实施例3CRISPR/Cas9在骨髓间质干细胞中的应用
基于含BSD-fsEGFP融合基因pBGN质粒的CRISPR/Cas9编辑载体
(1)BSD-fsEGFP融合基因:利用常规PCR,扩增公知的BSD基因,5’-PCR引物带HindIII位点,3’-PCR引物引入I-SceI和EcoRI位点。将PCR产物(BSD)插入EGFP质粒(EGFP核苷酸序列为本领域公知的序列,例如CN105647968A中的序列1和序列2所示)中CMV驱动子和EGFP编码区的之间的HindIII和EcoRI位点,生成含BSD-fsEGFP融合基因的质粒pBGN,BSD-fsEGFP融合基因核苷酸序列为如CN105647968A中的序列3和序列4所示)。该融合基因由CMV驱动子或PGK驱动子驱动,但EGFP由于移码而无活性,因此称fsEGFP。
5’-PCR引物为CTCAAGCTTAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAG,
3’-PCR引物为AGAATTCCAGTAGGGATAACAGGGTAATGCCAGGTCCGCCCTCCCACACATAACCAGAG。
(2)将测试质粒pBGN,实施例1制备的表达质粒共转染骨髓间质干细胞。以未转染实施例2的增效基因的干细胞作为阳性对照,同时,将常规用的GFP表达质粒平行转染细胞以测定转染效率,利用转染效率校正获取的CRISPR/Cas9基因编辑相对效率。
(4)转染2-3天后,利用流式细胞仪测量GFP+细胞的频率。
(5)计算特定sgRNA介导的CRISPR/Cas9基因编辑相对效率。这个相对效率由GFP阳性细胞频率与转染效率的比值代表,结果如图3所示。我们发现未转染实施例2的增效蛋白的干细胞中GFP阳性细胞频率明显低于转染了实施例2的增效蛋白的细胞。其中在14个sgRNA中,只有CASP3-sgRNA6具有较好的基因编辑效果,效率达到90%以上,这也是申请人付出了艰辛的劳动才筛选得到的具有较好编辑效果的sgRNA。采用空载体制备的阴性对照没有GFP阳性细胞,其中P值均小于0.01,具有统计学意义。
将骨髓间质干细胞CASP3-sgRNA6(即采用CASP3-sgRNA6进行CRISPR编辑后获得的干细胞)和将骨髓间质干细胞CASP3-sgRNA6进行稳定传代培养,培养43代之后,通过针对CASP3PCR测序,发现该基因仍然是突变失活的,保持了基因被敲除的效果。这充分说明,本发明的敲出系统具有较好的稳定性。
最后所应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围。
序列表
<110> 洛阳轩智生物科技有限公司
<120> 使用CRISPR-CAS系统对间充质干细胞进行CASP3基因敲除的方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1578
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
atgcaggaga acctggcccc ctggggcgag ctggccaccg acaacatcat cctgaccgtg 60
cccaccacca acctgcaggc cctgaaggac cccgagcccg tgctgaggct gtgggacgag 120
atgatgcagg ccgtggccag gctggccgcc gagcccttcc ccttcaggag gcccgagagg 180
atcgtggccg acgtgcagat cagcgccggc tggatgcaca gcggctaccc catcatgtgc 240
cacctggaga gcgtgaagga gatcatcaac gagatggaca tgaggagcag gggcgtgtgg 300
ggccccatcc acgagctggg ccacaaccag cagaggcacg gctgggagtt ccccccccac 360
accaccgagg ccacctgcaa cctgtggagc gtgtacgtgc acgagaccgt gctgggcatc 420
cccagggccc aggcccacga ggccctgagc ccccccgaga gggagaggag gatcaaggcc 480
cacctgggca agggcgcccc cctgtgcgac tggaacgtgt ggaccgccct ggagacctac 540
ctgcaggtgc tgagcaggaa cagcggcagg aggggcgtgg acggcaggct ggtgcacacc 600
tgcatcaagg ccggcgccgt gaggtggctg gccaggggcc agaccggcaa ggtgggcgtg 660
aacaccaacc tgaaggacct gtgccccctg ctgagcgagc acggcctgca gtgcagcctg 720
gagccccacc tgaacagcga cctgtgcgtg tactgctgca aggcctacag cgacaaggag 780
gccaagcagc tgcaggagtt cgtggccgag ggcggcggcc tgctgatcgg cggccaggcc 840
tggtggtggg ccagccagaa ccccggccac tgccccctgg ccggcttccc cggcaacatc 900
atcctgaact gcttcggcct gagcatcctg ccccagaccc tgaaggccgg ctgcttcccc 960
gtgcccaccc ccgagatgag gagctaccac ttcaggaagg ccctgagcca gttccaggcc1020
atcctgaacc acgagaacgg caacctggag aagagctgcc tggccaagct gagggtggac1080
ggcgccgcct tcctgcagat ccccgccgag ggcgtgcccg cctacatcag cctgcacagg1140
ctgctgagga agatgctgag gggcagcggc ctgcccgccg tgagcaggga gaaccccgtg1200
gccagcgaca gctacgaggc cgccgtgctg agcctggcca ccggcctggc ccacagcggc1260
accgactgca gccagctggc ccagggcctg ggcacctgga cctgcagcag cagcctgtac1320
cccagcaagc accccatcac cgtggagatc aacggcatca accccggcaa caacgactgc1380
tgggtgagca ccggcctgta cctgctggag ggccagaacg ccgaggtgag cctgagcgag1440
gccgccgcca gcgccggcct gagggtgcag atcggctgcc acaccgacga cctgaccaag1500
gccaggaagc tgagcagggc ccccatggtg acccaccagt gctggatgga caggaccgag1560
aggagcgtga gctgcctg 1578
<210> 2
<211> 526
<212> PRT
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
Met Gln Glu Asn Leu Ala Pro Trp Gly Glu Leu Ala Thr Asp Asn Ile
1 5 10 15
Ile Leu Thr Val Pro Thr Thr Asn Leu Gln Ala Leu Lys Asp Pro Glu
20 25 30
Pro Val Leu Arg Leu Trp Asp Glu Met Met Gln Ala Val Ala Arg Leu
35 40 45
Ala Ala Glu Pro Phe Pro Phe Arg Arg Pro Glu Arg Ile Val Ala Asp
50 55 60
Val Gln Ile Ser Ala Gly Trp Met His Ser Gly Tyr Pro Ile Met Cys
65 70 75 80
His Leu Glu Ser Val Lys Glu Ile Ile Asn Glu Met Asp Met Arg Ser
85 90 95
Arg Gly Val Trp Gly Pro Ile His Glu Leu Gly His Asn Gln Gln Arg
100 105 110
His Gly Trp Glu Phe Pro Pro His Thr Thr Glu Ala Thr Cys Asn Leu
115 120 125
Trp Ser Val Tyr Val His Glu Thr Val Leu Gly Ile Pro Arg Ala Gln
130 135 140
Ala His Glu Ala Leu Ser Pro Pro Glu Arg Glu Arg Arg Ile Lys Ala
145 150 155 160
His Leu Gly Lys Gly Ala Pro Leu Cys Asp Trp Asn Val Trp Thr Ala
165 170 175
Leu Glu Thr Tyr Leu Gln Val Leu Ser Arg Asn Ser Gly Arg Arg Gly
180 185 190
Val Asp Gly Arg Leu Val His Thr Cys Ile Lys Ala Gly Ala Val Arg
195 200 205
Trp Leu Ala Arg Gly Gln Thr Gly Lys Val Gly Val Asn Thr Asn Leu
210 215 220
Lys Asp Leu Cys Pro Leu Leu Ser Glu His Gly Leu Gln Cys Ser Leu
225 230 235 240
Glu Pro His Leu Asn Ser Asp Leu Cys Val Tyr Cys Cys Lys Ala Tyr
245 250 255
Ser Asp Lys Glu Ala Lys Gln Leu Gln Glu Phe Val Ala Glu Gly Gly
260 265 270
Gly Leu Leu Ile Gly Gly Gln Ala Trp Trp Trp Ala Ser Gln Asn Pro
275 280 285
Gly His Cys Pro Leu Ala Gly Phe Pro Gly Asn Ile Ile Leu Asn Cys
290 295 300
Phe Gly Leu Ser Ile Leu Pro Gln Thr Leu Lys Ala Gly Cys Phe Pro
305 310 315 320
Val Pro Thr Pro Glu Met Arg Ser Tyr His Phe Arg Lys Ala Leu Ser
325 330 335
Gln Phe Gln Ala Ile Leu Asn His Glu Asn Gly Asn Leu Glu Lys Ser
340 345 350
Cys Leu Ala Lys Leu Arg Val Asp Gly Ala Ala Phe Leu Gln Ile Pro
355 360 365
Ala Glu Gly Val Pro Ala Tyr Ile Ser Leu His Arg Leu Leu Arg Lys
370 375 380
Met Leu Arg Gly Ser Gly Leu Pro Ala Val Ser Arg Glu Asn Pro Val
385 390 395 400
Ala Ser Asp Ser Tyr Glu Ala Ala Val Leu Ser Leu Ala Thr Gly Leu
405 410 415
Ala His Ser Gly Thr Asp Cys Ser Gln Leu Ala Gln Gly Leu Gly Thr
420 425 430
Trp Thr Cys Ser Ser Ser Leu Tyr Pro Ser Lys His Pro Ile Thr Val
435 440 445
Glu Ile Asn Gly Ile Asn Pro Gly Asn Asn Asp Cys Trp Val Ser Thr
450 455 460
Gly Leu Tyr Leu Leu Glu Gly Gln Asn Ala Glu Val Ser Leu Ser Glu
465 470 475 480
Ala Ala Ala Ser Ala Gly Leu Arg Val Gln Ile Gly Cys His Thr Asp
485 490 495
Asp Leu Thr Lys Ala Arg Lys Leu Ser Arg Ala Pro Met Val Thr His
500 505 510
Gln Cys Trp Met Asp Arg Thr Glu Arg Ser Val Ser Cys Leu
515 520 525
<210> 3
<211> 8506
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900
gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960
ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc1020
tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg1080
accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag1140
ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac1200
ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata1260
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga1320
tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt1380
acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt1440
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga1500
agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga1560
agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga1620
tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct1680
tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg1740
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca1800
gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc1860
ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt1920
tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg1980
gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc2040
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga2100
gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc2160
agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc2220
agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca2280
tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat2340
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg2400
agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg2460
gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg2520
gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct2580
tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc2640
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga2700
ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga2760
tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg2820
gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg2880
agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga2940
ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga3000
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga3060
aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag3120
atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg3180
acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac3240
tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg3300
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga3360
agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt3420
ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta3480
aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg3540
ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg3600
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca3660
gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg3720
aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc3780
agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg3840
accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga3900
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg3960
gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc4020
agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga4080
gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc4140
ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg4200
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg4260
atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc4320
acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg4380
aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga4440
gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact4500
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga4560
caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga4620
aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct4680
tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg4740
actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg4800
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg4860
ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca4920
agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg4980
agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg5040
aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc5100
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact5160
acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg5220
ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc5280
aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca5340
agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg5400
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc5460
tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt5520
aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt5580
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc5640
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg5700
tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga5760
gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct5820
cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt5880
gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca5940
tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc6000
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc6060
ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc6120
cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc6180
gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg6240
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact6300
ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt6360
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa6420
atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag6480
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc6540
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt6600
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag6660
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg6720
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga6780
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat6840
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca6900
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc6960
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca7020
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg7080
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca7140
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata7200
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag7260
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg7320
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca7380
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta7440
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct7500
ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca7560
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag7620
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat7680
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt7740
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa7800
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga7860
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg7920
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc7980
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag8040
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc8100
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg8160
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac8220
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga8280
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt8340
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag8400
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg8460
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt 8506
<210> 4
<211> 23
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
atgcaggaga acctggcccc ctg 23
<210> 5
<211> 22
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
caggcagctc acgctcctct cg 22
<210> 6
<211> 20
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
ggaataattt ttggaacaaa 20