Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | lenti dCAS-VP64_Blast | Antibiotic Resistance | Ampicillin |
| Length | 14085 bp | Type | CRISPR Plasmids |
| Source | Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena | Copy Number | High copy number |
lenti dCAS-VP64_Blast vector Vector Map
lenti dCAS-VP64_Blast vector Sequence
LOCUS Exported 14085 bp ds-DNA circular SYN 06-1-2016
DEFINITION Zhang lab lentiviral vector encoding a blasticidin resistance marker
plus catalytically inactive dCas9 fused to the VP64 transcriptional
activation domain.
ACCESSION .
VERSION .
KEYWORDS lenti dCAS-VP64_Blast
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14085)
AUTHORS Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena
C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F.
TITLE Genome-scale transcriptional activation by an engineered CRISPR-Cas9
complex.
JOURNAL Nature 2015;517:583-8.
PUBMED 25494202
REFERENCE 2 (bases 1 to 14085)
AUTHORS Zhang Lab / Addgene #61425
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..14085
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
enhancer 238..617
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2440..2557
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2687..3865
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2918..3856
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
CDS 3878..3880
/codon_start=1
/product="start codon"
/note="ATG"
/translation="M"
CDS 3881..3928
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 3929..8029
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/note="dCas9"
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the N863A mutation in the HNH
catalytic domain"
/translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8084..8104
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/protein_id="
"
/translation="PKKKRKV"
CDS 8129..8278
/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 8300..8353
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/protein_id="
"
/translation="EGRGSLLTCGDVEENPGP"
CDS 8354..8752
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/note="BSD"
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
misc_feature 8777..9365
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9890..10070
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
polyA_signal 10102..10326
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 10372..10800
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10814..11143
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 10994..11129
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 11191..11238
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 11257..11631
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="BleoR"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 11761..11882
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(12333..12921)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13092..13952)
/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(13953..14057)
/gene="bla"
/note="AmpR promoter"
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