Basic Vector Information
pLVX-EF1α-AcGFP1-C1 is an HIV-1-based, lentiviral expression vector designed to constitutively express a protein of interest fused to the C-terminus of AcGFP1, a green fluorescent protein derived from Aequorea coerulescens. The excitation and emission maxima of native AcGFP1 are 475 nm and 505 nm, respectively. Stable, constitutive expression of the fusion protein is driven by the EF1α promoter (PEF1α), which continues to be constitutively active even after the vector integrates into the host cell genome (1). pLVX-EF1α-AcGFP1-C1 contains all of the viral processing elements necessary for the production of replicationincompetent 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 (2), 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 (3). Finally, pLVX-EF1α-AcGFP1-C1 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 (4). In addition to lentiviral elements, pLVX-EF1α-AcGFP-C1 contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
pLVX-EF1α-AcGFP1-C1 vector Vector Map
Plasmid Resuspension 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 ℃.
pLVX-EF1α-AcGFP1-C1 vector Sequence
LOCUS pLVX-EF1-alpha-A 9519 bp DNA circular SYN 08-APR-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pLVX-EF1-alpha-AcGFP1-C1.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9519)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9519)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9519
/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"
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"
CDS 3539..4255
/label=AcGFP1
/note="Aequorea coerulescens GFP"
misc_feature 4256..4321
/label=MCS
/note="multiple cloning site"
promoter 4354..4853
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 4874..5470
/label=PuroR
/note="puromycin N-acetyltransferase"
misc_feature 5487..6075
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6282..6915
/label=5' LTR
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(7044..7060)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(7068..7084)
/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(7092..7122)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(7137..7158)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7446..8034)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8208..9065)
/label=AmpR
/note="beta-lactamase"
promoter complement(9066..9170)
/label=AmpR promoter
polyA_signal 9218..9352
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
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