Summary of the invention
The invention discloses a kind of gene orders for effectively inhibiting II type PRRSV infection, and are prepared for containing this section of gene
The clone pig of sequence.The experimental results showed that the PAMs replaced after the 7th exon of pig CD163 with this section of gene order can be effective
Inhibit the infection of II type PRRSV in vitro.
A kind of gene order effectively inhibiting II type PRRSV infection disclosed by the invention, it is characterised in that:
Gene order is as shown in SEQ ID 1;CD163 gene order after gene modification as: shown in SEQ ID 2;Can effectively it expand
Increase the upstream primer sequence F1 of the target gene as: shown in SEQ ID 3, downstream primer sequence R1 as: shown in SEQ ID 4;Energy
Effectively amplification the 7th exon upstream gene 5-HA of CD163 upstream primer sequence F2 as: shown in SEQ ID 5, downstream primer
Sequence R2 as: shown in SEQ ID 6, the gene order 5-HA that amplifies as: shown in SEQ ID 7;CD163 can effectively be expanded
The upstream primer sequence F3 of seven exon downstream gene 3-HA is such as: SEQ ID 8, downstream primer sequence R3 such as 9 institute of SEQ ID
Show, the gene order amplified as: shown in SEQ ID 10.By SEQ ID 1, SEQ ID 7, SEQ ID 10 recombination connection are obtained
Sequence as shown in SEQ ID 19.
The present invention can effectively edit the DNA sequence dna of the sgRNA of pig CD163 gene cluster, it is characterised in that: the DNA of sgRNA1
Sequence as shown in SEQ ID 11, the DNA sequence dna of complementary strand as: shown in SEQ ID 12, the RNA sequence of sgRNA1 such as SEQ ID
Shown in 13, the RNA sequence of complementary strand as: shown in SEQ ID 14;The DNA sequence dna of sgRNA2 is as shown in SEQ ID 15, complementary strand
DNA sequence dna as: shown in SEQ ID 16, the RNA sequence of sgRNA1 as shown in SEQ ID 17, the RNA sequence of complementary strand such as:
Shown in SEQ ID 18.
A kind of preparation method of gene order effectively inhibiting II type PRRSV infection of the present invention, including following step
It is rapid:
1) pig CD163 gene order is recalled in NCBI gene pool;
2) base sequence such as SEQ ID 3 respectively, upstream primer F1 shown in SEQ ID 4 are obtained using Primer5 software design
With downstream primer R1;
3) base sequence objective gene sequence as shown in SEQ ID 1 is obtained using PCR amplification in vitro method;
4) pig CD163 gene order is recalled in NCBI gene pool;
5) base sequence is obtained respectively such as SEQ ID 5, SEQ ID 6, SEQ ID 8, SEQ ID using Primer5 software design
Upstream primer F2 shown in 9, R2 and downstream primer F3, R3;
6) base sequence such as SEQ ID 7 is obtained using PCR amplification in vitro method;Target gene upstream and downstream shown in SEQ ID 10
Gene;
7) by above-mentioned base sequence obtained such as SEQ ID 1, SEQ ID 7, gene of SEQ ID 10 etc. is than being added containing SEQ
In the PCR system of ID 5, SEQ ID 9;
8) by round pcr, vitro recombination connects these three segments, obtains sequence homologous replacement as shown in SEQ ID 19 and carries
Body;
9) by the sgRNA plasmid of building: SEQ ID 11, SEQ ID 12, SEQ ID 14, SEQ ID 15 and homologous targeting vector
Into porcine fetus fibroblasts PFF, and by limiting dilution assay, screening and identification obtains positive ho-mology and replaces 19 cotransfection of SEQ ID
The porcine fetus fibroblasts changed, positive fibroblasts CD163 gene order is as shown in SEQ ID 2.
A kind of gene order effectively inhibiting II type PRRSV infection of the present invention.It is characterized by: the gene
Gene order is obtained as shown in SEQ ID 2 after fixed point replacement the 7th exon of pig CD163 of sequence SEQ ID 1.Isolated
CD163 gene order primary pulmonary alveolar macrophage of positive colony pig as shown in SEQ ID 2 can effectively inhibit II type PRRSV
Duplication, and illustrate PRRSV infection Mechanism Study to be subsequent and the swinery of PRRSV infection can be effectively suppressed provide reason to obtain
By basis.
The positive effect of the present invention is: CRISPR/Cas9 technology is utilized, by gene order pig as shown in SEQ ID 1
The 5th exon genes sequence of CD163 successfully replaces the gene order of the 7th exon of pig CD163, and obtains CD163
The pulmonary alveolar macrophage of the positive colony pig of gene order such as SEQ ID 2;Can be used for illustrating PRRSV infection molecular mechanism and
Effectively inhibit the pig population of PRRSV infection to provide theoretical basis for subsequent acquisition, reduces because PRRSV is huge to pig breeding industry bring
Big loss.
Embodiment 4
The preparation of CD1163 genotype positive colony porcine alveolar macrophage as shown in SEQ ID 2
Electrotransfection buffering is added when after washing 2 ~ 3 times with DPBS, abandon supernatant close to when covering in recovery porcine fetus fibroblasts
Then PX330 plasmid and sequence such as sequence plasmid as shown in SEQ ID 19 are proportionally added into cell and buffer by liquid
In, after being mixed gently with pipettor, electric revolving cup is put on electroporation apparatus and carries out by moving into mixed liquor in pole cup gently
Electric shock operation.After the completion of electric shock, electric revolving cup is stood after ten minutes, and the mixed liquor in electric revolving cup is transferred in Tissue Culture Dish.Most
The Tissue Culture Dish is placed in 37 DEG C of carbon dioxide incubators afterwards and is cultivated.After culture 12 hours, liquid is changed.After electrotransfection 72h, make
Pig PFF cell is taped against in 100mm Tissue Culture Dish by the method for Method of Limited Dilution with trypsin digestion cell, replacement one in 2 ~ 3 days
Secondary cell culture fluid.After 8 ~ 10 days after cell clone grows up to, cell clone is uniformly marked under fluorescence microscope, so
Afterwards these labeled clone's pickings are entered in 24 porocyte culture plates to be then incubated for.It is long to the cell in 24 orifice plates after 2 ~ 3 days
To certain convergence degree, pass through the side of PCR sequencing and RFLP after a quarter of these cells is cracked with NP40 lysate again
Method further verifies pig measurement the 7th exon of pig CD163 fixed point replacement event.
The positive PFFs that above-mentioned screening is obtained obtains external reconstructed embryo by SCNT, and is implanted into replace-conceive sow
In utero, positive F is obtained after about 114 days0Clone pig.Part tail tissue is taken to mention genome identification (such as resulting offspring's clone pig
Shown in Fig. 2).Fig. 2's the result shows that the present invention has been successfully prepared 3 positive colony pigs.After clone pig long to 3 weeks, not
Entire lung is removed in the case where scratching tracheae and lung, and gas tube orifice is tightened immediately, it is closed, it puts into clean sack, low temperature
Transport laboratory back.Entire lung is cleaned with sterile PBS in super-clean bench, removes dirt.By upper 2/3rds part of tracheae
Excision;It is injected with the PBS of 50ML from gas tube orifice, in triplicate.Recovered liquid is blown and beaten repeatedly with suction pipe, keeps its cell mass fastly and glutinous
The dispersion of liquid block.Filtering, 500g, 15min.Supernatant is removed, precipitating is resuspended with PBS.Resulting cell be exactly CD1163 genotype such as
Positive colony porcine alveolar macrophage shown in SEQ ID 2.
In summary: the present invention is successfully obtained using Crispr/cas9 gene editing technology and somatic cell nuclear transfer technique
It is consistent containing sequence such as SEQ ID 2, the cell and individual of the gene order of II type PRRSV infection can be effectively suppressed.
Experimental example 1
The positive external II type PRRSV infection test of CD163 SRCR3 fixed point the 7th exon porcine alveolar macrophage of replacement:
Dual anti-, the 1mM sodium by above-mentioned the obtained primary pulmonary alveolar macrophage cell RPMI-1640,10%FBS, 1% of pig
1% glutamine of pyruvate, 1% nonessential amino acid culture.It is thin that cell obtained by certain density is inoculated in 12 holes
In born of the same parents' culture plate, after 4 hour cells are adherent, inhale and abandon not adherent cell.After cell adherent 12 hours, it is inoculated with II type
PRRSV virus (MOI=1) and cell are incubated for 3 hours altogether;Virus liquid is then discarded, PBS is cleaned three times, and the new culture of 1ml is added
Liquid is put into cell incubator culture.Cell and 250 microlitres of culture solution supernatant are collected after 72 hours, and 750 microlitres of TRIZOL are added,
Extract cell and supernatant total serum IgE.Quantitative RNA amount is 1 microgram, using Bo company cDNA the first chain synthetic agent box to total serum IgE
Carry out reverse transcription.The said firm BioEasy SYBR Green I fluorescent PCR kit and bio-rad iQ5 fluorescence are used later
Quantitative PCR apparatus carries out fluorogenic quantitative detection.Pass through the quantitative result pair of cellular control unit and water control group with not virus inoculation
Than determining the antitoxic effect of cell.(referring to Fig. 3, absolute fluorescence quantitative PCR result)
The experiment of Fig. 3 cell in vitro virus infection shows can with the PAMs after this section of gene order replacement the 7th exon of pig CD163
The infection or even antiviral effect for effectively inhibiting II type PRRSV in vitro become apparent from than deleting the PAMs of the 7th exon.
Sequence table
<110>Jilin University
<120>a kind of gene order and its application for effectively inhibiting II type PRRSV infection
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 315
<212> DNA
<213>pig (Sus scrofa)
<400> 1
cccacaggca gctgagagtg gtagatggag tcactgaatg ttcaggaaga ttggaagtga 60
aattccaagg agaatgggga acaatctgtg atgatggctg ggatagtgat gatgccgctg 120
tggcatgtaa gcaactggga tgtccaactg ctgtcactgc cattggtcga gttaacgcca 180
gtgagggaac tggacacatt tggcttgaca gtgtttcttg ccatggacac gagtctgctc 240
tctggcagtg tagacaccat gaatggggaa agcattattg caatcataat gaagatgctg 300
gtgtgacatg ttcta 315
<210> 2
<211> 3348
<212> DNA
<213>pig (Sus scrofa)
<400> 2
atggacaaac tcagaatggt gctacatgaa aactctggat ctgcagactt tagaagatgt 60
tctgcccatt taagttcctt cacttttgct gtagtcgctg ttctcagtgc ctgcttggtc 120
actagttctc ttggaggaaa agacaaggag ctgaggctaa cgggtggtga aaacaagtgc 180
tctggaagag tggaggtgaa agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg 240
gacatggatg tggtctctgt tgtttgtagg cagctgggat gtccaactgc tatcaaagcc 300
actggatggg ctaattttag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360
cgagggaatg agtcagctct ctgggactgc aaacatgatg gatggggaaa gcataactgt 420
actcaccaac aggatgctgg agtaacctgc tcagatggat ctgatttaga gatgaggctg 480
gtgaatggag gaaaccggtg cttaggaaga atagaagtca aatttcaagg acggtgggga 540
acagtgtgtg atgataactt caacataaat catgcttctg tggtttgtaa acaacttgaa 600
tgtggaagtg ctgtcagttt ctctggttca gctaattttg gagaaggttc tggaccaatc 660
tggtttgatg atcttgtatg caatggaaat gagtcagctc tctggaactg caaacatgaa 720
ggatggggaa agcacaattg cgatcatgct gaggatgctg gagtgatttg cttaaatgga 780
gcagacctga aactgagagt ggtagatgga gtcactgaat gttcaggaag attggaagtg 840
aaattccaag gagaatgggg aacaatctgt gatgatggct gggatagtga tgatgccgct 900
gtggcatgta agcaactggg atgtccaact gctgtcactg ccattggtcg agttaacgcc 960
agtgagggaa ctggacacat ttggcttgac agtgtttctt gccatggaca cgagtctgct 1020
ctctggcagt gtagacacca tgaatgggga aagcattatt gcaatcataa tgaagatgct 1080
ggtgtgacat gttctgatgg atcagatctg gaactgagac ttaaaggtgg aggcagccac 1140
tgtgctggga cagtggaggt ggaaattcag aaactggtag gaaaagtgtg tgatagaagc 1200
tggggactga aagaagctga tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa 1260
acatcatatc aagtttattc caaaaccaag gcaacaaaca catggctgtt tgtaagcagc 1320
tgtaatggaa atgaaacttc tctttgggac tgcaagaatt ggcagtgggg tggacttagt 1380
tgtgatcact atgacgaagc caaaattacc tgctcagccc acaggcagct gagagtggta 1440
gatggagtca ctgaatgttc aggaagattg gaagtgaaat tccaaggaga atggggaaca 1500
atctgtgatg atggctggga tagtgatgat gccgctgtgg catgtaagca actgggatgt 1560
ccaactgctg tcactgccat tggtcgagtt aacgccagtg agggaactgg acacatttgg 1620
cttgacagtg tttcttgcca tggacacgag tctgctctct ggcagtgtag acaccatgaa 1680
tggggaaagc attattgcaa tcataatgaa gatgctggtg tgacatgttc tagatacaca 1740
caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 1800
gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 1860
cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 1920
agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catgggagat 1980
tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 2040
tgctcaggga accagagtca gacactatcc ccgtgcaatt catcatcctc ggacccatca 2100
agctctatta tttcagaaga aaatggtgtt gcctgcatag ggagtggtca acttcgcctg 2160
gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 2220
accatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 2280
tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 2340
tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 2400
ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcggagttc 2460
atgtctctca gactgatcag tgaaagcagc agagagacct gtgcagggcg cctggaagtt 2520
ttttacaacg gagcttgggg cagcgttggc aagaatagca tgtctccagc cacagtgggg 2580
gtggtatgca ggcagctggg ctgtgcagac agaggggaca tcagccctgc atcttcagac 2640
aagacagtgt ccaggcacat gtgggtggac aatgttcagt gtcctaaagg acctgacacc 2700
ctatggcagt gcccatcatc tccatggaag aagagactgg ccagcccctc agaggagaca 2760
tggatcacat gtgccaacaa aataagactt caagaaggaa acactaattg ttctggacgt 2820
gtggagatct ggtacggagg ttcctggggc actgtgtgtg acgactcctg ggaccttgaa 2880
gatgctcagg tggtgtgccg acagctgggc tgtggctcag ctttggaggc aggaaaagag 2940
gccgcatttg gccaggggac tgggcccata tggctcaatg aagtgaagtg caaggggaat 3000
gaaacctcct tgtgggattg tcctgccaga tcctggggcc acagtgactg tggacacaag 3060
gaggatgctg ctgtgacgtg ttcagaaatt gcaaagagcc gagaatccct acatgccaca 3120
ggtcgctcat cttttgttgc acttgcaatc tttggggtca ttctgttggc ctgtctcatc 3180
gcattcctca tttggactca gaagcgaaga cagaggcagc ggctctcagt tttctcagga 3240
ggagagaatt ctgtccatca aattcaatac cgggagatga attcttgcct gaaagcagat 3300
gaaacggata tgctaaatcc ctcaggagac cactctgaag tacaatga 3348
<210> 3
<211> 32
<212> DNA
<213>pig (Sus scrofa)
<400> 3
cccacaggaa actgagagtg gtagatggag tc 32
<210> 4
<211> 34
<212> DNA
<213>pig (Sus scrofa)
<400> 4
tccctgggtc tcactagaac atgtcaccag catc 34
<210> 5
<211> 22
<212> DNA
<213>pig (Sus scrofa)
<400> 5
gatgattgcg ctcttaaacc tg 22
<210> 6
<211> 19
<212> DNA
<213>pig (Sus scrofa)
<400> 6
ctcagtttcc tgtgggctg 19
<210> 7
<211> 418
<212> DNA
<213>pig (Sus scrofa)
<400> 7
gatgattgcg ctcttaacct ggcaaagatt gtctttaaaa tctgagctcc atgtcttctg 60
ctttatttct ggtgtgcctt tgactccaga ttacagtaaa tggaggactg agtatagggc 120
taaaaagtag agagaatgga tgcatattat ctgtggtctc caatgtgatg aatgaagtag 180
gcaaatactc aaaggaaaga gaaagcatgc tccaagaatt atgggttcca gaaggcaaag 240
tcccagaatt gtctccaggg aaggacaggg aggtctagaa tcggctaagc ccactgtagg 300
cagaaaaacc aagaggcatg aatggcttcc ctttctcact tttcaccctc tggcttactc 360
ctatcatgaa ggaaaatatt ggaatcatat tctccctcac cgaaatgcta tttttcag 418
<210> 8
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 8
agtgagaccc agggaatgtg 20
<210> 9
<211> 21
<212> DNA
<213>pig (Sus scrofa)
<400> 9
tcagcctaca ccacaatcac a 21
<210> 10
<211> 1452
<212> DNA
<213>pig (Sus scrofa)
<400> 10
accatgaatg gggaaagcat tattgcaatc ataatgaaga tgctggtgtg acatgttcta 60
gtgagaccca gggaatgtgt tcactttgtt cccatgccat gaagagggta gggttaggta 120
gtcacagaca tctttttaaa gccctgtctc cttccaggat acacacaaat ccgcttggtg 180
aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 240
ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 300
ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 360
aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 420
ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc aggtaagaga 480
ataagggcag ccagtgatga gccactcatg acggtgcctt aagagtgggt gtacctagga 540
gttcccattg tggctcagtg gtaacaaact cgactggtat ccatgagggt atgggtttga 600
tccctggcct tgctcaatgg gttaaggatc cagcattgct gtgagctgtg gtataggttg 660
cagactctgc tcaggtccca tgttgctgtg attgtggtgt aggctgactg ctgcagcttc 720
aatttgaccc ctagcccggg aatttccata ggccacacgt gcagcactaa ggaaggaaaa 780
aaaaaaaaaa aaaaaaaaga gtgggtgtgc ctatagtgaa gaacagatgt aaaagggaag 840
tgaaagggat tcccccattc tgagggattg tgagaagtgt gccagaatat taacttcatt 900
tgacttgtta cagggaaagt aaacttgact ttcacggacc tcctagttac ctggtgctta 960
ctatatgtct tctcagagta cctgattcat tcccagcctg gttgacccat ccccctatct 1020
ctatggctat gtttatccag agcacatcta tctaacactc cagctgatct tcctgacaca 1080
gctgtggcaa ccctggatcc tttaaccaac tgtgccaggc tggagatcaa acctaagcct 1140
ctgcagcaac ccaagctgct gcagtcagat ttttaacccc ctgtgccact gtgggtatct 1200
ccgatatttt gtatcttctg tgactgagtg gtttgctgtt tgcagggaac cagagtcaga 1260
cactatcccc gtgcaattca tcatcctcgg acccatcaag ctctattatt tcagaagaaa 1320
atggtgttgc ctgcataggt gagaatcagt gaccaaccta tgaaaatgat ctcaatcctc 1380
tgaaatgcat tttattcatg ttttatttcc tctttgcagg gagtggtcaa cttcgcctgg 1440
tcgatggagg tg 1452
<210> 11
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 11
ggaaacccag gctggttgga 20
<210> 12
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 12
tccaaccagc ctgggtttcc 20
<210> 13
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 13
ggaaacccag gcugguugga 20
<210> 14
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 14
uccaaccagc cuggguuucc 20
<210> 15
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 15
gagtagcacc ccgccctgac 20
<210> 16
<211> 20
<212> DNA
<213>pig (Sus scrofa)
<400> 16
gtcagggcgg ggtgctactc 20
<210> 17
<211> 20
<212> RNA
<213>pig (Sus scrofa)
<400> 17
gaguagcacc ccgcccugac 20
<210> 18
<211> 2125
<212> DNA
<213>pig (Sus scrofa)
<400> 18
gatgattgcg ctcttaacct ggcaaagatt gtctttaaaa tctgagctcc atgtcttctg 60
ctttatttct ggtgtgcctt tgactccaga ttacagtaaa tggaggactg agtatagggc 120
taaaaagtag agagaatgga tgcatattat ctgtggtctc caatgtgatg aatgaagtag 180
gcaaatactc aaaggaaaga gaaagcatgc tccaagaatt atgggttcca gaaggcaaag 240
tcccagaatt gtctccaggg aaggacaggg aggtctagaa tcggctaagc ccactgtagg 300
cagaaaaacc aagaggcatg aatggcttcc ctttctcact tttcaccctc tggcttactc 360
ctatcatgaa ggaaaatatt ggaatcatat tctccctcac cgaaatgcta tttttcagcc 420
cacaggcagc tgagagtggt agatggagtc actgaatgtt caggaagatt ggaagtgaaa 480
ttccaaggag aatggggaac aatctgtgat gatggctggg atagtgatga tgccgctgtg 540
gcatgtaagc aactgggatg tccaactgct gtcactgcca ttggtcgagt taacgccagt 600
gagggaactg gacacatttg gcttgacagt gtttcttgcc atggacacga gtctgctctc 660
tggcagtgta gacaccatga atggggaaag cattattgca atcataatga agatgctggt 720
gtgacatgtt ctagtgagac ccagggaatg tgttcacttt gttcccatgc catgaagagg 780
gtagggttag gtagtcacag acatcttttt aaagccctgt ctccttccag gatacacaca 840
aatccgcttg gtgaatggca agaccccatg tgaaggaaga gtggagctca acattcttgg 900
gtcctggggg tccctctgca actctcactg ggacatggaa gatgcccatg ttttatgcca 960
gcagcttaaa tgtggagttg ccctttctat cccgggagga gcaccttttg ggaaaggaag 1020
tgagcaggtc tggaggcaca tgtttcactg cactgggact gagaagcaca tgggagattg 1080
ttccgtcact gctctgggcg catcactctg ttcttcaggg caagtggcct ctgtaatctg 1140
ctcaggtaag agaataaggg cagccagtga tgagccactc atgacggtgc cttaagagtg 1200
ggtgtaccta ggagttccca ttgtggctca gtggtaacaa actcgactgg tatccatgag 1260
ggtatgggtt tgatccctgg ccttgctcaa tgggttaagg atccagcatt gctgtgagct 1320
gtggtatagg ttgcagactc tgctcaggtc ccatgttgct gtgattgtgg tgtaggctga 1380
ctgctgcagc ttcaatttga cccctagccc gggaatttcc ataggccaca cgtgcagcac 1440
taaggaagga aaaaaaaaaa aaaaaaaaaa agagtgggtg tgcctatagt gaagaacaga 1500
tgtaaaaggg aagtgaaagg gattccccca ttctgaggga ttgtgagaag tgtgccagaa 1560
tattaacttc atttgacttg ttacagggaa agtaaacttg actttcacgg acctcctagt 1620
tacctggtgc ttactatatg tcttctcaga gtacctgatt cattcccagc ctggttgacc 1680
catcccccta tctctatggc tatgtttatc cagagcacat ctatctaaca ctccagctga 1740
tcttcctgac acagctgtgg caaccctgga tcctttaacc aactgtgcca ggctggagat 1800
caaacctaag cctctgcagc aacccaagct gctgcagtca gatttttaac cccctgtgcc 1860
actgtgggta tctccgatat tttgtatctt ctgtgactga gtggtttgct gtttgcaggg 1920
aaccagagtc agacactatc cccgtgcaat tcatcatcct cggacccatc aagctctatt 1980
atttcagaag aaaatggtgt tgcctgcata ggtgagaatc agtgaccaac ctatgaaaat 2040
gatctcaatc ctctgaaatg cattttattc atgttttatt tcctctttgc agggagtggt 2100
caacttcgcc tggtcgatgg aggtg 2125