Basic Vector Information
pLVX-EF1α-IRES-ZsGreen1 is an HIV-1-based, lentiviral expression vector designed to simultaneously and constitutively express a protein of interest and the green fluorescent protein ZsGreen1 from a bicistronic transcript in mammalian cells. ZsGreen1 is a human-codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein (ZsGreen) that has been engineered for brighter fluorescence (1–3). The excitation and emission maxima of native ZsGreen1 are 493 nm and 505 nm, respectively. Simultaneous expression of a protein of interest and ZsGreen1 is made possible by the presence of an encephalomyo- carditis virus internal ribosome entry site (IRES; 4, 5) positioned between the multiple cloning site (MCS) and the ZsGreen1 gene. The IRES allows a protein of interest and ZsGreen1 to be translated from a single bicistronic mRNA. Stable, constitutive expression of the bicistronic transcript is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after vector integration into the host cell genome (6). pLVX-EF1α-IRES-ZsGreen1 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (7), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (9). Finally, pLVX-EF1α-IRES-ZsGreen1 also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (9). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
- Vector Name:
- pLVX-EF1α-IRES-ZsGreen1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8889 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Clontech
- Copy Number:
- High copy number
- Promoter:
- EF-1α
- Cloning Method:
- Enzyme Cut
- Expression Method:
- Constiutive, Stable
pLVX-EF1α-IRES-ZsGreen1 vector Map
pLVX-EF1α-IRES-ZsGreen1 vector Sequence
LOCUS V012695 8889 bp DNA circular SYN 08-APR-2021
DEFINITION Exported.
ACCESSION V012695
VERSION V012695
KEYWORDS pLVX-EF1a-IRES-ZsGreen1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8889)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8889)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8889
/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
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2027..2144
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2338..3519
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature 3575..4154
/label="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 4149..4841
/label="ZsGreen1"
/note="Zoanthus green fluorescent protein"
misc_feature 4858..5446
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5653..6286
/label="5' LTR"
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6414..6430)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6438..6454)
/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(6462..6492)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6507..6528)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6816..7404)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7578..8435)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8436..8540)
/label="AmpR promoter"
polyA_signal 8588..8722
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
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