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Plasmid Files

pTet-On

Vector for expressing the rtTA transactivator in the Tet-On® system.

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pTet-On.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Clontech (TaKaRa)
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BstZ17I (7350) SfiI (6896) StuI (6847) AvrII (6842) BmtI (6828) NheI (6824) XcmI (6527) BglII (6502) EagI (6432) MscI (6260) RsrII (5823) BstBI (5658) NruI (5487) Bsu36I (5463) BspEI * (5332) PflMI (5184) PspXI (4470) XmnI (4077) ScaI (3958) PvuI (3848) BsrGI (47) SpeI (103) SnaBI (444) EcoRI (766) XbaI (777) BstXI (873) EcoNI (982) MluI (1159) AfeI (1178) AleI (1312) MauBI (1405) BsiWI (1410) SalI (1538) PshAI (1557) BmgBI (1598) PpuMI (2076) BstEII (2172) AhdI (3478) BsaI (3539) pTet-On 7400 bp
BstZ17I  (7350)
1 site
G T A T A C C A T A T G
SfiI  (6896)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
StuI  (6847)
1 site
A G G C C T T C C G G A
AvrII  (6842)
1 site
C C T A G G G G A T C C
BmtI  (6828)
1 site
G C T A G C C G A T C G
NheI  (6824)
1 site
G C T A G C C G A T C G
XcmI  (6527)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BglII  (6502)
1 site
A G A T C T T C T A G A
EagI  (6432)
1 site
C G G C C G G C C G G C
MscI  (6260)
1 site
T G G C C A A C C G G T
RsrII  (5823)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BstBI  (5658)
1 site
T T C G A A A A G C T T
NruI  (5487)
1 site
T C G C G A A G C G C T
Bsu36I  (5463)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BspEI  (5332)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PflMI  (5184)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
PspXI  (4470)
1 site
V C T C G A G B B G A G C T C V
XmnI  (4077)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3958)
1 site
A G T A C T T C A T G A
PvuI  (3848)
1 site
C G A T C G G C T A G C
BsrGI  (47)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SpeI  (103)
1 site
A C T A G T T G A T C A
SnaBI  (444)
1 site
T A C G T A A T G C A T
EcoRI  (766)
1 site
G A A T T C C T T A A G
XbaI  (777)
1 site
T C T A G A A G A T C T
BstXI  (873)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoNI  (982)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MluI  (1159)
1 site
A C G C G T T G C G C A
AfeI  (1178)
1 site
A G C G C T T C G C G A
AleI  (1312)
1 site
C A C N N N N G T G G T G N N N N C A C
MauBI  (1405)
1 site
C G C G C G C G G C G C G C G C
BsiWI  (1410)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SalI  (1538)
1 site
G T C G A C C A G C T G
PshAI  (1557)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BmgBI  (1598)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
PpuMI  (2076)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BstEII  (2172)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
AhdI  (3478)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BsaI  (3539)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
rtTA
774 .. 1781  =  1008 bp
335 amino acids  =  36.9 kDa
   Segment 1:  rTetR  
   774 .. 1394  =  621 bp
   207 amino acids  =  23.4 kDa
Product: tetracycline-controlled transactivator, comprising a fusion of the reverse tetracycline repressor rTetR with the C-terminal activation domain of herpes simplex virus VP16
In the Tet-On® system, rtTA binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline.
rtTA
774 .. 1781  =  1008 bp
335 amino acids  =  36.9 kDa
   Segment 2:  VP16 activation domain  
   1395 .. 1781  =  387 bp
   128 amino acids  =  13.6 kDa
Product: tetracycline-controlled transactivator, comprising a fusion of the reverse tetracycline repressor rTetR with the C-terminal activation domain of herpes simplex virus VP16
In the Tet-On® system, rtTA binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline.
rtTA
774 .. 1781  =  1008 bp
335 amino acids  =  36.9 kDa
2 segments
Product: tetracycline-controlled transactivator, comprising a fusion of the reverse tetracycline repressor rTetR with the C-terminal activation domain of herpes simplex virus VP16
In the Tet-On® system, rtTA binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline.
AmpR
3405 .. 4265  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   3405 .. 4196  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3405 .. 4265  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   4197 .. 4265  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3405 .. 4265  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
NeoR/KanR
5677 .. 6471  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamyc