Basic Vector Information
AAV-U6gRNA1-U6gRNA2-TnT-Cre 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-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"
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