Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V016980 | SFG.wtCNa_opt.IRES.eGFP | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
SFG.wtCNa_opt.IRES.eGFP is a retroviral expression vector that confers resistance to cyclosporine A inhibition in T cells via a codon-optimised wild-type calcineurin A (CNA) gene, whilst utilising an IRES element to enable synchronous eGFP expression for cell tracking. This vector is employed to generate anti-immunosuppression EB virus-specific T cells, sustaining their proliferative and cytotoxic functions within immunosuppressed environments.
- Vector Name:
- SFG.wtCNa_opt.IRES.eGFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9235 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Retrovirus
- Promoter:
- MMLV
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
SFG.wtCNa_opt.IRES.eGFP vector Map
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
- Brewin J, Mancao C, Straathof K, Karlsson H, Samarasinghe S, Amrolia PJ, Pule M. Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease. Blood. 2009 Nov 26;114(23):4792-803. doi: 10.1182/blood-2009-07-228387. Epub 2009 Sep 21. PMID: 19770360.
SFG.wtCNa_opt.IRES.eGFP vector Sequence
LOCUS Exported 9235 bp DNA circular SYN 06-NOV-2025
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS SFG.wtCNa_opt.IRES.eGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9235)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9235
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 1879..3441
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 3497..4058
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 651..1008
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1082..1489
/codon_start=1
/label=truncated gag
/translation="VTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTFNVG
WPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKPPPP
LPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
misc_feature 1499..1873
/label=pol region
/note="Moloney murine leukemia virus (MMLV) pol region
containing the splice acceptor site"
CDS 1879..3441
/gene="PPP3CA"
/label=Protein phosphatase 3 catalytic subunit alpha
/note="Protein phosphatase 3 catalytic subunit alpha from
Homo sapiens. Accession#: Q08209"
misc_feature 3497..4058
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 4060..4779
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
LTR 5045..5514
/note="long terminal repeat from Moloney murine leukemia
virus"
primer_bind complement(6210..6226)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 6700..6804
/gene="bla"
/label=AmpR promoter
CDS 6805..7665
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 7836..8424
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 8712..8733
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8748..8778
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8786..8802
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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 8810..8826
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
LTR join(9234..9235,1..588)
/note="long terminal repeat from Moloney murine leukemia
virus"