质粒名称: | MSP1101,pMSP1101-SpCas9VRER |
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出品公司: | 爱迪基因 |
目录编号: | 65773 |
存储实验室: | Keith Joung |
载体骨架基本信息 |
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载体名称: | JDS246 |
载体出品公司: | Joung lab (Addgene Plasmid # 43861) |
载体抗性: | 氨苄青霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 哺乳细胞表达,CRISPR |
筛选标记: | -- |
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: | mammalian codon-optimized Streptococcus pyogenes Cas9 (D1135V/G1218R/R1335E/T1337R)-NLS-3XFlag |
物种来源: | -- |
插入大小: | -- |
基因突变: | D1135V, G1218R, R1335E, and T1337R mutations in Cas9 |
GenBank编号 | -- |
启动子 | CMV & T7 |
标签/融合蛋白: |
NLS(插接件上的C端子)
3x FLAG(插接件上的C端子)
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克隆方法: | Gibson克隆 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | CMV-F |
3' 测序引物 | BGH-rev |
文献引用方法 |
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材料和方法部分: | MSP1101 was a gift from Keith Joung (Addgene plasmid # 65773 ; http://n2t.net/addgene:65773 ; RRID:Addgene_65773) |
参考文献部分: | Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369 |
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载体基本信息
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载体序列
LOCUS MSP1101 7614 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Human expression vector for SpCas9 VRER variant: CMV-T7-humanSpCas9(D1135V/G1218R/R1335E/T1337R)-NLS-3xFLAG (VRER variant). ACCESSION . VERSION . KEYWORDS MSP1101 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7614) AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities. JOURNAL Nature. 2015 Jun 22. doi: 10.1038/nature14592. PUBMED 26098369 REFERENCE 2 (bases 1 to 7614) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7614 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 403..412 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 409..4512 /codon_start=1 /product="Streptococcus pyogenes Cas9 endonuclease with the D1135V/G1218R/R1335E/T1337R mutations" /label=Cas9 VRER /note="has an altered recognition specificity for the protospacer adjacent motif (PAM) (Kleinstiver et al., 2015)" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 4519..4539 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4546..4611 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4629..4646 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4669..4686) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4675..4899 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4970..4986) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4970..4986) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4983..5005) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4994..5010 /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(5018..5048) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5063..5084 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5201..5218) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5372..5960) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5452..5471) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6131..6991) /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 6754..6773 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6992..7096) /gene="bla" /label=AmpR promoter primer_bind complement(7175..7194) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 7366..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag 421 tattctattg gtttagacat cggcactaat tccgttggat gggctgtcat aaccgatgaa 481 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 541 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 601 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 661 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 721 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 781 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 841 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 901 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 961 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1021 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1081 ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1141 tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1201 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 1261 cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 1321 atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 1381 tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 1441 gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 1501 ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 1561 gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 1621 ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 1681 aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 1741 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 1801 atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 1861 ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 1921 gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 1981 acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2041 aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2101 aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2161 gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 2221 gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 2281 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 2341 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 2401 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 2461 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 2521 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 2581 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 2641 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 2701 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 2761 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 2821 caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 2881 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa 2941 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3001 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3061 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3121 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3181 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 3241 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 3301 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 3361 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 3421 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 3481 agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 3541 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 3601 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 3661 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 3721 ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 3781 gactgggacc cgaaaaagta cggtggcttc gtgagcccta cagttgccta ttctgtccta 3841 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 3901 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 3961 aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4021 gagttagaaa atggccgaaa acggatgttg gctagcgcca gagagcttca aaaggggaac 4081 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4141 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4201 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 4261 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 4321 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 4381 aagtattttg acacaacgat agatcgcaaa gagtacagat ctaccaagga ggtgctagac 4441 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 4501 cttgggggtg acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat 4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga 4621 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc 4681 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 4741 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4801 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 4861 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 4921 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 4981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 5041 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 5161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 5221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 5281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 5341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 6061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 6121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 6181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 6241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 6301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 7021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 7081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 7141 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt 7201 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa 7261 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta 7321 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga 7381 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 7441 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 7501 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 7561 aatgggtgga ctatttacgg taaactgccc acttggcagt acatcaagtg tatc //
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