AAV-ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR vector (V016599)

MSDS

Basic Vector Information

Vector Name:: AAV-ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR

Antibiotic Resistance:: Ampicillin

Length:: 6562 bp

Type:: Gene knockout

Replication origin:: ori

Host:: Mammalian cells, Adeno-associated virus

Promoter:: SYN1

Growth Strain(s):: Stbl3

Growth Temperature:: 37℃

AAV-ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR vector Vector Map

View Map in NovoBuilder

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)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       62056_221        6562 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 6562)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        156..396
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        423..498
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        532..979
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             1020..2048
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             2049..2075
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             2145..2858
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    3150..3738
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     repeat_region   3825..3965
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4040..4495
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4777..4881
                     /label=AmpR promoter
     CDS             4882..5739
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5913..6501
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"

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