Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V013723 | pAAV-U6-sgRNA-EnCMV-Cre-WPRE | 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:
- pAAV-U6-sgRNA-EnCMV-Cre-WPRE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5982 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Adeno-associated virus
- Promoter:
- U6
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pAAV-U6-sgRNA-EnCMV-Cre-WPRE 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.
pAAV-U6-sgRNA-EnCMV-Cre-WPRE vector Sequence
LOCUS 62056_2555 5982 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5982)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5982
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(462..473)
/codon_start=1
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
/translation="IEGR"
polyA_signal complement(622..743)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1271..1859)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2033..2890)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(2891..2995)
/label=AmpR promoter
rep_origin complement(3021..3476)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
protein_bind 3728..3748
/label=attB4
/note="core recombination site for the Gateway(R) BP
reaction"
promoter 3780..4020
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4030..4059
/label=RfxCas13d DR30
/note="5' processed 30-base direct repeat for an RfxCas13d
gRNA (Konermann et al., 2018)"
enhancer 4141..4444
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4445..4648
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 4695..4719
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 4765..4785
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 4804..5832
/codon_start=1
/label=Cre
/note="site-specific recombinase"
/translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
DGD"
CDS 5851..5898
/codon_start=1
/label=nucleoplasmin NLS
/note="bipartite nuclear localization signal from
nucleoplasmin"
/translation="KRPAATKKAGQAKKKK"
protein_bind complement(5933..5957)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 5973..5982
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"