pTet-Off-Advanced vector (V011072)

Basic Vector Information

In the Tet-Off expression system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from the Tc resistance operon of Escherichia coli transposon Tn10 fused to the strong transactivating domain(sequence: PTDALDDFDLDML) of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter. In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive. The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. Like tTA, rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change(E19G, A56P, D148E, H179R) in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the TRE of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

Vector Name:: pTet-Off-Advanced

Antibiotic Resistance:: Ampicillin

Length:: 7140 bp

Type:: Mammalian Expression Vectors

Replication origin:: ori

Source/Author:: Clontech

Selection Marker:: Neomycin/G418(Geneticin)

Copy Number:: High copy number

Promoter:: SV40

pTet-Off-Advanced vector Map

Copy Sequence View Map

pTet-Off-Advanced vector Sequence

LOCUS       pTet-Off-Advance        7140 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector for expressing the tTA-Advanced transactivator in the 
            Tet-Off(R) Advanced system.
ACCESSION   .
VERSION     .
KEYWORDS    pTet-Off-Advanced
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7140)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7140
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        89..468
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        469..672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             775..1518
                     /label=tTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
     polyA_signal    1531..1665
                     /label=SV40 poly(A) signal
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2385..2973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3147..4004)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(4005..4109)
                     /label=AmpR promoter
     polyA_signal    complement(4222..4356)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4781..4801)
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     intron          complement(4931..4996)
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(5419..6210)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     promoter        complement(6571..6900)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"

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