Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012347 | dCAS9-VP64_GFP | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- dCAS9-VP64_GFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14382 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena
- Copy Number:
- High copy number
- Promoter:
- EF-1α
dCAS9-VP64_GFP vector Map
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
dCAS9-VP64_GFP vector Sequence
LOCUS V012347 14382 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V012347
VERSION V012347
KEYWORDS dCAS9-VP64_GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14382)
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 14382)
AUTHORS Zhang Lab / Addgene #61422
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14382)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature";
date: "2015"; volume: "517"; pages: "583-8"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14382
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 238..617
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 618..820
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 2291..2332
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2440..2557
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2682..3859
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3872..3874
/codon_start=1
/product="start codon"
/label="start codon"
/note="ATG"
/translation="M"
CDS 3878..3898
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 3908..8008
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 8063..8083
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 8108..8257
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label="tetrameric repeat of the minimal activation dom"
/note="VP64"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 8279..8332
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="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."
/translation="EGRGSLLTCGDVEENPGP"
CDS 8333..9046
/label="EGFP"
/note="enhanced GFP"
misc_feature 9074..9662
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10187..10367
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10399..10623
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 10669..11097
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11111..11440
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 11488..11535
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 11554..11925
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 12058..12191
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(12276..12306)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(12321..12342)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(12630..13218)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13392..14249)
/label="AmpR"
/note="beta-lactamase"
promoter complement(14250..14354)
/label="AmpR promoter"