Basic Vector Information
- Vector Name:
- 3xFLAG-dCas9/pCMV-7.1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8848 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Fujita T, Fujii H.
- Copy Number:
- High copy number
- Promoter:
- SV40
3xFLAG-dCas9/pCMV-7.1 vector Vector Map
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 ℃.
3xFLAG-dCas9/pCMV-7.1 vector Sequence
LOCUS 3xFLAG-dCas9_pCM 8848 bp DNA circular SYN 01-JAN-1980
DEFINITION Plasmid for expression in mammalian cells of FLAG(R)-tagged
catalytically inactive dCas9, for engineered ChIP (enChIP)
purification of specific genomic regions.
ACCESSION .
VERSION .
KEYWORDS 3xFLAG-dCas9 pCMV-7.1.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8848)
AUTHORS Fujita T, Fujii H.
TITLE Efficient isolation of specific genomic regions and identification
of associated proteins by engineered DNA-binding molecule-mediated
chromatin immunoprecipitation (enChIP) using CRISPR.
JOURNAL Biochem. Biophys. Res. Commun. 2013;439:132-6.
PUBMED 23942116
REFERENCE 2 (bases 1 to 8848)
AUTHORS Fujii Lab / Addgene #47948
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8848)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Biochem.
Biophys. Res. Commun."; date: "2013"; volume: "439"; pages: "132-6"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8848
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 141..157
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
enhancer 318..697
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 698..901
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 928..930
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 931..996
/label=3xFLAG
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
CDS 1000..5103
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5116..5136
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
polyA_signal 5189..5811
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
promoter 5840..6169
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
promoter complement(6208..6226)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6240..6256)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6264..6280)
/label=lac operator
/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(6288..6318)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6333..6354)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6642..7230)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7404..8261)
/label=AmpR
/note="beta-lactamase"
promoter complement(8262..8366)
/label=AmpR promoter
rep_origin 8393..8848
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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