Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012785 | MSCV-IRES-GFP | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The MSCV-IRES-GFP vector is a widely used retroviral construct designed to express a bicistronic mRNA encoding both the gene of interest and a GFP marker. This vector enables simultaneous expression of the transgene and fluorescent reporter, allowing for direct visualization and sorting of transduced cells. Prior to retroviral transduction, target cells are pre-activated by culturing in cytokine-supplemented media for two days to enhance receptivity to viral infection and integration.
- Vector Name:
- MSCV-IRES-GFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6488 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Tannishtha Reya
- Copy Number:
- High copy number
- Promoter:
- MSCV
- 5' Primer:
- pLXSN5; pBABE5
- 3' Primer:
- pCDH-rev (GCATTCCTTTGGCGAGAG)
- Fusion Tag:
- EGFP
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
MSCV-IRES-GFP 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
- Sarma NJ, Takeda A, Yaseen NR. Colony forming cell (CFC) assay for human hematopoietic cells. J Vis Exp. 2010 Dec 18;(46):2195. doi: 10.3791/2195. PMID: 21252854; PMCID: PMC3159642.
MSCV-IRES-GFP vector Sequence
LOCUS 40924_2064 6488 bp DNA circular SYN 16-NOV-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6488)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6488)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6488
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(458..1315)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1316..1420)
/label=AmpR promoter
LTR 2248..2764
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
misc_feature 2828..3169
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 3236..3652
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
misc_feature 3685..4246
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 4249..4965
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
LTR 5130..5644
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
protein_bind complement(5806..5822)
/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)."
promoter complement(5830..5860)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5875..5896)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(join(6184..6488,1..284))
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"