pscAAV-CAG-GFP vector (Cat. No.: V000865)
Note: The pscAAV-CAG-GFP is a recombinant AAV vector packaging self-complementary GFP under the CAG promoter
- Name:
- pscAAV-CAG-GFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4799 bp
- Type:
- Mammalian Expression, AAV
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- chicken β-actin
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- GTTATTGTGCTGTCTCATC
- 3' Primer:
- CCACACCTCCCCCTGAAC
- Growth Strain(s):
- DH10b
- Growth Temperature:
- 37℃
Resources
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Pekrun K, De Alencastro G, Luo QJ, Liu J, Kim Y, Nygaard S, Galivo F, Zhang F, Song R, Tiffany MR, Xu J, Hebrok M, Grompe M, Kay MA. Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors. JCI Insight. 2019 Nov 14;4(22):e131610.
pscAAV-CAG-GFP vector (Cat. No.: V000865) Sequence
LOCUS pscAAV-CAG-GFP 4799 bp DNA circular SYN 26-DEC-2025
DEFINITION recombinant AAV vector packaging self-complementary GFP under the
CAG promoter.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4799)
AUTHORS Pekrun K, De Alencastro G, Luo QJ, Liu J, Kim Y, Nygaard S, Galivo
F, Zhang F, Song R, Tiffany MR, Xu J, Hebrok M, Grompe M, Kay MA
TITLE Using a barcoded AAV capsid library to select for clinically
relevant gene therapy vectors.
JOURNAL JCI Insight. 2019 Nov 14;4(22). pii: 131610. doi:
10.1172/jci.insight.131610.
PUBMED 31723052
REFERENCE 2 (bases 1 to 4799)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4799)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4799)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "JCI
Insight."; date: "2019-11-14"; pages: " 10.1172/jci.insight.131610"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4799
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 417..694
/label=chicken beta-actin promoter
primer_bind 973..995
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1057..1076
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
CDS 1122..1838
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind 1885..1904
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2154..2170)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2379..2398)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2498..2520
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2558..2576)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2644..2748
/label=AmpR promoter
CDS 2749..3606
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3780..4368
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4522..4539
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4656..4677
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4692..4722
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4730..4746
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 4754..4770
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"