Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001295 | pSAG1::CAS9-U6::sgUPRT | 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
Encodes CAS9 nuclease (GFP fusion) under control of the Toxoplasma gondii SAG1 promotor. Vector also provides U6 controlled expression of a single guide RNA for disruption of genes by CRISPR.
- Vector Name:
- pSAG1::CAS9-U6::sgUPRT
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9674 bp
- Type:
- CRISPR/Cas system, Toxoplasma gondii expression ve
- Replication origin:
- ori
- Promoter:
- SAG1
pSAG1::CAS9-U6::sgUPRT 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.
pSAG1::CAS9-U6::sgUPRT vector Sequence
LOCUS 40924_38443 9674 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9674)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9674)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9674
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_RNA 559..633
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(658..676)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(697..713)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(721..737)
/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(745..775)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(790..811)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(1099..1687)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1861..2718)
/label=AmpR
/note="beta-lactamase"
promoter complement(2719..2823)
/label=AmpR promoter
rep_origin complement(2849..3304)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 3446..3462
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 3472..3490
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 3516..3532
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
CDS 4358..8461
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 8465..8491
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/note="HA"
/translation="YPYDVPDYA"
CDS 8510..8530
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 8537..8557
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 8597..9310
/label=superfolder GFP
/note="GFP variant that folds robustly even when fused to
poorly folded proteins (Pedelacq et al., 2006)"
primer_bind complement(9635..9651)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"