactga ctaggctgta gggaccacaa 21360

cacttcaccc ccaaatgctc ccatgtgcag ttagggctga tagctgaaag tcaaggccac 21420

tctgcttcac aataaccaag gatcccaaaa gcctatcaag gctggggagt gctctactca 21480

cagtaacaag ggtccagtgc acacaggact taacagtatt tcattcatgg aaagacgtga 21540

ctattgaagc tggacgtact gtccacatct gcagtccttg cacttggagg gactgaacca 21600

cgaagattgt gggtttaggc tctcatgtgt gccttgctgc accattaatt tatggctcca 21660

ctggacagga aatgaactca gtaccactgt ccagggaagg gagccggagc tgtctgtcca 21720

tggctcagag gccaggtgac cttaaggaag aaacagtctg taagtcaccc tttctttctc 21780

tctacagtgg agttccaggt gacatcagag acccaaaccc tgaaccacct gctggggtcc 21840

tctgtctccc tgcactgcag tttc 21864

<210>23

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

gcagactcaa gtaccagtga aggg 24

<210>24

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

tagtgcctct ctgggcagct ctt 23

<210>25

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

cctattacag cccaaagatc cct 23

<210>26

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

agcactcccc agccttgata g 21

<210>27

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

catgaggtag ctggtaggac ct 22

<210>28

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

ctttgttgag gggctcagag gat 23

<210>29

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

ccaaatgctc ccatgtgcag t 21

<210>30

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

acctggaact ccactgtaga gag 23

<210>31

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

tcttatcatg tctggatccg gg 22

<210>32

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

tgtcaaagag agaatgtcaa aggtc 25

<210>33

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

cagtgactcg gtacaagcag ttg 23

<210>34

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

gtcacagtccttcacgagga atg 23

<210>35

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

gaagatgggt gggctgaatg ag 22

<210>36

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

gggacaaatt ccttcttggc atc 23

<210>37

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

aactgcacag tcactgccaa tg 22

<210>38

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

gaaacagaca gagcgagtca caag 24

<210>39

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

caagugugua agacga 16

<210>40

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

ucuggucagu cagcgug 17

<210>41

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

ggacacagaa agatgccttg tt 22

<210>42

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

actaaggact tcccctgggc t 21

<210>43

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

caggaggttc ccctaccttt 20

<210>44

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

cttggttccc catctccaac 20

<210>45

<211>15858

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

gtgaggaccc tatccatgtg tgtaatatct caagttctgt gtgtctccat tgctcagtga 60

aggaagaatg tgggcacacg tccggtagat aagggggtgc tgtatcatgc caggggcaca 120

tggtaacatg aagcttacct gaagctacat ggatgggatg gtttagccca gaatccctgc 180

agaggctggc ccttttcctg ggctatccaa gggccctcaa cattgctgct ccttcagggg 240

acccatctca gtcacctgta ctgcctgact tgggctagac ccttccggtt ccagaatcac 300

tggctctaga gactcacatg catgtccctg tacggacatc tcgatataaa caagcatggg 360

tgtccaccaa cctaaaaatg taggggaatc caagcttccg aaggcctgga atgtgtccgc 420

catcactttg tgctgaggca gctgggaccg tcttcctttc gctgcattgc actgcactgt 480

tgctcaatgg atctccaggt tccctgcagc tctgctcagt agctgccttc ccagggcatg 540

gctaccttcc tcctctctgc ttgccagccc agatccatcc ctacccatcc ctgcccacgc 600

cacaagaaag agatgacaac tctgtttgta gaacaaagaa cactggagtc aggggtggga 660

actttgaaca tttcacatca cgggaccatt cataaaaggc tgttctgcaa cctcggtggc 720

cccacaatgg ggtctgagct ggatcatgag atgcttgact cctctgcagg gcatatgaga 780

cagctttctc tcctggttct gaaatggact cctggcatca tgcagagtga catggaccgg 840

gaaactgcct atggcttctt catgctgggt agcctgtaca taatgtaggg tcgagcctcg 900

tgtgtagact gctggggacc caggcaactg tggcttgcag gtcctgctgc tggtatgagg 960

gcagtgttgg cttccagctt gttctgggct tgtgctatag gcctctacat ggaccagtta 1020

aaactggact ggacatggaa ttggtgcctg aggcagccag tacagaaggc cctaatgaag 1080

caaggctgtc aggactggct ggggctgaag actgggacat taaaacctga cccttcagaa 1140

agaaaaagaa acattctgca aggtttcctt ggatttaaaa gtcatcatga gggctgagag 1200

ctttaggagc cttcctggaa ctgggtgatg ctcccagagg cagcagcctc acactgaaag 1260

gtagctagct aggagctagc ttagttaagg gatcctataa gaagagttgt gctgagggag 1320

agttgaagaa aagtaggaaa ttcccaaaat tgagcttctt ggaagctagt tgggaatagt 1380

actcctgccc cttggcaact agcatctcct accccgcccc tccccatggg aggcagcagc 1440

ctctcacccc atgagagttg gtacacagtc actctgttgt cacaacaact gtcatttaag 1500

caaataatct aggtgcttgc gaatggttcc ctctagcaca ggaactcata cctgcttcct 1560

aagactgtga tcatgaccca aggatccgtc gttacaagat ggttaaagag gaatttagac 1620

acagtgcacc ccagggaata ggggtggctg tttctgtgtc ctttgctgaa ctcattgtgc 1680

tgctaagtat gcaaagatgg cagtaagcag aagggctgcg cgctgccggt cccttctctc 1740

tgtgattgag gtctagccaa cctcaagaag ctatagccat gtttactttc aaacgctacc 1800

tcagagtttc caagctatgt gacagccaca gcccaaacat tctcagggca ggcccgggca 1860

gtgtctgggt ggggtctggg agtcacagga ggggcccaga gccaagcgga gcctgaggtg 1920

gagggaggga gggctgggtg gggctggacc tgtgtacaca accacaccta ctatcgggga 1980

gtagctgtgt ccagtcactg agcagaggcc aactagtgtt tcttcatctc cctgggaaaa 2040

gcagcagaca ggatggcctg gtggaaagcc tgggtaagtt gaaatggtct ggggagatgg 2100

gtccctagtc cttgttggga gtgtggagga cacaaaaagt aaagctgaac ctgcgaggcc 2160

aagtcccggt gaggaaactg ggagaaaaac cctgtccacg cttagaaaag ttaaggtttg 2220

aaaggtaaga cagttaaaaa taaaacaaga acattttttt ttttaatttt taactgcaaa 2280

gagaaagcta aaggattcga gtgtccagag ggagacttct gtaggggaaa agctacttta 2340

gtgcctctgt ggatatgtgc aatgtgtgag tttgtgcttg gagagactct agcaggctgc 2400

tccttgcaga agtagcagtg tgtgaaaggc gctagggggc ccaggaagtt gactttggaa 2460

aacattgccc tgtttgagca tttgaatgtt cagatgggtt ggatctaggt ctggagagga 2520

tcccggatgg aaggaacatc gagggacttg aatgacaggc taaggggttg gagctcgact 2580

tccaaacagt acggagccac ctctacccac atgtacagcc gaaacacctc atgggcaggc 2640

acacggctac atgtgctatg caccttcctc gtcccagctg gggcactcaa agagggaaaa 2700

gctgaggttc tgggacacca ggtagcttgc tcaggtccct cggacagctt agactagaga 2760

aagcccaaag tctctgcaca gtctccgggc tccttatagg cagaagatcc aagttggccc 2820

agcttcagga aaatctttgt tgctgtgcca ccacccagag agaagctgta ggtgtagatg 2880

ccccgggaag gggggggggg cgtctctgga cagataggac agactagaag gcaggaagag 2940

atgagctatc atcctgaccc tcaggagaaa gcagcccgag cagtggctgt gcatagcttc 3000

acagcagctg ctggcaatac gccttgtgca ggtgcacaca gcagggctct gagtgttggg 3060

cagttcctgc agtcagatgc tggccaaaat agcctgattc taggccctgc actccatagg 3120

gcaggggcag gggccatcct cttggggacc tgattggatt ggttggtact ctcagaacct 3180

tctgtatgga tggggacact cctgtcggca tctctcacca ctgtcactgg aacctcacag 3240

caagtctgta ggatgggtgg ctttacacac aagggtggaa atggagactc cagtaccaca 3300

gctgccacac tagtaagggt ttaggtctgg gcagcctggg tccggatgca ctcagtcatc 3360

cagcatctct gacccttggg agagcagact cagggcagca aggcttctgt atctgtccag 3420

gctgccagtg aggaccaaat gtcccctgag tacaaagttg gattaaatac aggactggag 3480

aggtcaaaag agtgacagag agtgcctgtc ttcaaaacag gttccccctt tgtggtgaag 3540

agatcagaca aaagtatgcc agatcctgca tgcccatgac agacggtgtt ctagacatag 3600

catccaaaat tgcctgaatt gcctttttcc tgggtgtgtg atggtctgga atcactggcc 3660

tcagggtagc tccatcagaa gcctgaggct gacatggaag ctgggataga tgcctcctaa 3720

tctccactct aggcagtgtc ccatccttgg cacccagaac gctgatctct ttctatagaa 3780

ccttagcaag ggaagagaga caggcttctc aatgtggtga tatctgagcc acccctggag 3840

ctgctgacaa atacaggtcc cagaatggta tgggggacac tcctcaatga ctaagtttct 3900

gacacttctt tgggtcaggc agagaaacta ggaagtgaga acttaaatgc tgcccacccc 3960

acacccctca gaaaacccac atgtctatgg gtgaggctct ctgcagtgaa acattccaat 4020

aaaacccagt gtctggaaaa gtgtgaatca tctgaagagt gggggaggta gacagagctc 4080

aggttgtgaa ccaggctgtg ggtgaagtgc cagcttacta gccaagccac caagcccagg 4140

ctcctaatac gactgctgga gaaggtggca ggctgagcaa catgcttaac gcactttaca 4200

gagaatgctc aagtagactt ctagtagtag cagtgtgtga cctcagaagc aaggagtgtg 4260

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tggcagcatt tggctcactg gagaaacttt 4320

ccctaggaat tatttctaac agttcctgtt ataaagcatg gtgcaataaa tgggaaggga 4380

aactctattt ttggacacgt gaaaatcaac caaaggacac agaaagatgc cttgttcctt 4440

tctcagaagc aggaccctta tggaggtggg gacctgttgt gtatttccag ggtctagcct 4500

gaggcttgag gggacagaca atgagtgtag ggagaaaagg aaagataggt ggagggtgct 4560

tgagaagaaa ctaagagaaa aggaggcaaa cgggggagga gaggagacgg aaagaaggaa 4620

gccggacgaa ggggaagggc aggcaggtgg gtgtaaggac cctgcagcct gggtagagaa 4680

ttggtgaatg gttgggcgaa cgtgtgaggt ggaaagcagg tgggtggatg gggaacaaca 4740

gaagagctgt gtgagcaggt gggtgaacga ccagaggggt tcgactaaac tctccatctg 4800

aaggaaccag gtctcccctc aaccctaccc ctagttcccc ccactcttca ccacccctcc 4860

ccctaaaaca cacagggtcc tgcatcctaa gcatctgata aattatgcgg agcacacaga 4920

taagtggcct ttacactggc cccgcctact gcgactatag agatatcaac cactttgtac 4980

aagaaagctg ggtgtcgaca agcttgatat aacttcgtat agcatacatt atacgaagtt 5040

attaacttta aataatgcca attatttaaa gttactcgac gcggtacccc cggatccaga 5100

catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg 5160

ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa 5220

acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga 5280

ggttttttaa agcaagtaaa acctctacag atgtgatatg gctgattatg atcattactt 5340

atctagagct tagatccggg gggtgggcga agaactccag catgagatcc ccgcgctgga 5400

ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg 5460

cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc 5520

ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc 5580

gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc 5640

agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc 5700

acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc 5760

gccatgggtc acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag 5820

ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc 5880

ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt 5940

agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc 6000

aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc 6060

ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag 6120

ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt 6180

gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc 6240

gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc 6300

tgcgtgcaat ccatcttgtt caatggccga tcccatggtt tagttcctca ccttgtcgta 6360

ttatactatg ccgatatact atgccgatga ttaattgtca acaggctgca ggtcgaaagg 6420

cccggagatg aggaagagga gaacagcgcg gcagacgtgc gcttttgaag cgtgcagaat 6480

gccgggcctc cggaggacct tcgggcgccc gccccgcccc tgagcccgcc cctgagcccg 6540

cccccggacc caccccttcc cagcctctga gcccagaaag cgaaggagca aagctgctat 6600

tggccgctgc cccaaaggcc tacccgcttc cattgctcag cggtgctgtc catctgcacg 6660

agactagtga gacgtgctac ttccatttgt cacgtcctgc acgacgcgag ctgcggggcg 6720

ggggggaact tcctgactag gggaggagta gaaggtggcg cgaaggggcc accaaagaac 6780

ggagccggtt ggcgcctacc ggtggatgtg gaatgtgtgc gaggccagag gccacttgtg 6840

tagcgccaag tgcccagcgg ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc 6900

gcctccccta cccggtagaa tttcgacgac ctgcagccaa gcggttccgg atcagcttga 6960

tggggatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 7020

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 7080

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 7140

ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta 7200

tgatcctcta gagtcgcaga tccagacatg ataagataca ttgatgagtt tggacaaacc 7260

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 7320

tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 7380

tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 7440

ggtatggctg attatgatcc tctagagtcg cagatccaga catgataaga tacattgatg 7500

agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg 7560

atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 7620

gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa 7680

acctctacaa atgtggtatg gctgattatg atcctctaga gtcgcagatc ctctagagtc 7740

gcagatctgc aagctttcat ttattcatcg cgggcctacg tggcctcaca ctttcctctt 7800

cttcttagga ccggcgccgc tagcggagtc catcagccta gagttggctc ccacagacac 7860

tcctcccctc ctcaggtatc tcatcagtgt ctcaggcttt ttccaggtgc cctcctgcat 7920

gatctccacc atggacactt gcttctcagc catgtcgatg gcagctccca ctctagcaga 7980

gtggccggtc caggtccggt agcggccctt gttaggggtg gcatccctct tgttcagcag 8040

cacccaggca tcgctaaaaa tcttctccat ggcaggggcg gtcagagggg tggtggtgat 8100

cctagccttg ttgctccggt ggataggagg gaacagcaca gcgtcagggt gctctctcag 8160

gccagacaca tccagccagt cattcagcac agcggtggtc ctcctagaca gcactttgtc 8220

caggccagca gcggtggtga tggtcttggt gtgggagatg tgcagggtca cggtgtctcc 8280

ggtctggtcc aggtctccca ctctgatcct agagatctca gacatcctca tcagggtgtt 8340

gtaggccaca aacaggaagg ccctattcct caggtccacc agtctctcag atctagacag 8400

cagcacatcc agcagcttca ggtcatccca tctcagaggg atggcctggc cggttctctc 8460

tcccttctcg gtggcagcct ctctccggat tctcctcatg gccaggctca cagacttgtc 8520

atcagacaga ggaggcaggc cacagtggga cagcagcatg ttcagcatgg cgtagtgctt 8580

gtcgatggtg gtagaggcca ggtcagcatc gtgcagctga aggaagtact ccctagccat 8640

ctcaggagag atagggaacc aggccagctg tctagcgtgg caccatctag cccaggagtg 8700

gaacaccagc ctcaggtctc tcagggtgtt aggggcgtag gctccctggt cattcatgaa 8760

tctcatgaag ttctcagcag cctcctggta ctccttgccg atgttcctca ggaatcctcc 8820

agaggagcca gagatgatca gctcgctagc acccatggtg gcggcgcgcc gctggcttgg 8880

ccgggagctg gctcagagca ggggacacca cctgggtcga gccagccaac ctgtgagcag 8940

gtggaatttt gtgggctgtg gcctgggagc cagcaccctc ttcctcttat agatactagt 9000

ggcccctagg aattatgaag tcaaagagga ccaggacctc acagaccatg gccagtgagg 9060

acctgtacca tgtccaaata tgggcatgag aggggtgggc agggctttgg catcaggagt 9120

tgcttgtgtc acagtcaaga agtgacaaag atggcatcca cttgagtgtt cagttagtca 9180

ctcagcttag gtgttaagtg ccacacacct gcttctaggc taggtcctga tagataaccc 9240

aaggccaggc aggtgggtga aacagccaca tggatttgaa ctgtgaaaag cacacatctt 9300

cagactgctc agagaatgct gctgagggaa cttgaccttt taagaaatta tccaacgccc 9360

cagtgaggca ctgacagaca aatccagagg gtctcagagt tgcagggggg tgggctctag 9420

taaaacattg aggccccatc aagtgcttca ggtataaatg ggagccacat ggatgcagag 9480

cagtgtttgg actgagggag gtgttggaca ttactagaca gaaggtggac gtgggtgctg 9540

ctactggccc ctgctcccag cctgagaggg aagctgaaag gtggacagga aacagaggga 9600

ggctcaggag gtgagtgggg cagcaacaag gaattggctg aggtggagtg ggacctcacg 9660

gtgcccccat gaacttttgg tgtatagagg attgcagttc atattagact tggcatctgg 9720

tcaagagaag tagtatagga tgaagaggct ttgttgtgct ttctgccaac cagagaggcc 9780

caccatgtcc tgaggataca caggtaggca gaagggtgat aggcaccttt tttttggggg 9840

ggttaattaa taactttaaa taatgccaat tatttaaagt taatcacgcg tagatctgca 9900

tgcgttccgg cgccagcctg cttttttgta caaacttgtt gatatcgtgg tatcgttatg 9960

cgccttaata ttctggattg caagtgtgta agacgagggt ctgggtactt tggttatcaa 10020

gtttattcca taatccatag aatatccatc ctacagttct ctctaaactt aaccctctgc 10080

ccaatacgat cccatggtct tgcaaaccac ctgagctttg gggatgctca gcgtgtctcc 10140

tggccctctg aaatagccac acctcaccac aggaaagacc agaataggct tagaaccttc 10200

ctcacaagaa aaaaaaaatg tccagatgcc acatccagac acaaagccac tccttgtgag 10260

tttatttcca cccattctgc tgtacacggt gcggatgaga actgtgtgtg accagtccgc 10320

tatctgccct cagccaacag gtaatttctt tttataagct ctggtcccag ctgcaccagt 10380

ctattagttg gagcacccag tcacaccctg caaggctcca tctggtctca gagcccaggg 10440

gaagtcctta gtttaacaaa gtagactaca tggtaagcca ggaggttccc ctacctttcc 10500

tgtattttcc atcatgagca gacctgtttg tcttgcaggt tgaacaggag ggcgtcagcg 10560

tgaagggtag ttctcatttc aacccagacc ctgatgcaga gaccctctac aaagccatga 10620

aggggattgg tgagttacca cccatggcct ggccgctggg gcctcacatc ccacatcctc 10680

agaaagtgga tgccagggac agtcaggcat agacggggcc cccattttac taagtgactt 10740

agttacttac agcatcacac cggctctgag ttgagtggct gttccttccc aagtgacaat 10800

gacagggaag gccatcatta atctgaagtt tctcacttgt aaaacggctg ccaggactct 10860

ggactcgata gttctcttct gcttcataaa cacacacttc ccaatccatc aggggaagat 10920

ggattggcac caggcccact gtgggcatgg gatgggagtt taggtgtggc actgaggcca 10980

gggcgatcag ccacatggat cctgcagctt gtcctctacc ccacgctgac tgaccagaca 11040

gtcagctgct tcccgctcta gaactagtgg atcccctcga gggacctaat aacttcgtat 11100

agcatacatt atacgaagtt atattaaggg ttattgaata tgatcggaat tgggctgccc 11160

atacctcaca tgagcgtgag tgggaaggga ccggtttacc ctaggatttg gtctcgctcc 11220

aggtaggagt catctctcag ctggctctgc ccagtgagtc aggacactaa agagaactgc 11280

cgtgcccaaa ggggcctaga ctgcatcctc atacccactg tttccatgaa aactttactg 11340

ttatccccac ttctaatata agcaaatgca ggctccagga aatgagcctt ctcgtccatg 11400

acatacaagg cagactctca gacccaatgg tctagaattc gctgtgatat tcctctaatg 11460

tgaccaacac aagggaaaga cttaaaatat ctcccttggg catgcttcaa aggtccacag 11520

ttgttgtcca aagctccctg cccttggttg ctatgctatg aagtcaagcc tgagccatac 11580

ccaacactat gtaagttgga gatggggaac caagaggtgc actacctgga tgccttgttt 11640

acaccatgga aattggacgt ttgcagctcg ccccccttca gtcttttctg atgccccccc 11700

ccagaggtag aagggattct catactgtac actcctacag gaaccaatga gcaggccatc 11760

atagacgtgc tcaccaagag gagcaatgtg cagaggcagc agattgccaa gtccttcaag 11820

gctcaatttg gaaaggtaag caggttgggt gccaaagacc aggcagttcc tcccagaatg 11880

gaaatgtgta catagggcat attgcattaa aacaatactt ccattcatct gtggtcaagt 11940

ttctctggct gttctggttt tttgtttgtt tgtttgtttg tttgcttttt tggttttttg 12000

gttttttggt ttttttttgt ttttttgttt ttttttctgc aagagccatt tatagctccc 12060

aagccccagc atgacattag ccttgcatgg tctagcccca tttcttccct gttctctctc 12120

cagctttgct gtcccactac ctgaactgac cttccatatc cctgctccct ggcaacacct 12180

ggctcccttc tgtttcacag cacaggcaca tgcttctcac aggggagtta cttgtgtgtt 12240

cctctcctgc ctgtggatac tctaggggca ctttggctgc tcccagccct tagcacagaa 12300

ccagaggcgc acagtgaatg ccagaggttt gtgcataggc atgtgggtgc ctagagtctc 12360

cccatttgct cacagattta acacaaaagc atcagtgatg aagtttagtg aagccatatg 12420

taactccttt cccctccaag ctacacatta tcctccgagt tcaacatcag aaaggttgag 12480

taatttccta aggctccaga gctcttaagt gacagtctga cagccctgta caaatcagct 12540

gggtgtgacc tagtggcttc ttgtagttgt gtgtgccctc aggaagagtc aggttgagac 12600

agaatcttgg aatctctttc tttctttccc ttcctcctct ctctccaacc ccctctttcc 12660

ctctcctcac tgaatcatgt aataagttaa gcatggagct gccaccctcc gatcttgcag 12720

tcccgaaagc aatggcaatc ctattttcct gagtttagaa tgtgcccagt actggactgt 12780

gtgctgagct gtcacatgtc tgtgtcatca caccaacagg acagagtcca tgtgggaagg 12840

aagaaagagt ccccaggcca gccttaacca cctgcctgca ctgcagtggg cttagaagat 12900

aagtcatctc gacctttctt tctgcctgtc ttaggacctc actgagacct taaagtcaga 12960

gctgagtggc aaatttgaga gactcatcgt ggccctcatg tacccgccat acagctacga 13020

ggccaaggag ctgcacgatg ccatgaaggt actccacttt gccataaatc agagtggcca 13080

cctgggcctg actgacctaa tgagcttgca acctgctcca acagccgccc agcagtgggg 13140

ctgaccagtc acaataaagg tctagatcag tacttcttag aatagggttg gaccagcagg 13200

agtgcagggg aatcggaaat ccatctctgc agtctgagca ctgccccttg gccatccttt 13260

gcttcatcat tcctgttact ctcacagatt aggtggtact ggagacccag aagccacaac 13320

atccccacta aaaggatact cagtacagtc tatgccaggc attttacagc tgtgacctct 13380

aaggccacct tcagggtagg cttgcctcat tatcctcact aacagatggc agtgcttaag 13440

ggtgggaagg tggagccacc taggtgagac cacccagcta aggaaagaga gctgaggtag 13500

cttacagcat tagctgtcct aagaaacaat ggtggggatt caatatcacc ctccaacaga 13560

agagggcaga agaggcagtg gccatagctt cttagaagat agggcaggcg gagcaggaga 13620

ccaagggcag agcactccag aacacagagg tctctgtccc cagggcttag gaaccaaaga 13680

aggagtcatc attgagatcc tagcttctcg gaccaagaac cagctgagag agataatgaa 13740

ggcatatgag gaaggtaagg ggttgcaaag atggtggggt ggaaacctag acatggccta 13800

cgttttggca acctgatagg gaaaaggagt caggcagttc ccaagggaac tcaaccagag 13860

tggaaaggag cctttggaag gtcttctgag ataaagtcca actagtgccc acgggtgcca 13920

aatcctgtac ccaacagtaa ttgagcatgg ctcagacatg gggagagtga tgtgtgccca 13980

gatgagctca tgtcccttaa tgctgtggca gtgctgggga cgggggcagc agtgggcaag 14040

agtacttgtt ggctgcacat gggttgaccc agaacactag agagggtcat ctgccagggc 14100

cccattatgc taggggagcc ccactggctt actctcctat atagtgccta gccttccagg 14160

caagcaaagg ccagccagtc ctccatgttg tgcttctggc caagggagtc taaaggctct 14220

ggctttgctc aacttggcaa tgaaatagtc tcagtctgca ggctctggtc tggctctctg 14280

gcctgatcta tttctctctc tagcttggtt gcacatttat gtcaacatct gaatgattac 14340

ctagctagat gtctgccaat ttggacatct aaatggggtg tacaccttct gtaccaagaa 14400

cttgtccgcc cgttgggaga agtgtgggaa gcaggtctgg aggaaggaag tggaccctga 14460

caggccctgg gtcactcact gtccattacc tctggttctg tttttctgca gactatggat 14520

ctaccctgga agaagacatc caaggagaca cgagtggcta tctggagcgg attctggtgt 14580

gtctcctgca ggtaacacaa tccagattcc agaactctgt ggcttaaaga tccaaagaga 14640

gccatcccag cacagccctt gagtcatgac tcccagtgag ggctgggcta tggggaaacc 14700

cagcgatctg gaagcaccat ctgtgtgtca gcacaaaagg agggtcctcc ctgagagcct 14760

catgcctctc tttccttatc ctagtgggct ctgctcacta gtgtggctca ctgaatccac 14820

cattccaatg aagtagctca tctactttcg tggtacaaat gccaacagtg aggcatggtg 14880

aggatggctg ctggcttgca ggtctcatgg atcagaagca atagtctttt cctacctgcc 14940

ctctgtaccc cactgtcccc tcagcccagt gttctgagtc ctgacctgcc ttacagagtg 15000

attttctctg atcctagggc agcagagatg atgtgagcgg ctttgtggat ccaggactgg 15060

tcctccaaga tgcacaggta aaacggtacc ttgaccttac cacctgaact ctgagcttta 15120

tgatcccctt tgccaaggag agctttcttt aatatagacc cgtctctgca gcctccacca 15180

tgtgttgact aaggaggaaa gcaggatatc tgggaaactc aatggaagaa aaggaaaaat 15240

ggagaagctg ggaagggagg agctaaggct gtcaggcaga gcgagcctcc ctgagcccag 15300

agcttgctca tgtgcacatc tctgtgtatc tattggtaga gtgcttgtta ctcagtgctc 15360

ccagagacaa tgggggaagg agaattttct aaagaagctg atggcccagg acagaggaga 15420

gccaacacta ttagaggaac aggagttcag gacagtgagc caccatgtta ggctcactga 15480

tggtgagcta gaattctctc tcacctccac ctcctcctgt cccaattcta ggccctgcat 15540

gaagcgggtg agaagattat gggaaccgat gagatgaagt tcatcactat cctgtgcaca 15600

cgcagtgcca ctcacctgat gagaggtaca gggcagaagt gggacacgca ggggtgtgtg 15660

gctgccctag ccacgtggcc tctgcctttg tgtggaaggt ctgatgtgta gtcttaagca 15720

gcttagactt ggggagagag ccatggagac cttgactctt ccccatgtgc caagatggag 15780

gcagaaatgt gctcatactg cactcaggtg cagcgcgtaa tctacgtatc ccttacgtgg 15840

atgcttctct ccatagtg 15858

<210>46

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

cctaagtaaa cttctgccag cctc 24

<210>47

<211>24

<212>DNA

<213> Artificial sequence (artificacial sequence)

<400>47

cacccagctt tcttgtacaa agtg 24

<210>48

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

atgggatggg agtttaggtg tg 22

<210>49

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

aaacagtggg tatgaggatg cag 23

<210>50

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

gagtgttacc tcagtgactc caggc 25

<210>51

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

catagggata gagggaggag gt 22

<210>52

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

gtgggttcat tgacatcact tg 22

<210>53

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

gtgtgtgatg atgggtcagg 20

<210>54

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

ccatgtccat agcacatctt gg 22

<210>55

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

gagggtgact gaggctttaa g 21

<210>56

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

acacacactt cccaatccat cag 23

<210>57

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

gtgggtatga ggatgcagtc tagg 24

<210>58

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

tctccatctg aaggaaccag gtc 23

<210>59

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

tcttatcatg tctggatccg gg 22

<210>60

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

tctccatctg aaggaaccag gtc 23

<210>61

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

atcgtattgg gcagagggtt aag 23

Claims

1. A method for gene targeting by using a CRISPR/Cas9 system is characterized in that based on the CRISPR/Cas9 system, a mutant of Cas9 with DNA single-strand cutting capacity is selected, a tracrRNA/crRNA binary complex or an sgRNA is used for each targeting site, and the mutant of Cas9 recognizes conserved spacer adjacent motifs near the targeting site to generate DNA single-strand cuts without generating DNA double-strand breaks and induce single-strand cut homologous recombination repair.

2. The method of claim 1, wherein said gene targeting comprises gene knock-out, gene knock-in.

3. The method of claim 1, characterized in that the mutant of Cas9 is selected from Cas9n D10A and/or H840A.

4. The method according to claim 1, wherein at least 2 targeting sites are designed for each target gene, and each targeting site is spaced by not less than 100 bp.

5. A method for preparing gene targeting ES cells by using a CRISPR/Cas9 system is characterized by comprising the following steps:

(2) preparing a mutant of Cas9 or an expression vector thereof, designing and preparing sgRNA or tracrRNA/crRNA binary complex or respective expression vectors according to a target gene and a targeting site by using the method of any one of claims 1 to 4;

(3) transfecting the mutant of Cas9, sgRNA or tracrRNA/crRNA binary complex prepared in the step (1), or respective expression vectors or co-expression vectors into ES cells, selecting and simultaneously transfecting homologous DNA templates according to the type of gene targeting to delete bases of a targeting site or insert exogenous DNA into a genome;

6. The method according to claim 5, wherein the step (2) further comprises detecting the cleavage efficiency of the sgRNA or the binary complex of crRNA and tracrRNA produced.

7. The method of claim 6, wherein the detection comprises endonuclease detection method, SSA reporter vector detection method, Sanger sequencing method, Digenome-Seq technique.

8. Method according to claim 7, characterized in that said endonuclease is selected from the group consisting of SURVEYOR enzymes, T7EN1 enzymes.

9. The method of claim 6, wherein a wild-type Cas9 protein, sgRNA or tracrRNA/crRNA binary complex, or respective expression vectors or co-expression vectors are transfected into ES cells, cell genomes are extracted, target cutting regions are amplified by specific primers, the obtained PCR products are subjected to T7EN1 enzyme digestion, electrophoresis detection is performed after enzyme digestion, the detection result shows that one or more bands are more than those of a control group and the PCR products are regarded as effective cutting, or Sanger sequencing is performed on the obtained PCR products, and the comparison of the sequencing result shows that double peaks appear in the sgRNA regions and the sgRNA regions are regarded as effective cutting; and screening high-efficiency sgRNA or crRNA and tracrRNA.

10. The method according to any one of claims 5 to 9, wherein the method of transfecting ES cells comprises viral transfection, lipofection, or electrotransfection.

11. A method for preparing a gene targeting animal model by using a CRISPR/Cas9 mediated ES targeting technology is characterized in that a gene targeting positive ES cell prepared by the method of any one of claims 5 to 10 is injected into an animal blastocyst, the blastocyst is respectively transplanted into a surrogate mother body for breeding, the produced offspring is an F0 generation chimeric animal, the F1 generation is obtained by breeding with a background wild animal after the F0 generation is sexually mature, and the positive F1 generation animal identified by the gene is the gene targeting animal model.

12. The method of claim 11, wherein the animal model of gene targeting is a mammal.

13. The method of claim 12, wherein the gene targeting animal model is selected from the group consisting of mouse and rat.

14. The method of claim 11, wherein the animal model of gene targeting is a KO, CKO, KI, KOfirst, TM mouse model.

15. The method of claim 14, wherein the animal model of gene targeting is PRKAA1-KI, CD27-KI, ANXA8-CKO animal model.

16. A method for improving screening efficiency of target-hitting ES positive clones based on a CRISPR/Cas9 system is characterized in that a CRISPR/Cas9 system is utilized to select a mutant of Cas9 with DNA single-strand cutting capacity, a tracrRNA/crRNA binary complex or an sgRNA is used for each target hitting site, the mutant of Cas9 recognizes conserved spacer adjacent motifs near the target hitting site, DNA single-strand cuts are generated, double-strand breaks are not generated, and single-strand cut homologous recombination repair is induced.