Basic Vector Information
- Vector Name:
- AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6752 bp
- Type:
- Mammalian Expression, Mouse Targeting, AAV, Cre/Lo
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- EFS
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CCAATGCTATTGTTGAAGGTGCC
- 3' Primer:
- gtatccacatagcgtaaaaggagc
AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-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)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR vector Sequence
LOCUS 40924_150 6752 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses Cre recombinase and Renilla luciferase from the EFS
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 6752)
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 6752)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6752)
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..6752
/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"
promoter 548..759
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 773..1705
/codon_start=1
/label=Rluc
/note="luciferase from the anthozoan coelenterate Renilla
reniformis (sea pansy)"
/translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
CDS 1721..1777
/codon_start=1
/label=P2A
/note="2A peptide from porcine teschovirus-1 polyprotein"
/translation="ATNFSLLKQAGDVEENPGP"
CDS 1778..1798
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1796..2824
/codon_start=1
/label=Cre
/note="site-specific recombinase"
/translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
DGD"
CDS 2825..2851
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
misc_feature 2879..3467
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
polyA_signal 3499..3975
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
repeat_region 4015..4155
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
rep_origin 4230..4685
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4702..4721)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4821..4843
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4881..4899)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4967..5071
/label=AmpR promoter
CDS 5072..5929
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6103..6691
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
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