阅读说明：本技术 一种敲除鸭hnRNPA3基因的sgRNA、细胞系及其构建方法和应用 (sgRNA and cell line with duck hnRNPA3 gene knocked out, and construction method and application thereof ) 是由 陈舜 胡涛 吴震 陈维琼 汪铭书 程安春 于 2021-10-14 设计创作，主要内容包括：本发明公开了一种敲除鸭hnRNPA3基因的sgRNA,所述sgRNA位于鸭hnRNPA3基因第五外显子上,所述sgRNA的核苷酸序列如SEQ ID NO.1所示；所述sgRNA对应的上游引物核苷酸序列如SEQ ID NO.2所示,下游引物核苷酸序列如SEQ ID NO.3所示。本发明还提供了一种包含上述sgRNA的表达载体,所述表达载体为CRISPR/Cas9重组慢病毒载体,所述表达载体命名为lentiCRISPR-v2-Blast。本发明进一步提供了一种敲除鸭hnRNPA3基因的细胞系及其构建方法和应用。本发明首次得到敲除鸭hnRNPA3基因的稳定传代的鸭源细胞系,本发明中的hnRNPA3基因敲除细胞系能够成为鸭源病毒扩增和研究的重要工具,尤其是有利于研究hnRNPA3基因参与TMUV生命周期中发挥的功能。(The invention discloses a sgRNA for knocking out duck hnRNPA3 gene, wherein the sgRNA is positioned on the fifth exon of duck hnRNPA3 gene, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1; the upstream primer nucleotide sequence corresponding to the sgRNA is shown as SEQ ID NO.2, and the downstream primer nucleotide sequence is shown as SEQ ID NO. 3. The invention also provides an expression vector containing the sgRNA, wherein the expression vector is a CRISPR/Cas9 recombinant lentiviral vector and is named lentiCRISPR-v 2-Blast. The invention further provides a duck hnRNPA3 gene knockout cell line and a construction method and application thereof. The stably passaged duck source cell line with the duck hnRNPA3 gene knocked out is obtained for the first time, and the hnRNPA3 gene knocked-out cell line can become an important tool for amplification and research of duck source viruses, and is particularly favorable for researching the function of the hnRNPA3 gene participating in TMUV life cycle.)

1. An sgRNA for knocking out duck hnRNPA3 gene is characterized in that the sgRNA is positioned on the fifth exon of duck hnRNPA3 gene, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1; the upstream primer nucleotide sequence corresponding to the sgRNA is shown as SEQ ID NO.2, and the downstream primer nucleotide sequence is shown as SEQ ID NO. 3.

2. An expression vector comprising the sgRNA of claim 1, wherein the expression vector is a CRISPR/Cas9 recombinant lentiviral vector designated lentiCRISPR-v 2-Blast.

3. The expression vector according to claim 2, wherein the expression vector is constructed by a method comprising: artificially synthesizing an sgRNA sequence and upstream and downstream primer sequences corresponding to the sgRNA, and annealing to form double-stranded DNA; connecting the double-stranded DNA with a vector lentiCRISPR-v2 cut by BsmBI I to obtain an expression vector lentiCRISPR-v 2-Blast; the nucleotide sequence of the expression vector lentiCRISPR-v2-Blast is shown in SEQ ID NO. 6.

4. A duck hnRNPA3 gene knockout cell line, which is a monoclonal cell line obtained by knocking out duck hnRNPA3 gene by using the expression vector lentiCRISPR-v2-Blast of claim 3, and is a stably passaged duck embryo fibroblast cell line.

5. A method for constructing a duck hnRNPA3 gene knockout cell line is characterized by comprising the following steps:

s1, construction, transformation, screening, PCR identification and sequencing verification of an expression vector lentiCRISPR-v 2-Blast; the nucleotide sequences of the upstream primer and the downstream primer identified by the PCR are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

s2, co-transfecting HEK293T cells with an expression vector lentiCRISPR-v2-Blast constructed in S1, a helper plasmid psPAX2 and a helper plasmid pCMV-VSV-G to obtain recombinant lentiviruses;

s3, determination of concentration of Blasticidin S (BS) for screening gene knockout positive cells;

s4, infecting the immortalized duck embryo fibroblast with the recombinant lentivirus obtained in the S2 and screening monoclonal cells.

6. The method for constructing a recombinant plasmid according to claim 5, wherein in S2, the mass ratio of the expression vector lentiCRISPR-v2-Blast, the helper plasmid psPAX2 and the helper plasmid pCMV-VSV-G is 4: 3: 1.

7. the method according to claim 5, wherein in S3, the concentration of BS positive for gene knockout screening is determined by culturing immortalized duck embryo fibroblasts with BS at a concentration of 0. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml, 6. mu.g/ml, or 8. mu.g/ml, respectively.

8. The method for constructing a recombinant duck embryo fibroblast cell line as claimed in claim 5, wherein in S4, the immortalized duck embryo fibroblast cell line is infected with the recombinant lentivirus obtained in S2, and after 48h of infection, BS with a determined screening concentration in S3 is added for pressure screening; diluting the screened and surviving cells, carrying out monoclonal screening, and carrying out western blotting verification on the screened positive monoclonal cells; and meanwhile, continuously using the BS to maintain the screening of the positive monoclonal cells, thereby obtaining the duck embryo fibroblast line with the stable passage of the duck hnRNPA3 gene knocked out.

9. The use of the duck hnRNPA3 gene knock-out cell line of claim 4 in the production of a continuous duck cell line.

10. The use of the duck hnRNPA3 gene knock-out cell line of claim 4 in the preparation of a cell model in which hnRNPA3 gene is involved in duck tembusu virus (TMUV) life cycle studies.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to sgRNA and a cell line for knocking out duck hnRNPA3 gene, and a construction method and application thereof.

Background

The CRISPR/Cas system is an acquired immune system found in bacteria and archaea, which enables site-directed cleavage of specific genes. Among them, the streptococcus-based type ii CRISPR/Cas9 system is simple in component composition [ functional units: cas9 protein and sgRNA (single-guide RNA) ], which are relatively high in efficiency, are widely used gene editing systems at present. When double-stranded DNA breaks (DSBs) caused by CRISPR/Cas9 system appear in organism genome, cells can repair damaged double-stranded DNA by homologous recombination (HDR) or Non-homologous end joining (NHEJ) of their own repair mechanism, and because mutations may be introduced at junctions during repair, it is usually used to implement gene editing, especially gene knockout, on target genes.

The lentivirus system is a gene transduction tool developed based on HIV-1. This system is capable of mediating efficient transduction and stable expression of both dividing and non-dividing cells in vitro and in vivo. Since the stable expression of the Cas9 protein and sgRNA is required for the CRISPR/Cas9 system to function, the CRISPR/Cas9 system is integrated into a host genome by using a lentiviral vector, so that the function of CRISPR/Cas9 can be stably exerted, and the target gene can be stably knocked out.

Duck tembusu virus (TMUV) belongs to the flaviviridae genus of the flaviviridae family, and mainly infects ducks to cause duck tembusu virus disease of the ducks. The infection of the ducks shows clinical symptoms of decreased feed intake, green and thin manure, rapid decrease of egg production and the like. The duck Tembusu virus disease is characterized by acute disease onset and rapid spread, and causes great economic loss for the duck breeding industry. Therefore, the research on duck tembusu virus is very important. However, the existing duck-derived gene knockout cell lines are few, and usually only gene knock-down and gene overexpression can be realized, but the genes cannot be completely knocked out, so that the duck-derived gene knockout cell line for virus in vitro research cannot be provided. And because the amplification level of the duck tembusu virus in the existing cell line is low, the research on a duck cell model infected by the duck tembusu virus is seriously influenced without a proper gene knockout cell line.

Disclosure of Invention

The invention aims to solve the problems that no duck source gene knockout cell line suitable for duck tembusu virus research exists in the prior art and deep research on duck tembusu virus is not facilitated, and provides a sgRNA and a cell line for knocking out duck hnRNPA3 gene, and a construction method and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the invention provides a sgRNA for knocking out duck hnRNPA3 gene, wherein the sgRNA is positioned on the fifth exon of duck hnRNPA3 gene, and the nucleotide sequence of the sgRNA is shown as SEQ ID No. 1; the upstream primer nucleotide sequence corresponding to the sgRNA is shown as SEQ ID NO.2, and the downstream primer nucleotide sequence is shown as SEQ ID NO. 3.

The invention also provides an expression vector containing the sgRNA, wherein the expression vector is a CRISPR/Cas9 recombinant lentiviral vector and is named lentiCRISPR-v 2-Blast.

Preferably, the construction method of the expression vector comprises the following steps: artificially synthesizing an sgRNA sequence and upstream and downstream primer sequences corresponding to the sgRNA, and annealing to form double-stranded DNA; connecting the double-stranded DNA with a vector lentiCRISPR-v2 cut by BsmBI I to obtain an expression vector lentiCRISPR-v 2-Blast; the nucleotide sequence of the expression vector lentiCRISPR-v2-Blast is shown in SEQ ID NO. 6.

The invention also provides a cell line for knocking out duck hnRNPA3 gene, which is a monoclonal cell strain obtained by knocking out duck hnRNPA3 gene by using the expression vector lentiCRISPR-v2-Blast, and the cell line is a duck embryo fibroblast line with stable passage.

The invention further provides a method for constructing a duck hnRNPA3 gene knockout cell line, which comprises the following steps:

s1, construction, transformation, screening, PCR identification and sequencing verification of an expression vector lentiCRISPR-v 2-Blast; the nucleotide sequences of the upstream primer and the downstream primer identified by the PCR are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

s2, co-transfecting HEK293T cells with an expression vector lentiCRISPR-v2-Blast constructed in S1, a helper plasmid psPAX2 and a helper plasmid pCMV-VSV-G to obtain recombinant lentiviruses;

s3, determination of concentration of Blasticidin S (BS) for screening gene knockout positive cells;

s4, infecting the immortalized duck embryo fibroblast with the recombinant lentivirus obtained in the S2 and screening monoclonal cells.

Preferably, in the S2, the mass ratio of the expression vector lentiCRISPR-v2-Blast, the helper plasmid psPAX2 and the helper plasmid pCMV-VSV-G is 4: 3: 1.

preferably, in S3, the concentration of BS screening positive cells for gene knockout is determined by culturing immortalized duck embryo fibroblasts with BS at the concentration of 0. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml, 6. mu.g/ml and 8. mu.g/ml, respectively.

Further preferably, in S4, the immortalized duck embryo fibroblast is infected by the recombinant lentivirus obtained in S2, and BS with the determined screening concentration in S3 is added for pressurized screening after 48 hours of infection; diluting the screened and surviving cells, carrying out monoclonal screening, and carrying out western blotting verification on the screened positive monoclonal cells; and meanwhile, continuously using the BS to maintain the screening of the positive monoclonal cells, thereby obtaining the duck embryo fibroblast line with the stable passage of the duck hnRNPA3 gene knocked out.

The invention further provides application of the cell line with the duck hnRNPA3 gene knocked-out in production of a duck-derived continuous cell line.

The invention further provides application of the cell line with the duck hnRNPA3 gene knocked out in preparation of a cell model with a hnRNPA3 gene participating in duck tembusu virus (TMUV) life cycle research.

The invention has the following beneficial effects:

firstly), the invention utilizes a CRISPR/Cas9 system to knock out duck hnRNPA3 gene from passaged duck embryo fibroblasts, tests of gene and protein level prove that the duck hnRNPA3 gene is successfully knocked out, a duck source cell line with stable passage of the duck hnRNPA3 gene knocked out is obtained for the first time, and the hnRNPA3 gene knocked-out cell line can become an important tool for duck source virus amplification and research.

Secondly), RT-qPCR shows that the virus genome level of the hnRNPA3 gene knockout cell line constructed by the invention is higher than that of a parent duck embryo fibroblast after the hnRNPA3 gene knockout cell line is infected with duck tembusu virus (TMUV), which indicates that the cell line with the hnRNPA3 gene knockout can obviously improve the amplification level of the duck tembusu virus (TMUV); the TMUV translation system is used for indicating that the hnRNPA3 gene knockout cell line can promote the translation of the TMUV genome, and is favorable for researching the function of the hnRNPA3 gene participating in the TMUV life cycle.

Drawings

FIG. 1 is a schematic diagram of sgRNA targeting duck hnRNPA3 gene and expression vector lentiCRISPR-v2-Blast plasmid;

FIG. 2 is a diagram showing the cell growth of blasticidin at various concentrations for gene knockout positive cell screening;

FIG. 3 shows verification of positive monoclonal cell immunoblotting (WB) of hnRNPA3 gene of a knock-out duck;

FIG. 4 is a graph showing the results of RT-qPCR evaluation of the effect of duck hnRNPA3 gene knockout cells on TMUV proliferation;

FIG. 5 is a graph showing the results of evaluating the effect of duck hnRNPA3 gene knock-out on TMUV RNA translation using TMUV translation system.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

Example 1

Plasmid, cell, virus: the lentiCRISPR-v2 plasmid was purchased from addge, accession # 83480; plasmid pCMV-VSV-G was purchased from Addgene, accession # 8454; plasmid psPAX2 was purchased from Addgene, accession # 12260; immortalized Duck Embryo Fibroblasts (DEF) were purchased from pluberculus siebold (shanghai) biotechnology limited; DH5 alpha competent cells were obtained from the poultry disease prevention and treatment center of the animal medical college of Sichuan university of agriculture, HEK293T cells were purchased from Wuhan Punuoise Life technologies, Inc.; duck Tembusu virus (TMUV) is provided by the poultry disease control center of the animal medical college of Sichuan university of agriculture.

1. Construction of expression vector lentiCRISPR-v2-Blast plasmid

Schematic diagrams of sgRNA of targeted duck hnRNPA3 gene and expression vector lentiCRISPR-v2-Blast plasmid are shown in FIG. 1. The lentiCRISPR-v2 vector was linearized with BsmBI endonuclease, the linearization system and procedure were as follows: BsmBI 1 uL, lentiCRISPR-v2 plasmid 1 ug, NEB buffer 3.15ul, make up to 50ul with sterilized water; the enzyme digestion condition is 55 ℃ for 30 min; and after enzyme digestion is finished, recovering for later use.

Artificially synthesizing an sgRNA sequence SEQ ID NO.1 and upstream and downstream primer sequences SEQ ID NO.2 and SEQ ID NO.3 corresponding to the sgRNA; annealing the sgRNA oligonucleotide to double strand. The DNA ligase is used for being connected with a linearized vector lentiCRISPR-v2-Blast, and the reaction system and the program are as follows: 1 mu L of vector plasmid, 1 mu L of annealed sgRNA, 5 mu L of Solution I and 3 mu L of sterilized water; the reaction conditions were 16 ℃ for 1h, and the ligation products were transformed into DH 5. alpha. competent cells and plated in LB solid medium containing ampicillin resistance and cultured overnight at 37 ℃. Then picking a single colony to a solid culture medium containing benzyl resistance, carrying out PCR amplification by taking sequences SEQ ID NO.4 and SEQ ID NO.5 as primers, carrying out electrophoresis identification on the colony, carrying out amplification culture on the colony with positive electrophoresis in an LB liquid culture medium containing ampicillin resistance, then extracting plasmids and carrying out sequencing verification to obtain an expression vector lentiCRISPR-v2-Blast, wherein the nucleotide sequence of the expression vector is shown as SEQ ID NO. 6. The helper plasmids pCMV-VSV-G and psPAX2 were extracted simultaneously. sgRNA and primer sequences are detailed in table 1.

TABLE 1

2. Recombinant lentivirus packaging and concentration

HEK293T cells are inoculated to a 10cm cell culture dish, when the cells grow to 70% -80% for transfection, the constructed lentiCRISPR-v2-blast plasmid is used for transfecting HEK293T cells together with helper plasmids pCMV-VSV-G and psPAX2, wherein the mass ratio of lentiCRISPR-v2-blast, psPAX2 and pCMV-VSV-G is 4: 3: 1, replacing the liquid after 6-8h of transfection. After 60h of transfection, the supernatant from the cell culture dish was collected, filtered through a filter to remove cellular impurities, and centrifuged at 12000rpm for 3h at 4 ℃. After centrifugation, the supernatant was discarded and the viral pellet was resuspended in 200ul of Opti-MEM medium to obtain a recombinant lentivirus suspension for subsequent infection.

3. Determination of the concentration of Blasticidin S (BS) for screening Gene knockout Positive cells

To subsequently screen for positive cells for potential gene knock-outs using BS, a BS screening curve in immortalized Duck Embryo Fibroblasts (DEF) was first determined. Immortalized DEF is transferred to a 6-well plate, and when the cell density reaches 70% -80%, BS (0 mug/ml, 2 mug/ml, 4 mug/ml, 6 mug/ml and 8 mug/ml) with different concentrations is added into each well in sequence. The survival of the cells was observed every 24h, from which the optimal concentration of BS to be used was determined. Dead cells float on the culture medium in a circular shape, and living cells grow in a fusiform adherent manner. The screening results showed that 8 μ g/ml BS can kill all DEF cells after three days dosing, so this concentration was used as the subsequent screening concentration. The results are shown in FIG. 2.

4. Immortalized duck embryo fibroblast infected by recombinant lentivirus and screening monoclonal cell

And setting a test group and a control group, wherein the test group and the control group are inoculated with well-grown immortalized DEF on a 6cm cell culture dish. When the cell density of the test group reaches 80%, lentivirus is inoculated according to the virus titer; the control group was not infected with virus. After 48h, the test and control groups were replaced with DMEM medium containing 5% FBS, and 8. mu.g/ml BS was added for selection, and the solution was changed every 3 days until all the cells in the control group died.

And digesting the immortalized DEF infected with the recombinant lentivirus in the test group into cell suspension, taking 100ul of the cell suspension, diluting the cell suspension by 10 times by using DMEM, taking 20 ul of the diluted cell suspension, adding an equal volume of 0.4% trypan blue staining solution, uniformly mixing, adding into a cell counting chamber, standing for several minutes, and counting the living cells. Cells were diluted to 100 cells/10 ml, seeded into 96-well plates, and the cells were changed every 3 days, and monoclonal cells were passaged to 24-well plates 7 days later. After the cells in the 24-well plate are overgrown, the cells are expanded and cultured into two 24-well plates, wherein one 24-well plate is used for verifying protein immunoblotting (WB) and is a 9-1clone group; the control group was set up as parental duck embryo fibroblast (nasal DEF) group without recombinant lentivirus infection, and the WB validation test results are shown in FIG. 3. And using the other 24-pore plate for subsequent amplification culture, carrying out passage from the 24-pore plate to a 6-pore plate, adding BS (beta-lactamase) for pressurization maintenance screening when the monoclonal cells grow in the 6-pore plate, continuously carrying out amplification culture and freezing and storing after the 6-pore plate is full, and obtaining the passaged duck embryo fibroblast line with the duck hnRNPA3 gene knocked out.

Example 2

Verification of TMUV proliferation on hnRNPA3 knockout cell line

Test groups 9-1clone and a control group DEF (IM) were set, and the test groups inoculated the DEF knocked out by hnRNPA3 gene prepared in example 1 into 12-well plates, and three replicates were set for each group; control groups an equal amount of parental duck embryo fibroblasts (naive DEF) were seeded into 12-well plates, with three replicates per group. When the cell density of the test group and the control group reaches 80-90%, the test group and the control group are respectively infected with TMUV with the same amount, after 24 hours of infection, the supernatant is discarded, the total RNA is extracted by using a Trizol method, and the total RNA is reversely transcribed into cDNA. Detecting the expression conditions of the TMUV E gene in the test group and the control group relative to the reference gene beta-actin by utilizing fluorescent quantitative PCR (RT-qPCR), and taking sequences SEQ ID NO.7 and SEQ ID NO.8 as primers of the reference gene beta-actin; SEQ ID NO.9 and SEQ ID NO.10 are used as primers of TMUV E gene. RT-qPCR primers are shown in Table 2. The results are shown in fig. 4, that hnRNPA3 gene knock-out cells significantly promoted the proliferation of TMUV on cell models.

TABLE 2

Example 3

Verification of TMUV viral RNA translation on hnRNPA3 knockout cell line

Test groups 9-1clone and a control group DEF (IM) were set up, and the test groups inoculated the HNRNPA3 knock-out DEF prepared in example 1 into 24-well plates, with four replicates per group; control groups inoculated equal amounts of naive DEF into 24-well plates, with four replicates per group. When the density reaches 80-90%, respectively transfecting a 5 'UTR-Rluc-3' UTR of a TMUV translation system, after transfecting for 4h, discarding the supernatant, collecting a cell sample, and detecting the Rluc level in the cell, namely representing the translation condition of a TMUV genome. The results show that, as shown in fig. 5, the knock-out of hnRNPA3 gene can significantly promote the level of TMUV RNA translation on duck embryo fibroblasts.

In conclusion, the duck hnRNPA3 gene is knocked out from passaged duck embryo fibroblasts by using a CRISPR/Cas9 system, and the duck source cell line with the duck hnRNPA3 gene knocked out and stably passaged is obtained for the first time. The RT-qPCR result in the embodiment 2 shows that after the duck tembusu virus (TMUV) with the same quantity is infected, the hnRNPA3 gene knockout cell line constructed by the invention has higher virus genome level than that of a parental duck embryo fibroblast, which indicates that the cell line with hnRNPA3 gene knockout can obviously improve the amplification level of the duck tembusu virus (TMUV); in example 3, a TMUV translation system is used to show that the hnRNPA3 gene knockout cell line can promote the translation of a TMUV genome, and the hnRNPA3 gene knockout cell line provided by the invention is beneficial to researching the functions of the hnRNPA3 gene participating in the TMUV life cycle.

The present specification and figures are to be regarded as illustrative rather than restrictive, and it is intended that all such alterations and modifications that fall within the true spirit and scope of the invention, and that all such modifications and variations are included within the scope of the invention as determined by the appended claims without the use of inventive faculty.

Sequence listing

<110> Sichuan university of agriculture

<120> sgRNA and cell line for knocking out duck hnRNPA3 gene, and construction method and application thereof

<141> 2021-10-14

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acgcgtccat caaccttatg tgg 23

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caccgacgcg tccatcaacc ttatg 25

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaaccataag gttgatggac gcgtc 25

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagggcctat ttcccatgat t 21

<210> 5

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggctccgcc gtcaat 16

<210> 6

<211> 12816

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaaggag tgggaattgg 120

ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg 180

aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga 240

tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt 300

agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta 360

gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt 420

gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg 480

caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg 540

cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa 600

gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag 660

cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca 720

ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta 780

ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct 840

ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc 900

cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt 960

cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact 1020

gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg 1080

cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt 1140

cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga 1200

caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct 1260

gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc 1320

cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa 1380

agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa 1440

gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat 1500

caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga 1560

ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct 1620

gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa 1680

ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc 1740

caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg 1800

gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac 1860

caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga 1920

gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa 1980

gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa 2040

ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga 2100

ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga 2160

tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct 2220

ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct 2280

gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata 2340

caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg 2400

caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct 2460

gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca 2520

gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg 2580

catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc 2640

cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa 2700

cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct 2760

gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct 2820

gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta 2880

cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt 2940

gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt 3000

gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa 3060

gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt 3120

catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga 3180

ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat 3240

caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg 3300

cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc 3360

cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta 3420

cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta 3480

cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga 3540

gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa 3600

gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa 3660

aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag 3720

cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag 3780

ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa 3840

actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa 3900

gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat 3960

caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc 4020

tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct 4080

gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca 4140

gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt 4200

ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa 4260

gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac 4320

caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta 4380

caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta 4440

cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa 4500

ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc 4560

aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccggccaa 4620

gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat 4680

ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt 4740

cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg 4800

cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa 4860

caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg 4920

gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt 4980

gctgccctct ggttatgtgt gggagggcta aacgcgttaa gtcgacaatc aacctctgga 5040

ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 5100

tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 5160

ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 5220

gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 5280

caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 5340

actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 5400

ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 5460

ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 5520

tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 5580

gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 5640

aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 5700

cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 5760

atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 5820

ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 5880

tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg 5940

atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 6000

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 6060

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 6120

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 6180

tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 6240

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 6300

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 6360

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 6420

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 6480

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 6540

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 6600

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 6660

aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 6720

agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 6780

agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 6840

cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 6900

ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 6960

ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 7020

ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat 7080

aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca 7140

ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 7200

acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 7260

cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 7320

acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 7380

cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 7440

ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg 7500

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 7560

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 7620

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 7680

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 7740

gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 7800

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 7860

cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 7920

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 7980

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 8040

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 8100

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 8160

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 8220

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 8280

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 8340

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 8400

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 8460

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 8520

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 8580

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 8640

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 8700

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 8760

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 8820

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 8880

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 8940

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 9000

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 9060

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 9120

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 9180

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 9240

acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 9300

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 9360

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 9420

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 9480

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 9540

gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 9600

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 9660

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 9720

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 9780

cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 9840

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 9900

ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga 9960

tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 10020

gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 10080

aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 10140

gcttcgcgat gtacgggcca gatatacgcg cgcgttgaca ttgattattg actagttatt 10200

aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 10260

aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 10320

taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 10380

agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 10440

cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 10500

tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 10560

tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 10620

gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 10680

caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 10740

aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga 10800

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 10860

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 10920

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 10980

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 11040

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 11100

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 11160

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 11220

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 11280

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 11340

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 11400

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 11460

aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa 11520

ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag 11580

agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc 11640

ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga 11700

caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa 11760

cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct 11820

gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc 11880

atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt 11940

tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata 12000

cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa 12060

ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata 12120

aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt 12180

tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca 12240

accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga 12300

gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat 12360

ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 12420

aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 12480

aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt ggttaattaa 12540

ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 12600

agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 12660

cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 12720

tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 12780

aaaggacgaa acaccgacgc gtccatcaac cttatg 12816

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatcacagcc ctggcacc 18

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggattcatc atactcctgc tt 22

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aatggctgtg gcttgtttgg 20

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gggcgttatc acgaatcta 19