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.
| 载体基本信息 | |||
| 载体名称 | pLVX-EF1α-IRES-ZsGreen1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 8889 bp | 载体类型 | Viral Expression & Packaging Vectors |
| 载体来源 | Clontech | 拷贝数 | High copy number |
| 启动子 | EF1α/ EF1a | 克隆方法 | Enzyme Cut |
| 表达方法 | Constiutive, Stable | ||
pLVX-EF1α-IRES-ZsGreen1 载体载体图谱
pLVX-EF1α-IRES-ZsGreen1 载体载体序列
LOCUS Exported 8889 bp ds-DNA circular SYN 08-APR-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLVX-EF1a-IRES-ZsGreen1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8889)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8889
/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 3575..4147
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 3598..4149
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 4149..4844
/codon_start=1
/product="Zoanthus green fluorescent protein"
/note="ZsGreen1"
/note="mammalian codon-optimized"
/translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
AIASGSALP"
misc_feature 4858..5446
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5329..5340)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 5653..6286
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
primer_bind complement(6414..6430)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 6438..6454
/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(6462..6492)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6507..6528
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6816..7404)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7575..8435)
/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(8436..8540)
/gene="bla"
/note="AmpR promoter"
polyA_signal 8588..8722
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
