pHAGE-EF1a-MCS-IRES-ZsGreen1 vector (Cat. No.: V013929)
Note: pHAGE-EF1a-MCS-IRES-ZsGreen1 (8.3 kb) is a third‑generation lentiviral vector for bicistronic gene expression. The EF‑1α promoter drives transcription of a gene of interest cloned into the MCS, followed by an IRES (internal ribosome entry site) and the ZsGreen1 fluorescent reporter . Lentiviral elements include HIV‑1 Ψ (packaging signal), RRE (Rev response element), cPPT/CTS (central polypurine tract), gp41 peptide, Protein Tat, WPRE (woodchuck hepatitis post‑transcriptional regulatory element), and 3‘ ΔU3 LTR (self‑inactivating) for efficient transduction and high expression . The vector also carries AmpR for bacterial selection, a pUC origin (high‑copy replication), and SV40 ori.
- Name:
- pHAGE-EF1a-MCS-IRES-ZsGreen1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8342 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Promoter:
- EF-1α
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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.
pHAGE-EF1a-MCS-IRES-ZsGreen1 vector (Cat. No.: V013929) Sequence
LOCUS pHAGE-EF1a-MCS-I 8342 bp DNA circular SYN 14-MAY-2026
DEFINITION Exported.
ACCESSION V013929
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8342)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8342)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8342
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/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 1914..1955
/label=Protein Tat
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
misc_feature 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2224..3401
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature 3441..4019
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 4023..4715
/label=ZsGreen1
/note="Zoanthus green fluorescent protein"
misc_feature 4732..5320
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5395..5628
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter 5833..5937
/label=AmpR promoter
CDS 5938..6795
/label=AmpR
/note="beta-lactamase"
rep_origin 6969..7557
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 7968..7989
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 8004..8034
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8042..8058
/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)."
primer_bind 8066..8082
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"