Basic Vector Information
- Vector Name:
- AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6362 bp
- Type:
- Mammalian Expression, Mouse Targeting, AAV, Cre/Lo
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CBh
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- ctgagcaagaggtaagggtttaagg
- 3' Primer:
- cacatagcgtaaaaggagcaacatag
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR vector Vector Map
Plasmid Resuspension 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 ℃.
AAV:ITR-U6-sgRNA(backbone)-pCBh-Cre-WPRE-hGHpA-ITR vector Sequence
LOCUS 40924_145 6362 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses Cre recombinase from the Cbh promoter and one U6-driven
sgRNA. AAV backbone with SapI spacer for sgRNA cloning..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6362)
AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE,
Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S,
Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A,
Feng G, Sharp PA, Zhang F
TITLE CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.
JOURNAL Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi:
10.1016/j.cell.2014.09.014.
PUBMED 25263330
REFERENCE 2 (bases 1 to 6362)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6362)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2014
Sep 24. pii: S0092-8674(14)01163-5. doi:
10.1016/j.cell.2014.09.014."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6362
/mol_type="other DNA"
/organism="synthetic DNA construct"
repeat_region 1..130
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
promoter 145..385
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 412..487
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
enhancer 577..862
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 864..1139
/label=chicken beta-actin promoter
intron 1140..1367
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 1388..1408
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1406..2434
/codon_start=1
/label=Cre
/note="site-specific recombinase"
/translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
DGD"
CDS 2435..2461
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
misc_feature 2489..3077
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
polyA_signal 3109..3585
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
repeat_region 3625..3765
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 3840..4295
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4312..4331)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4431..4453
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4491..4509)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4577..4681
/label=AmpR promoter
CDS 4682..5539
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5713..6301
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
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