Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012796 | pSLCAR-CD19-CD3z | 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
Modular CD3z alone CAR backbone, For Transient Expression or Lentiviral Production
- Vector Name:
- pSLCAR-CD19-CD3z
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8746 bp
- Type:
- Mammalian Expression Vectors
- Replication origin:
- ori
- Source/Author:
- Scott McComb
- Selection Marker:
- EGFP
- Copy Number:
- High copy number
- Promoter:
- CMV
- Expression Method:
- Constiutive, Stable / Transient
pSLCAR-CD19-CD3z 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.
pSLCAR-CD19-CD3z vector Sequence
LOCUS Exported 8746 bp DNA circular SYN 06-JAN-2022
DEFINITION Modular CD3z alone CAR backbone, For Transient Expression or
Lentiviral Production.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8746)
AUTHORS Bloemberg D, Nguyen T, MacLean S, Zafer A, Gadoury C, Gurnani K,
Chattopadhyay A, Ash J, Lippens J, Harcus D, Page M, Fortin A, Pon
RA, Gilbert R, Marcil A, Weeratna RD, McComb S
TITLE A High-Throughput Method for Characterizing Novel Chimeric Antigen
Receptors in Jurkat Cells.
JOURNAL Mol Ther Methods Clin Dev. 2020 Jan 31;16:238-254. doi:
10.1016/j.omtm.2020.01.012. eCollection 2020 Mar 13.
PUBMED 32083149
REFERENCE 2 (bases 1 to 8746)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8746)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther
Methods Clin Dev."; date: "2020-01-31"; volume: "16:238-254. doi";
pages: " 10.1016/j.omtm.2020.01.012. eCollection 2020 Mar 13"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8746
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..583
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label=5' LTR (truncated)
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
misc_feature 2128..2245
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2271..2482
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2508..2561
/codon_start=1
/label=signal peptide from CD28
/note="signal peptide from CD28"
/translation="MLRLLLALNLFPSIQVTG"
CDS 2613..2636
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 2637..2954
/codon_start=1
/label=anti-CD19 VL from FMC63
/note="anti-CD19 VL from FMC63"
/translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
EI"
CDS 3012..3371
/codon_start=1
/label=anti-CD19 VH FMC63
/note="anti-CD19 VH FMC63"
/translation="EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL
EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSS"
CDS 3387..3521
/codon_start=1
/label=CD8a hinge region
/note="CD8a hinge region"
/translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
"
CDS 3531..3626
/codon_start=1
/label=CD28 transmembrane region
/note="CD28 transmembrane region"
/translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR"
CDS 3633..3974
/codon_start=1
/label=CD247/CD-3-zeta cytoplasmic domain
/note="CD247/CD-3-zeta cytoplasmic domain"
/translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
ALHMQALPPR"
CDS 3981..4037
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=2A peptide from porcine teschovirus-1
polyprotein
/note="P2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 4038..4751
/label=EGFP
/note="enhanced GFP"
misc_feature 4769..5357
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5429..5662
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 5740..5874
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 5901..6036
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(6057..6075)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6085..6101)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 6243..6698
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6724..6828
/label=AmpR promoter
CDS 6829..7686
/label=AmpR
/note="beta-lactamase"
rep_origin 7860..8448
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"