pLVX-EF1α-AcGFP1-N1 is an HIV-1-based, lentiviral expression vector designed to constitutively express a protein of interest fused to the N-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-N1 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-N1 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-N1 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.
| 载体基本信息 | |||
| 载体名称 | pLVX-EF1α-AcGFP1-N1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 9519 bp | 载体类型 | Viral Expression & Packaging Vectors |
| 载体来源 | Clontech | 筛选标记 | Puromycin |
| 拷贝数 | High copy number | 启动子 | EF1α/ EF1a |
| 克隆方法 | Enzyme Cut | 载体标签 | AcGFP1 |
| 表达方法 | Constiutive, Stable | ||
pLVX-EF1α-AcGFP1-N1 载体载体图谱
pLVX-EF1α-AcGFP1-N1 载体载体序列
LOCUS Exported 9519 bp ds-DNA circular SYN 08-APR-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLVX-EF1-alpha-AcGFP1-N1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9519)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9519
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2028..2143
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)"
promoter 2338..3519
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2568..3510
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
misc_feature 3525..3605
/note="MCS"
/note="multiple cloning site"
CDS 3607..4326
/codon_start=1
/product="Aequorea coerulescens GFP"
/note="AcGFP1"
/note="mammalian codon-optimized"
/translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
GFVTAAAITHGMDELYK"
promoter 4354..4853
/note="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 4874..5473
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 5487..6075
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5958..5969)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 6282..6915
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
primer_bind complement(7044..7060)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7068..7084
/bound_moiety="lac repressor encoded by lacI"
/note="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)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 7137..7158
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7446..8034)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8205..9065)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9066..9170)
/gene="bla"
/note="AmpR promoter"
polyA_signal 9218..9352
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
