Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012306 | pAC152-dual-dCas9VP64-sgExpression | 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:
- pAC152-dual-dCas9VP64-sgExpression
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8625 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan
- Copy Number:
- High copy number
- Promoter:
- CBh
pAC152-dual-dCas9VP64-sgExpression 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.
pAC152-dual-dCas9VP64-sgExpression vector Sequence
LOCUS pAC152-dual-dCas 8625 bp DNA circular SYN 01-JAN-1980
DEFINITION CRISPR activation vector for co-expressing a single guide RNA
(sgRNA) with catalytically inactive dCas9 fused to the VP64
transcriptional activation domain.
ACCESSION .
VERSION .
KEYWORDS pAC152-dual-dCas9VP64-sgExpression
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8625)
AUTHORS Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan
S, Shivalila CS, Dadon DB, Jaenisch R.
TITLE Multiplexed activation of endogenous genes by CRISPR-on, an
RNA-guided transcriptional activator system.
JOURNAL Cell Res. 2013;23:1163-71.
PUBMED 23979020
REFERENCE 2 (bases 1 to 8625)
AUTHORS Jaenisch Lab / Addgene #48238
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8625)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell Res.";
date: "2013"; volume: "23"; pages: "1163-71"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8625
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..241
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 268..343
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 344..349
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
enhancer 440..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 727..1004
/label=chicken beta-actin promoter
intron 1005..1233
/note="hybrid intron"
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1251..1253
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 1254..1280
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/note="HA"
/translation="YPYDVPDYA"
CDS 1284..1304
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1314..5414
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5418..5438
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 5472..5621
/label=VP64
/note="tetrameric repeat of the minimal activation domain
of herpes simplex virus VP16 (Beerli et al., 1998)"
polyA_signal 5672..5879
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 5888..6028
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 6103..6558
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6840..6944
/label=AmpR promoter
CDS 6945..7802
/label=AmpR
/note="beta-lactamase"
rep_origin 7976..8564
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"