载体基本信息 | |||
载体名称 | pMSCV-LTR-dCas9-VP64-BFP | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 11369 bp | 载体类型 | Mammalian Expression, Retroviral, CRISPR |
筛选标记 | Puromycin | 拷贝数 | Low Copy |
启动子 | PGK | 5'引物 | CGGGCGCCCGAAGGTCCTCCG |
3'引物 | GAGACGTGCTACTTCCATTTGTC |
pMSCV-LTR-dCas9-VP64-BFP 载体载体图谱
pMSCV-LTR-dCas9-VP64-BFP 载体载体序列
LOCUS Exported 11369 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x NLS, VP64 and tagBFP. ACCESSION . VERSION . KEYWORDS pMSCV-LTR-dCas9-VP64-BFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11369) AUTHORS Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS TITLE CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. JOURNAL Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044. PUBMED 23849981 REFERENCE 2 (bases 1 to 11369) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044." FEATURES Location/Qualifiers source 1..11369 /organism="synthetic DNA construct" /mol_type="other DNA" LTR 1..517 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" misc_feature 581..922 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 989..1405 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1335..1357 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1373..1389 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" regulatory 1419..1428 /regulatory_class="other" /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1425..5528 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5538..5558 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5574..5594 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5601..5750 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 5757..5777 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5784..6479 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" promoter 6514..7013 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(6549..6572) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 6946..6965 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 7034..7633 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(7034..7053) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 7530..7550 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 7733..8247 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" primer_bind complement(8416..8432) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8416..8432) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8429..8451) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8440..8456 /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(8464..8494) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8509..8530 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(8647..8664) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8818..9406) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8898..8917) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9577..10437) /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 10200..10219 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10438..10542) /gene="bla" /label=AmpR promoter primer_bind 10610..10628 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(10666..10688) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 10788..10807 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 11001..11023 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 11151..11170 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer"
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