Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V007351 | pRevTet-Off | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
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.
- Vector Name:
- pRevTet-Off
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7847 bp
- Type:
- Tetracycline Regulatory System
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Promoter:
- CMV
- Cloning Method:
- Enzyme digestion and ligation
pRevTet-Off vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pRevTet-Off vector Sequence
LOCUS 40924_36888 7847 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7847)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7847)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7847
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 2..589
/label=5' LTR
/note="long terminal repeat from Moloney murine sarcoma
virus"
misc_feature 652..851
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1052..1468
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
CDS 1512..2303
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
enhancer 2788..3167
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 3168..3371
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3476..4480
/codon_start=1
/label=tTA
/note="tetracycline-controlled transactivator, comprising a
fusion of the tetracycline repressor TetR with the
C-terminal activation domain of herpes simplex virus VP16"
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
LTR 4749..5342
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
misc_feature 5553..5693
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(5879..6467)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6641..7498)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7499..7603)
/label=AmpR promoter