Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001322 | pCDH-EF1-copGFP-T2A-Puro | 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
It is a 3rd gen lenti-vector. The 3rd generation LV packaging plasmid includes gag, coding for the virion main structural proteins; pol, responsible for the retrovirus-specific enzymes; and RRE, a binding site for the Rev protein which facilitates export of the RNA from the nucleus. This plasmid does not include Rev. The third-generation packaging system offers maximal biosafety but is more cumbersome, as it involves the transfection of four different plasmids in the producer cells. If you wish to use the 3rd gen lentivector, you need to have a lentiviral vector with a chimeric 5' LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. Examples of these vectors include pLKO.1, pLL3.7, pLB, pLenti6, pSico, pCL and pCS. Most Aebischer and Trono lab lentiviral vectors CANNOT be used with this system. A lentiviral vector carrying a chimeric 5' LTR can be packaged with either the 2nd or 3rd generation packaging system.
- Vector Name:
- pCDH-EF1-copGFP-T2A-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8549 bp
- Type:
- Mammalian Expression, Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- GCACTTGATGTAATTCTCC
- 3' Primer:
- CAACACCACGGAATTGTCAG
pCDH-EF1-copGFP-T2A-Puro 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
- Maughan EF, Hynds RE, Pennycuick A, Nigro E, Gowers KHC, Denais C, Gómez-López S, Lazarus KA, Orr JC, Pearce DR, Clarke SE, Lee DDH, Woodall MNJ, Masonou T, Case KM, Teixeira VH, Hartley BE, Hewitt RJ, Al Yaghchi C, Sandhu GS, Birchall MA, O'Callaghan C, Smith CM, De Coppi P, Butler CR, Janes SM. Cell-intrinsic differences between human airway epithelial cells from children and adults. iScience. 2022 Oct 20;25(11):105409.
pCDH-EF1-copGFP-T2A-Puro vector Sequence
LOCUS V001322 8549 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001322
VERSION V001322
KEYWORDS pCDH-EF1-copGFP-T2A-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8549)
TITLE lenti vector
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 8549)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8549)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8549
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 6..233
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 234..414
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 458..583
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1076..1309
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1493..1537
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 1686..1727
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 1804..1921
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2013..3191
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 3228..3893
/label="CopGFP"
/note="green fluorescent protein 2 from Pontellina plumata,
also known as ppluGFP2 (Shagin et al., 2004)"
CDS 3978..4031
/label="T2A"
/note="2A peptide from Thosea asigna virus capsid protein"
CDS 4032..4628
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 4644..5232
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5306..5539
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 5611..5745
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 5751..5886
/label="SV40 ori"
/note="SV40 origin of replication"
primer_bind complement(5924..5940)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5948..5964)
/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(5972..6002)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6017..6038)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6155..6172)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(6326..6914)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7088..7945)
/label="AmpR"
/note="beta-lactamase"
promoter complement(7946..8050)
/label="AmpR promoter"
primer_bind 8118..8136
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(8174..8196)
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind 8296..8315
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8524..8540
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"