载体基本信息 | |||
载体名称 | hCas9 | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 9553 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, | 筛选标记 | Neomycin/G418(Geneticin) |
拷贝数 | High copy number |
hCas9 载体载体图谱
hCas9 载体载体序列
LOCUS Exported 9553 bp ds-DNA circular SYN 06-1-2016 DEFINITION Mammalian vector with a G418 resistance marker for expressing human codon-optimized Cas9. ACCESSION . VERSION . KEYWORDS hCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9553) AUTHORS Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. TITLE RNA-guided human genome engineering via Cas9. JOURNAL Science 2013;339:823-6. PUBMED 23287722 REFERENCE 2 (bases 1 to 9553) AUTHORS Church Lab / Addgene #41815 TITLE Direct Submission COMMENT Minor sequence errors were corrected using the Addgene sequencing results. FEATURES Location/Qualifiers source 1..9553 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" enhancer 50..429 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 430..633 /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" CDS 748..4851 /codon_start=1 /locus_tag=" " /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9" /note="generates RNA-guided double strand breaks in DNA" /protein_id=" " /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 4864..4884 /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" polyA_signal 4983..5031 /note="HSV TK poly(A) signal" /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5233..5661 /direction=RIGHT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 5675..6004 /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 5855..5990 /note="SV40 ori" /note="SV40 origin of replication" CDS 6071..6865 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /note="NeoR/KanR" /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" polyA_signal 7041..7162 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" rep_origin complement(7613..8201) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8372..9232) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(9233..9337) /gene="bla" /note="AmpR promoter"
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