质粒名称: | pCAS |
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出品公司: | 爱迪基因 |
目录编号: | 60847 |
存储实验室: | Jamie Cate |
载体骨架基本信息 |
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载体名称: | 2-micron/pUC |
载体出品公司: | -- |
载体抗性: | 卡那霉素 |
空载体大小: | -- |
完整质粒大小: | 8761 bp |
载体修饰: | -- |
载体类型: | 细菌表达,酵母表达,CRISPR,合成生物学 |
筛选标记: | G418 |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | S. pyogenese Cas9 |
物种来源: | 化脓性链球菌 |
插入大小: | 4104 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | NLS/His8(插入件上的C端子) |
克隆方法: | -- |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | -- |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pCAS was a gift from Jamie Cate (Addgene plasmid # 60847 ; http://n2t.net/addgene:60847 ; RRID:Addgene_60847) |
参考文献部分: | Selection of chromosomal DNA libraries using a multiplex CRISPR system. Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, DeLoache W, Dueber JE, Arkin AP, Cate JH. Elife. 2014 Aug 19;3. doi: 10.7554/eLife.03703. 10.7554/eLife.03703 PubMed 25139909 |
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载体基本信息
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载体序列
LOCUS pCAS 8743 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Expresses S. pyogenes Cas9 plus an HDV ribozyme-sgRNA for genome editing in yeast. ACCESSION . VERSION . KEYWORDS pCAS SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8743) AUTHORS Ryan OW, Skerker JM, Maurer MJ, Li X, Tsai JC, Poddar S, Lee ME, DeLoache W, Dueber JE, Arkin AP, Cate JH TITLE Selection of chromosomal DNA libraries using a multiplex CRISPR system. JOURNAL Elife. 2014 Aug 19;3. doi: 10.7554/eLife.03703. PUBMED 25139909 REFERENCE 2 (bases 1 to 8743) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8743 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind complement(749..796) /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." misc_RNA 1169..1244 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" gene 1378..2734 /label=kanMX /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 1378..1721 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 1722..2531 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(1789..1808) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" terminator 2537..2734 /label=TEF terminator /note="Ashbya gossypii TEF terminator" CDS 3497..7600 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 7610..7630 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 7646..7669 /codon_start=1 /product="8xHis affinity tag" /label=8xHis /translation="HHHHHHHH" terminator 7673..7862 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" rep_origin complement(7919..8507) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7999..8018) /label=pBR322ori-F /note="pBR322 origin, forward primer" rep_origin 8529..768 /label=2u ori /note="yeast 2u plasmid origin of replication" ORIGIN 1 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg 61 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt 121 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt 181 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc 241 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc 301 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata 361 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat 421 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt 481 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag 541 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat 601 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc 661 aatattttag tagcccgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca 721 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact 781 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc 841 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata 901 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtatat gtgttatgta 961 gtatactctt tcttcaacaa ttaaatactc tcggtagcca agttggttta aggcgcaaga 1021 ctgtaattta tcactacgaa atcttgagat cgggcgttcg actcgccccc gggagagatg 1081 gccggcatgg tcccagcctc ctcgctggcg ccggctgggc aacaccttcg ggtggcgaat 1141 gggactttca taataacgtc caataaatgt tttagagcta gaaatagcaa gttaaaataa 1201 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tattttttgt 1261 cactattgtt atgtaaaatg ccacctctga cagtatggaa cgcaaacttc tgtctagtgg 1321 atataggtct agagatctgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 1381 atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 1441 gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 1501 tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 1561 ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 1621 ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 1681 ctaggataca gttctcacat cacatccgaa cataaacaac catgggtaag gaaaagactc 1741 acgtttcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 1801 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 1861 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 1921 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 1981 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggcaaaaca gcattccagg 2041 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 2101 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc 2161 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg 2221 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 2281 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 2341 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 2401 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 2461 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg 2521 agtttttcta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt 2581 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt 2641 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg 2701 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca 2761 tccagtgtcg aaaacgagct ctcgagaacc cttaatagtc gaacaagaag caggcaaagt 2821 ttagagcact gcccctccgc actcaaaaaa gaaaaaacta ggaggaaaat aaaattctca 2881 accacacaaa cacataaaca catacaaata caaatacaag cttatttact tgacatcgcg 2941 cgatcttcca ctattcagcg ccgtccgccc tctctcgtgt tttttgttta cgcgacaact 3001 atgcgaaatc cggagcaacg ggcaaccgtt tggggaaaga ccacacccac gcgcgatcgc 3061 catggcaacg aggtcgcaca cgccccacac ccagacctcc ctgcgagcgg gcatgggtac 3121 aatgtccccg ttgccacaga caccacttcg tagcacagcg cagagcgtag cgtgttgttg 3181 ctgctgacaa aagaaaattt ttcttagcaa agcaaaggag gggaagcacg ggcagatagc 3241 accgtaccat acccttggaa actcgaaatg aacgaagcag gaaatgagag aatgagagtt 3301 ttgtaggtat atatagcggt agtgtttgcg cgttaccatc atcttctgga tctatctatt 3361 gttcttttcc tcatcacttt cccctttttc gctcttcttc ttgtctttta tttctttctt 3421 ttttttaatt gttccctcga ttggctatct accaaagaat ccaaacttaa tacacgtatt 3481 tatttgtcca attaccatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag 3541 cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct 3601 gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag 3661 tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg 3721 gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga 3781 tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg 3841 tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat 3901 ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta 3961 tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa 4021 tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt 4081 atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg 4141 attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa 4201 tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa 4261 ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt 4321 agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa 4381 tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc 4441 tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt 4501 aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc 4561 aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt 4621 tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg 4681 tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca 4741 cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga 4801 caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt 4861 ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc 4921 atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat 4981 gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta 5041 tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg 5101 aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac 5161 aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt 5221 tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca 5281 tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat 5341 cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag 5401 acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg 5461 ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc 5521 tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca 5581 gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg 5641 acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa 5701 aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa 5761 gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa 5821 aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat 5881 tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta 5941 tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga 6001 ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa 6061 ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt 6121 agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg 6181 taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg 6241 ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt 6301 ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt 6361 gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt 6421 acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac 6481 tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt 6541 ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa 6601 atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg 6661 agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga 6721 taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt 6781 caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa 6841 ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga 6901 tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa 6961 gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga 7021 aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat 7081 cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc 7141 tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt 7201 tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa 7261 acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga 7321 attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa 7381 caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt 7441 gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg 7501 atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct 7561 ttatgaaaca cgcattgatt tgagtcagct aggaggtgac ggtggagggc caaaaaagaa 7621 aagaaaagtt gaagatgctt ctgctcatca tcaccatcac catcaccatt aaatccgctc 7681 taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag 7741 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac 7801 gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga 7861 agtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 7921 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 7981 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 8041 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 8101 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 8161 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 8221 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 8281 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 8341 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 8401 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 8461 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 8521 tcttttctac cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc 8581 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag 8641 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag 8701 agcgctaatt tttcaaacaa agaatctgag ctgcattttt aca //
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载体质粒应用举例
eSpCas9-LentiCRISPR v2
2020-02-14