Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012151 | MSGV1-1D3-28Z.1-3 mut | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
MSGV1-1D3-28Z.1-3 mut contains an anti-mouse CD19 CAR with CD3 ITAMs mutated.
- Vector Name:
- MSGV1-1D3-28Z.1-3 mut
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6941 bp
- Type:
- Retroviral
- Replication origin:
- ori
- Promoter:
- MSCV
- Cloning Method:
- Restriction Enzyme
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
MSGV1-1D3-28Z.1-3 mut 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
- Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood. 2010 Nov 11;116(19):3875-86.
MSGV1-1D3-28Z.1-3 mut vector Sequence
LOCUS 40924_2079 6941 bp DNA circular SYN 13-MAY-2021
DEFINITION Anti-mouse CD19 CAR with CD3 ITAMs mutated.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6941)
AUTHORS Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA
TITLE Adoptive transfer of syngeneic T cells transduced with a chimeric
antigen receptor that recognizes murine CD19 can eradicate lymphoma
and normal B cells.
JOURNAL Blood. 2010 Nov 11;116(19):3875-86. doi:
10.1182/blood-2010-01-265041. Epub 2010 Jul 14.
PUBMED 20631379
REFERENCE 2 (bases 1 to 6941)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6941)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1182/blood-2010-01-265041"; journalName: "Blood"; date:
"2010-11-11- 11"; volume: "116"; issue: "19"; pages: "3875-86"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6941
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..517
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
CDS 1020..1427
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="VTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTFNVG
WPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKPPPP
LPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
misc_feature 1437..1807
/label=pol region
/note="Moloney murine leukemia virus (MMLV) pol region
containing the splice acceptor site"
regulatory 1822..1831
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
LTR 3326..3840
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
protein_bind complement(4012..4028)
/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(4036..4066)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4081..4102)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4219..4236)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4390..4978)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5152..6009)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6010..6114)
/label=AmpR promoter
primer_bind 6182..6200
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(6238..6260)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 6360..6379
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 6573..6595
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 6723..6742
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"