质粒名称: | pSU-araC-Cas9 |
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出品公司: | Addgene |
目录编号: | 120423 |
存储实验室: | Sheng Yang |
载体骨架基本信息 |
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载体名称: | p15A |
完整质粒大小: | 7795 bp |
载体类型: | Bacterial Expression, CRISPR |
载体抗性: | Chloramphenicol |
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | Cas9 |
启动子: | araBAD |
克隆方法: | Gibson Cloning |
5’测序引物 | pBAD Forward |
文献引用方法 |
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材料和方法部分: | pSU-araC-Cas9 was a gift from Sheng Yang (Addgene plasmid # 120423 ; http://n2t.net/addgene:120423 ; RRID:Addgene_120423) |
参考文献部分: | Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14. 10.1128/AEM.04023-14 PubMed 25636838 |
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LOCUS pSU-araC-Cas9 7795 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. ACCESSION . VERSION . KEYWORDS pSU-araC-Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7795) AUTHORS Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S TITLE Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system. JOURNAL Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14. PUBMED 25636838 REFERENCE 2 (bases 1 to 7795) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..7795 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 134..153 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(220..322) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" primer_bind 709..731 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 723..740 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 724..740 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS complement(855..1733) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 1760..2044 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 1967..1986 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" RBS 2071..2079 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 2088..6194 /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" primer_bind complement(6266..6282) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6266..6282) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6279..6301) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6290..6306 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6314..6344) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6359..6380 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6615..6632) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6749..7294) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(6829..6848) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7355..219) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" ORIGIN 1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgccattcg ccattcaggc 601 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 661 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 721 gttgtaaaac gacggccagt gccaagcttg catgcctgca gcatgagcgg atacatattt 781 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 841 cctgcatcga tttattatga caacttgacg gctacatcat tcactttttc ttcacaaccg 901 gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga gaagtagagt 961 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa 1021 agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac 1081 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg 1141 gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc 1201 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat 1261 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta 1321 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac 1381 cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga 1441 aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc 1501 tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt 1561 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt 1621 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga 1681 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc 1741 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac 1801 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt 1861 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac 1921 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt 1981 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct 2041 ccatacccgt ttttttggga attcgagctc taaggaggtt ataaaaaatg gataagaaat 2101 actcaatagg cttagatatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat 2161 ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa 2221 aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca 2281 aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga 2341 ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt 2401 ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg 2461 aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt 2521 ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc 2581 gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat 2641 ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg 2701 gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc 2761 tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc attgctttgt 2821 cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac 2881 agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa attggagatc 2941 aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata 3001 tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct 3061 acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag 3121 aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg 3181 ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg 3241 gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct 3301 ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa 3361 gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga 3421 cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga 3481 tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag 3541 gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg 3601 aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga 3661 caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga 3721 agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa 3781 aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag 3841 atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag 3901 attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct 3961 tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg 4021 ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa 4081 aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat 4141 cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta 4201 aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg 4261 caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg 4321 atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac 4381 gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg 4441 aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc 4501 aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg 4561 accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt gttccacaaa 4621 gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg 4681 gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac 4741 aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac 4801 gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc 4861 gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg 4921 aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg 4981 acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc 5041 atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg 5101 aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt 5161 ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact 5221 tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa 5281 ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca 5341 aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat 5401 tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag 5461 actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag 5521 tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag 5581 ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta 5641 aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg 5701 agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg 5761 agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt 5821 tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt 5881 atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg 5941 ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac 6001 aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta 6061 aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg 6121 ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc 6181 taggaggtga ctgaagtata ttttagatga agattatttc ttaatctaga ggatccccgg 6241 gtaccgagct cgaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc 6301 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 6361 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 6421 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 6481 ggcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt 6541 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg 6601 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg 6661 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg 6721 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc 6781 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg 6841 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg 6901 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag 6961 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc 7021 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca 7081 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta 7141 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga 7201 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga 7261 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa 7321 atatttctag gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc 7381 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat 7441 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg 7501 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac 7561 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg 7621 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat 7681 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt 7741 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacg //
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