Cas9m4-VP64 vector (V012350#)

Basic Vector Information

Basic Vector Information
Vector Name Cas9m4-VP64 Antibiotic Resistance Ampicillin
Length 9771 bp Type CRISPR Plasmids
Source Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang Selection Marker Neomycin/G418(Geneticin)
Copy Number High copy number

Cas9m4-VP64 vector Vector Map

Cas9m4-VP649771 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400880092009600SV40 poly(A) signalNeoR/KanRSV40 promoterf1 oriHSV TK poly(A) signalVP64SV40 NLSCas9m4SV40 NLSATGCMV promoterCMV enhancerAmpR promoterAmpRori

Plasmid Resuspension Protocol:

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5.Store the plasmid at -20 ℃.

Cas9m4-VP64 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                9771 bp ds-DNA    circular SYN 06-1-2016
DEFINITION  Plasmid for expression in mammalian cells of catalytically dead 
            Cas9m4 fused to the VP64 transcriptional activation domain.
ACCESSION   .
VERSION     .
KEYWORDS    Cas9m4-VP64
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9771)
  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 9771)
  AUTHORS   Church Lab / Addgene #47319
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9771
                     /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(2559..2708)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /note="VP64"
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(2733..2753)
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     "
                     /protein_id="
                     "
                     /translation="PKKKRKV"
     CDS             complement(2766..6869)
                     /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"
     CDS             complement(6879..6899)
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /note="
                     "
                     /protein_id="
                     "
                     /translation="PKKKRKV"
     CDS             complement(6900..6902)
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     promoter        complement(7017..7220)
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        7221..7600
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7866..7970
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             7971..8831
                     /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"
     rep_origin      9002..9590
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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