pRevTet-Off is a retroviral vector expressing the tetracycline-controlled transactivator (tTA) from the CMV promoter. This vector is derived from pLNCX, a retroviral vector created using elements of Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV) as described (1). tTA is a fusion of amino acids 1–207 of the tet repressor (TetR) and the negatively charged C-terminal activation domain (130 amino acids) of the VP16 protein of Herpes Simplex Virus. The 5 viral LTR controls expression of the transcript that contains Y+ (the extended viral packaging signal), and the neomycin resistance gene (Neor) for antibiotic selection in mammalian cells. tTA is derived from vectors described previously (2, 3). pRevTet-Off also includes the E. coli Ampr gene for antibiotic selection in bacteria.pRevTet-Off can be used to establish stable Tet-Off cell lines via retrovirus-mediated gene transfer (4). Retroviral gene transfer allows the highly efficient transduction of virtually all dividing cell types. The RevTet Systems are also suitable for establishing transgenic animals. In combination with the pRev-TRE retroviral expression vector, a gene of interest can be inducibly expressed at high levels in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline (Dox). tTA binds to the Tet-response element (TRE), thus activating transcription in the absence of Tc or Dox. As Tc or Dox is added to the culture medium, transcription from the inducible promoter is turned off in a highly dose-dependent manner. pRevTet-Off lacks the viral genes gag, pol, and env, which are supplied by the packaging cell line. It can be transfected into a high titer packaging cell line and thereby mediate production of infectious, replication-incompetent retroviral particles (1, 5–6).The transcript produced by the pRevTet-Off construct is recognized by the viral structural proteins expressed in a packaging cell line and packaged into infectious retroviral particles. Because the RNA transcript packaged in these particles does not contain the viral genes, it cannot replicate in the target cells that it infects.The level of induction in cell populations infected with this vector depends on the efficiency of infection, the site of integration, and the titer of the virus. Viral supernatants with titers >105 cfu/ml should be produced to achieve high-level induction.
| 载体基本信息 | |||
| 载体名称 | pRevTet-Off | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 7847 bp | 载体类型 | Tetracycline Regulatory System |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | ||
pRevTet-Off 载体载体图谱
pRevTet-Off 载体载体序列
LOCUS Exported 7847 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7847)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7847
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 414..589
/note="5 LTR (truncated)"
/note="truncated long terminal repeat from Moloney murine
sarcoma virus"
misc_feature 652..851
/note="MMLV Psi"
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1052..1468
/note="gag (truncated)"
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
CDS 1512..2306
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
enhancer 2788..3167
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 3168..3371
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3476..4483
/codon_start=1
/product="tetracycline-controlled transactivator,
comprising a fusion of the tetracycline repressor TetR with
the C-terminal activation domain of herpes simplex virus
VP16"
/note="tTA"
/note="In the Tet-Off(R) system, tTA binds to the
Tet-responsive element and stimulates transcription only in
the absence of tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
LTR 4749..5342
/note="long terminal repeat from Moloney murine leukemia
virus"
misc_feature 5553..5693
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(5879..6467)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6638..7498)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7499..7603)
/gene="bla"
/note="AmpR promoter"
