Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012308 | pAc-sgRNA-Cas9 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pAc-sgRNA-Cas9
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10663 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Bassett AR, Tibbit C, Ponting CP, Liu JL.
- Copy Number:
- High copy number
- Promoter:
- Ac5
pAc-sgRNA-Cas9 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.
pAc-sgRNA-Cas9 vector Sequence
LOCUS pAc-sgRNA-Cas9. 10663 bp DNA circular SYN 01-JAN-1980
DEFINITION Plasmid for expressing a single guide RNA (sgRNA) together with Cas9
in Drosophila cells.
ACCESSION .
VERSION .
KEYWORDS pAc-sgRNA-Cas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10663)
AUTHORS Bassett AR, Tibbit C, Ponting CP, Liu JL.
TITLE Mutagenesis and homologous recombination in Drosophila cell lines
using CRISPR/Cas9.
JOURNAL Biol Open 2014;3:42-9.
PUBMED 24326186
REFERENCE 2 (bases 1 to 10663)
AUTHORS Liu Lab / Addgene #49330
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10663)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Biol Open";
date: "2014"; volume: "3"; pages: "42-9"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10663
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator 7..12
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
misc_RNA complement(13..88)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(107..506)
/label=dU6-2 promoter
/note="RNA polymerase III promoter for Drosophila U6-2
snRNA (Das et al., 1987)"
promoter 507..2948
/label=Ac5 promoter
/note="Drosophila melanogaster actin 5c promoter"
CDS 2989..2991
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 2992..3057
/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 3064..3084
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 3109..7209
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 7210..7257
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 7273..7326
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 7351..7947
/label=PuroR
/note="puromycin N-acetyltransferase"
polyA_signal complement(7958..8092)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(8714..9302)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9476..10333)
/label=AmpR
/note="beta-lactamase"
promoter complement(10334..10438)
/label=AmpR promoter