Basic Vector Information
- Vector Name:
- AAV-ITR-U6-sgRNA(backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6752 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Adeno-associated virus
- Promoter:
- U6
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
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 62056_226 6752 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 6752) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..6752 /mol_type="other DNA" /organism="synthetic DNA construct" 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 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" 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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