| 质粒名称: | pCas9(D10A) |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 74495 |
| 存储实验室: | Xiao Wang |
|
载体骨架基本信息 |
|
| 载体名称: | pWTCas9 |
| 载体出品公司: | Derived from Addgene #44250 |
| 完整质粒大小: | 6932bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 细菌细胞表达、合成生物 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Cas9 D10A / Nicking Cas9 |
| 物种来源: | Synthetic |
| 基因突变: | D10A |
| 启动子: | pTeto |
| 克隆方法: | 未知 |
| 5’测序引物 | AAGCAGCTCTAATGCGCT |
|
文献引用方法 |
|
| 材料和方法部分: | pCas9(D10A) was a gift from Xiao Wang (Addgene plasmid # 74495 ; http://n2t.net/addgene:74495 ; RRID:Addgene_74495) |
| 参考文献部分: | Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases. Standage-Beier K, Zhang Q, Wang X. ACS Synth Biol. 2015 Nov 20;4(11):1217-25. doi: 10.1021/acssynbio.5b00132. Epub 2015 Oct 25. 10.1021/acssynbio.5b00132 PubMed 26451892 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
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载体简介
载体序列
LOCUS pCas9(D10A) 6932 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Nicking Cas9 derived from pWT Cas9 (Addgene #44250). Ampicillin resistance marker, the tetracycline repressor (TetR), a ColE1 origin of replication and Streptococcus pyogenes Cas9 D10A. ACCESSION . VERSION . KEYWORDS pCas9(D10A) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6932) AUTHORS Standage-Beier K, Zhang Q, Wang X TITLE Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases. JOURNAL ACS Synth Biol. 2015 Nov 20;4(11):1217-25. doi: 10.1021/acssynbio.5b00132. Epub 2015 Oct 25. PUBMED 26451892 REFERENCE 2 (bases 1 to 6932) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6932 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(7..630) /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind 538..557 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter 649..704 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 685..703 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" RBS 722..733 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 740..4846 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" terminator 4870..4941 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4957..4984 /label=T7Te terminator /note="phage T7 early transcription terminator" rep_origin complement(5151..5739) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5231..5250) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5827..5921 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5951..6811) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6574..6593 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6812..6916) /gene="bla" /label=AmpR promoter ORIGIN 1 gcatgcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta 661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc 721 taaagaggag aaaggatcta tggataagaa atactcaata ggcttagcta tcggcacaaa 781 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt 841 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga 901 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg 961 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga 1021 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga 1081 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac 1141 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat 1201 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt 1261 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca 1321 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc 1381 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa 1441 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc 1501 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga 1561 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa 1621 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa 1681 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct 1741 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca 1801 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa 1861 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa 1921 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat 1981 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa 2041 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc 2101 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac 2161 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg 2221 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct 2281 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat 2341 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa 2401 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg 2461 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta 2521 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga 2581 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga 2641 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg 2701 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca 2761 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat 2821 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc 2881 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa 2941 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca 3001 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca 3061 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca 3121 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta 3181 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag 3241 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa 3301 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga 3361 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca 3421 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc 3481 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat 3541 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa 3601 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa 3661 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg 3721 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa 3781 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc 3841 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa 3901 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg 3961 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat 4021 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag 4081 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt 4141 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc 4201 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt 4261 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt 4321 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct 4381 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa 4441 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca 4501 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag 4561 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata 4621 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac 4681 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa 4741 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg 4801 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaactcg agtaaggatc 4861 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 4921 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc 4981 tgcgtttata cctagggaga cgtcggcgtt cggctgcggc gagcggtatc agctcactca 5041 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 5101 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 5161 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 5221 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 5281 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 5341 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 5401 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5461 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5521 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5581 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 5641 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 5701 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 5761 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgactagt 5821 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag 5881 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgtaaactt 5941 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 6001 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 6061 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 6121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6241 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6541 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6901 aaataggggt tccgcgcaca tttccccgaa aa //
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