Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000045 | pSLCAR-CD19-28z | 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
- Vector Name:
- pSLCAR-CD19-28z
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8866 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- EF1a-short
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CGGGTTTGCCGCCA
- 3' Primer:
- caggagccTGCTCCTCTTACTcc
pSLCAR-CD19-28z 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-CD19-28z vector Sequence
LOCUS pSLCAR-CD19-28z 8866 bp DNA circular SYN 15-APR-2024
DEFINITION Modular CD28-CD3z CAR backbone, For Transient Expression or
Lentiviral Production.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8866)
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 8866)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8866)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 8866)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther
Methods Clin Dev."; date: "2020-01-31"; pages: "
10.1016/j.omtm.2020.01.012. eCollection 2020 Mar 13"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8866
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(52..71)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 243..622
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 623..825
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 840..1020
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1067..1192
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1685..1918
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2103..2147
/codon_start=1
/label=gp41 peptide
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
/translation="KNEQELLELDKWASL"
misc_feature 2370..2487
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2513..2724
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 2750..2803
/codon_start=1
/label=signal peptide from CD28
/note="signal peptide from CD28"
/translation="MLRLLLALNLFPSIQVTG"
CDS 2813..2878
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 2879..3196
/codon_start=1
/label=anti-CD19 VL from FMC63
/translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
EI"
CDS 3254..3613
/codon_start=1
/label=anti-CD19 VH FMC63
/translation="EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL
EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSS"
CDS 3629..3763
/codon_start=1
/label=CD8a hinge region
/translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
"
CDS 3773..3868
/codon_start=1
/label=CD28 transmembrane region
/note="CD28 transmembrane region"
/translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR"
CDS 3869..3988
/codon_start=1
/label=CD28 costim.
/translation="SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS"
CDS 3995..4336
/codon_start=1
/label=CD3z stim domains
/translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
ALHMQALPPR"
CDS 4343..4399
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 4400..5113
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
misc_feature 5131..5719
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5791..6024
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 6102..6236
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 6263..6398
/label=SV40 ori
/note="SV40 origin of replication"
promoter complement(6419..6437)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6447..6463)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 6605..7060
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 7086..7190
/label=AmpR promoter
CDS 7191..8048
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 8222..8810
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"