pRevTRE is a retroviral Tet response vector that expresses a gene of interest from the Tet-response element (TRE). 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). The TRE contains seven direct repeats of the tetO operator sequence, upstream of a minimal CMV promoter, which can be bound by the tTA and rtTA transactivators. The 5 viral LTR controls expression of the transcript that contains Y + (the extended viral packaging signal) and the hygromycin resistance (Hyg r ) gene for antibiotic selection in mammalian cells. The TRE is derived from vectors described previously (2, 3). pRevTRE also includes the E. coli Amp r gene for antibiotic selection in bacteria.The complete RevTet-Off and RevTet-On Systems also include the control vector pRevTRE-Luc, which was constructed by cloning the firefly luciferase gene into the Hin d III/ Cla I sites in the MCS of pRevTRE.pRevTRE can be used to establish inducible Tet Systems via retrovirus-mediated gene transfer (4). Retroviral gene transfer allows the highly efficient transduction of virtually all dividing cell types. The RevTet TM Systems are also suitable for establishing transgenic animals. In combination with the pRevTet-On or pRevTet-Off regulatory 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 and rtTA bind to the Tet-response element (TRE) and activate transcription from the minimal promoter in the absence or presence of Dox, respectively. pRevTRE 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, 6–7). The transcript produced by the pRevTRE 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 >10 5 cfu/ml should be produced to achieve high-level induction.
| 载体基本信息 | |||
| 载体名称 | pRevTR | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6487 bp | 载体类型 | Tetracycline Regulatory System |
| 筛选标记 | Hygromycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | ||
pRevTR 载体载体图谱
pRevTR 载体载体序列
LOCUS Exported 6487 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 6487)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6487
/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 1524..2543
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/note="HygR"
/note="confers resistance to hygromycin"
/translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
protein_bind 2847..3117
/bound_moiety="tetracycline repressor TetR"
/note="tetracycline response element"
/note="contains seven copies of the tetracycline operator
tetO"
promoter 3150..3187
/note="minimal CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 3281..3337
/note="MCS"
/note="pUC18/19 multiple cloning site"
LTR 3389..3982
/note="long terminal repeat from Moloney murine leukemia
virus"
misc_feature 4193..4333
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(4519..5107)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5278..6138)
/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(6139..6243)
/gene="bla"
/note="AmpR promoter"
