载体基本信息 | |||
载体名称 | Cas9m4 | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 9553 bp | 载体类型 | CRISPR Plasmids |
载体来源 | Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang | 筛选标记 | Neomycin/G418(Geneticin) |
拷贝数 | High copy number |
Cas9m4 载体载体图谱
Cas9m4 载体载体序列
LOCUS Exported 9553 bp ds-DNA circular SYN 06-1-2016 DEFINITION Plasmid for expression in mammalian cells of catalytically dead Cas9m4. ACCESSION . VERSION . KEYWORDS Cas9m4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9553) AUTHORS Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM. TITLE CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. JOURNAL Nat. Biotechnol. 2013;31:833-8. PUBMED 23907171 REFERENCE 2 (bases 1 to 9553) AUTHORS Church Lab / Addgene #47316 TITLE Direct Submission FEATURES Location/Qualifiers source 1..9553 /organism="synthetic DNA construct" /lab_host="Mammalian Cells" /mol_type="other DNA" polyA_signal 270..391 /note="SV40 poly(A) signal" /note="SV40 polyadenylation signal" CDS complement(567..1361) /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" promoter complement(1428..1757) /note="SV40 promoter" /note="SV40 enhancer and early promoter" rep_origin 1442..1577 /note="SV40 ori" /note="SV40 origin of replication" rep_origin complement(1771..2199) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" polyA_signal 2401..2449 /note="HSV TK poly(A) signal" /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" CDS complement(2548..2568) /codon_start=1 /locus_tag=" " /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /note=" " /protein_id=" " /translation="PKKKRKV" CDS complement(2581..6684) /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /note="Cas9m4" /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D10A, D839A, H840A, and N863A (Mali et al., 2013)" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" promoter complement(6799..7002) /note="CMV promoter" /note="human cytomegalovirus (CMV) immediate early promoter" enhancer 7003..7382 /note="CMV enhancer" /note="human cytomegalovirus immediate early enhancer" promoter 7648..7752 /gene="bla" /note="AmpR promoter" CDS 7753..8613 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGVRVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 8784..9372 /direction=RIGHT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
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