pLVX-shRNA2 is an HIV-1-based, lentiviral expression vector designed to express a small hairpin RNA (shRNA) for RNA interference (RNAi) studies. Expression of your shRNA is driven by the RNA Pol III-dependent, human U6 promoter (PU6), located just upstream of the MCS. pLVX-shRNA2 can be used as a plasmid expression vector and transfected into cells, or it can be packaged into viral particles and transduced into cells. Lentiviral particles derived from the vector allow the expression of shRNAs in virtually any cell type, including primary cells. In addition to expressing shRNAs, pLVX-shRNA2 also expresses the fluorescent protein ZsGreen1, a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen (1). Expression of ZsGreen1 is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE), allowing it to be used as an indicator of transfection or transduction efficiency, as well as a marker for cell sorting. pLVX-shRNA2 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer 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-shRNA2 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). 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-shRNA2 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7880 bp | 载体类型 | Viral Expression & Packaging Vectors |
| 载体来源 | Clontech | 筛选标记 | ZsGreen1 |
| 拷贝数 | High copy number | 克隆方法 | Enzyme Cut |
pLVX-shRNA2 载体载体图谱
pLVX-shRNA2 载体载体序列
LOCUS Exported 7880 bp ds-DNA circular SYN 08-APR-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLVX-shRNA2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7880)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7880
/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 2196..2436
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
enhancer 2522..2825
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2826..3029
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3074..3769
/codon_start=1
/product="Zoanthus green fluorescent protein"
/note="ZsGreen1"
/note="mammalian codon-optimized"
/translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
AIASGSALP"
misc_feature 3802..4390
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(4273..4284)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 4597..5230
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
primer_bind complement(5358..5374)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 5382..5398
/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(5406..5436)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 5451..5472
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5760..6348)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6519..7379)
/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(7380..7484)
/gene="bla"
/note="AmpR promoter"
polyA_signal 7532..7666
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
