Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012283 | pcDNA-dCas9-p300 Core (D1399Y) | 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:
- pcDNA-dCas9-p300 Core (D1399Y)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11475 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy
- Selection Marker:
- Neomycin/G418(Geneticin)
- Copy Number:
- High copy number
pcDNA-dCas9-p300 Core (D1399Y) vector 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.
pcDNA-dCas9-p300 Core (D1399Y) vector Sequence
LOCUS pcDNA-dCas9-p300 11475 bp DNA circular SYN 01-JAN-1980
DEFINITION Control plasmid for CRISPR activation, encoding catalytically
inactive dCas9 plus an inactivated form of the p300 core
transcriptional activation domain.
ACCESSION .
VERSION .
KEYWORDS pcDNA-dCas9-p300 Core (D1399Y)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11475)
AUTHORS Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy
TE, Gersbach CA.
TITLE Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates
genes from promoters and enhancers.
JOURNAL Nat. Biotechnol. 2015;33:510-7.
PUBMED 25849900
REFERENCE 2 (bases 1 to 11475)
AUTHORS Gersbach Lab / Addgene #61358
TITLE Direct Submission
REFERENCE 3 (bases 1 to 11475)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Biotechnol."; date: "2015"; volume: "33"; pages: "510-7"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11475
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 904..906
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 907..972
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 979..999
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1009..5112
/label=dCas9
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5137..5157
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 5167..7017
/label=p300 core (D1399Y)
/note="catalytic histone acetyltransferase core of the
human E1A-associated protein p300, inactivated by the
D1399Y point mutation (Hilton et al., 2015)"
CDS 7018..7044
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
polyA_signal 7075..7299
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 7345..7773
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7787..8116
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 8183..8974
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
polyA_signal 9151..9284
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter complement(9369..9399)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(9414..9435)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(9723..10308)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10482..11339)
/label=AmpR
/note="beta-lactamase"
promoter complement(11340..11444)
/label=AmpR promoter