pTet-tTS

Vector for expressing tTS, which represses expression from Tet-responsive promoters in the absence of doxycycline.

Sequence Author: Clontech (TaKaRa)

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PaeR7I - PspXI - XhoI (5) XmnI (3931) ScaI (3812) PvuI (3702) BsaI (3393) AhdI (3332) AlwNI (2855) PspFI (2747) BseYI (2743) DrdI (2547) AflIII - PciI (2439) BspQI - SapI (2323) PvuII (2263) Bpu10I (2136) BsrGI (51) SpeI (107) NdeI (342) SnaBI (448) Eco53kI (674) SacI (676) BsmBI - Esp3I (697) SacII (763) EcoRI (770) XbaI (782) PmlI (910) PfoI * (1014) BstXI (1061) NheI (1429) BmtI (1433) BspDI - ClaI (1629) PsiI (1760) HpaI (1780) MfeI (1789) BsaBI * (1881) KasI (1994) NarI (1995) SfoI (1996) PluTI (1998) CsiI - SexAI * (2011) BstEII (2026) NgoMIV (2070) NaeI (2072) HindIII (2113) pTet-tTS 4319 bp
PaeR7I  (5)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5)
1 site
C T C G A G G A G C T C
XmnI  (3931)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3812)
1 site
A G T A C T T C A T G A
PvuI  (3702)
1 site
C G A T C G G C T A G C
BsaI  (3393)
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.
AhdI  (3332)
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.
AlwNI  (2855)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PspFI  (2747)
1 site
C C C A G C G G G T C G
BseYI  (2743)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
DrdI  (2547)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AflIII  (2439)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2439)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2323)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2323)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
PvuII  (2263)
1 site
C A G C T G G T C G A C
Bpu10I  (2136)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BsrGI  (51)
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  (107)
1 site
A C T A G T T G A T C A
NdeI  (342)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
SnaBI  (448)
1 site
T A C G T A A T G C A T
Eco53kI  (674)
1 site
G A G C T C C T C G A G
SacI  (676)
1 site
G A G C T C C T C G A G
BsmBI  (697)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (697)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
SacII  (763)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
EcoRI  (770)
1 site
G A A T T C C T T A A G
XbaI  (782)
1 site
T C T A G A A G A T C T
PmlI  (910)
1 site
C A C G T G G T G C A C
PfoI  (1014)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BstXI  (1061)
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.
NheI  (1429)
1 site
G C T A G C C G A T C G
BmtI  (1433)
1 site
G C T A G C C G A T C G
BspDI  (1629)
1 site
A T C G A T T A G C T A
ClaI  (1629)
1 site
A T C G A T T A G C T A
PsiI  (1760)
1 site
T T A T A A A A T A T T
HpaI  (1780)
1 site
G T T A A C C A A T T G
MfeI  (1789)
1 site
C A A T T G G T T A A C
BsaBI  (1881)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
KasI  (1994)
1 site
G G C G C C C C G C G G
NarI  (1995)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (1996)
1 site
G G C G C C C C G C G G
PluTI  (1998)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
CsiI  (2011)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2011)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BstEII  (2026)
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.
NgoMIV  (2070)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (2072)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
HindIII  (2113)
1 site
A A G C T T T T C G A A
AmpR
3259 .. 4119  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3259 .. 4050  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3259 .. 4119  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4051 .. 4119  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3259 .. 4119  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
tTS
779 .. 1627  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 1:  TetR  
   779 .. 1411  =  633 bp
   211 amino acids  =  23.6 kDa
Product: fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain
silences Tet-responsive promoters in the absence but not the presence of doxycycline
tTS
779 .. 1627  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 2:  NLS  
   1412 .. 1432  =  21 bp
   7 amino acids  =  897.2 Da
Product: fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain
silences Tet-responsive promoters in the absence but not the presence of doxycycline
tTS
779 .. 1627  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 3:  KRAB silencing domain  
   1433 .. 1627  =  195 bp
   64 amino acids  =  7.4 kDa
Product: fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain
silences Tet-responsive promoters in the absence but not the presence of doxycycline
tTS
779 .. 1627  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
Product: fusion of a hybrid tetracycline repressor TetR with a nuclear localization signal and a KRAB silencing domain
silences Tet-responsive promoters in the absence but not the presence of doxycycline
ori
2500 .. 3088  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2500 .. 3088  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
CMV enhancer
93 .. 472  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
93 .. 472  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
473 .. 676  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
473 .. 676  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
1646 .. 1780  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1646 .. 1780  =  135 bp
SV40 polyadenylation signal
AmpR promoter
4120 .. 4224  =  105 bp
AmpR promoter
4120 .. 4224  =  105 bp
ORF:  632 .. 1627  =  996 bp
ORF:  331 amino acids  =  38.0 kDa
ORF:  1871 .. 2272  =  402 bp
ORF:  133 amino acids  =  14.7 kDa
ORF:  3389 .. 3655  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2049 .. 2399  =  351 bp
ORF:  116 amino acids  =  13.3 kDa
ORF:  1745 .. 2077  =  333 bp
ORF:  110 amino acids  =  12.0 kDa
ORF:  691 .. 939  =  249 bp
ORF:  82 amino acids  =  9.4 kDa
ORF:  3259 .. 4119  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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