Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007261 | AAV-U6gRNA1-U6gRNA2-TnT-Cre | 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:
- AAV-U6gRNA1-U6gRNA2-TnT-Cre
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5958 bp
- Type:
- AAV
- Replication origin:
- ori
- Promoter:
- cTnT
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- ACGACTCACTATAGGCTAGC
- 3' Primer:
- AGGGACTTCGGGCACAATCG
AAV-U6gRNA1-U6gRNA2-TnT-Cre 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.
AAV-U6gRNA1-U6gRNA2-TnT-Cre vector Sequence
LOCUS 40924_160 5958 bp DNA circular SYN 13-MAY-2021
DEFINITION AAV vector for U6 driven expression of two gRNAs, and cardiomyocyte
specific expression of Cre recombinase..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5958)
AUTHORS Guo Y, VanDusen NJ, Zhang L, Gu W, Sethi I, Guatimosim S, Ma Q,
Jardin BD, Ai Y, Zhang D, Chen B, Guo A, Yuan GC, Song LS, Pu WT
TITLE Analysis of Cardiac Myocyte Maturation Using CASAAV, A Platform for
Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.
JOURNAL Circ Res. 2017 Mar 29. pii: CIRCRESAHA.116.310283. doi:
10.1161/CIRCRESAHA.116.310283.
PUBMED 28356340
REFERENCE 2 (bases 1 to 5958)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5958)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Circ Res.
2017 Mar 29. pii: CIRCRESAHA.116.310283. doi:
10.1161/CIRCRESAHA.116.310283."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5958
/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 278..353
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 366..606
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 633..708
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 721..1133
/label=cTnT
/note="Chicken cardiac troponin T promoter"
intron 1260..1392
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 1437..1455
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1467..1487
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1485..2513
/codon_start=1
/label=Cre
/note="site-specific recombinase"
/translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
DGD"
polyA_signal 2554..3030
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
repeat_region 3070..3210
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 3285..3740
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3757..3776)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3876..3898
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(3936..3954)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4022..4126
/label=AmpR promoter
CDS 4127..4984
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5158..5746
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
repeat_region 5808..5937
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"