| 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"
我们不提供该载体，载体信息仅供查询和参考。