ptTS-Neo

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

Sequence Author: Clontech (TaKaRa)

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EcoNI (4) XmnI (5977) PvuI (5748) BsaI (5439) AhdI (5378) Bpu10I (4182) HindIII (4159) PflMI (3912) BglII (3705) Bsu36I (3635) ApaI (3535) PspOMI (3531) PsiI (3364) MfeI (3356) NsiI (61) SfiI (192) StuI (238) BsaBI * (276) EagI (323) KasI (416) NarI (417) SfoI (418) PluTI (420) PflFI - Tth111I (535) RsrII (933) BstBI (1099) BsrGI (1407) SpeI (1463) SnaBI (1804) Eco53kI (2030) SacI (2032) BsmBI - Esp3I (2053) BbsI (2098) SacII (2119) XbaI (2138) PmlI (2266) NheI (2785) BmtI (2789) AvaI - BsoBI (2829) BamHI (2971) KflI (3014) ptTS-Neo 6365 bp
EcoNI  (4)
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.
XmnI  (5977)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (5748)
1 site
C G A T C G G C T A G C
BsaI  (5439)
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  (5378)
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.
Bpu10I  (4182)
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.
HindIII  (4159)
1 site
A A G C T T T T C G A A
PflMI  (3912)
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.
BglII  (3705)
1 site
A G A T C T T C T A G A
Bsu36I  (3635)
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.
ApaI  (3535)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (3531)
1 site
G G G C C C C C C G G G
PsiI  (3364)
1 site
T T A T A A A A T A T T
MfeI  (3356)
1 site
C A A T T G G T T A A C
NsiI  (61)
1 site
A T G C A T T A C G T A
SfiI  (192)
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  (238)
1 site
A G G C C T T C C G G A
BsaBI  (276)
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.
EagI  (323)
1 site
C G G C C G G C C G G C
KasI  (416)
1 site
G G C G C C C C G C G G
NarI  (417)
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  (418)
1 site
G G C G C C C C G C G G
PluTI  (420)
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.
PflFI  (535)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (535)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
RsrII  (933)
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  (1099)
1 site
T T C G A A A A G C T T
BsrGI  (1407)
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  (1463)
1 site
A C T A G T T G A T C A
SnaBI  (1804)
1 site
T A C G T A A T G C A T
Eco53kI  (2030)
1 site
G A G C T C C T C G A G
SacI  (2032)
1 site
G A G C T C C T C G A G
BsmBI  (2053)
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  (2053)
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.
BbsI  (2098)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
SacII  (2119)
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.
XbaI  (2138)
1 site
T C T A G A A G A T C T
PmlI  (2266)
1 site
C A C G T G G T G C A C
NheI  (2785)
1 site
G C T A G C C G A T C G
BmtI  (2789)
1 site
G C T A G C C G A T C G
AvaI  (2829)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2829)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
BamHI  (2971)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
KflI  (3014)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
AmpR
5305 .. 6165  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   5305 .. 6096  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5305 .. 6165  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   6097 .. 6165  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5305 .. 6165  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
tTS
2135 .. 2983  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 1:  TetR  
   2135 .. 2767  =  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
2135 .. 2983  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 2:  NLS  
   2768 .. 2788  =  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
2135 .. 2983  =  849 bp
282 amino acids  =  31.9 kDa
3 segments
   Segment 3:  KRAB silencing domain  
   2789 .. 2983  =  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
2135 .. 2983  =  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
NeoR/KanR
289 .. 1083  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
289 .. 1083  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
ori
4546 .. 5134  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4546 .. 5134  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
β-globin intron
3004 .. 3576  =  573 bp
intron from rabbit β-globin gene
β-globin intron
3004 .. 3576  =  573 bp
intron from rabbit β-globin gene
CMV enhancer
1449 .. 1828  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
1449 .. 1828  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
1829 .. 2032  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
1829 .. 2032  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 promoter
58 .. 254  =  197 bp
SV40 early promoter
SV40 promoter
58 .. 254  =  197 bp
SV40 early promoter
AmpR promoter
6166 .. 6270  =  105 bp
AmpR promoter
6166 .. 6270  =  105 bp
β-globin poly(A) signal
3773 .. 3828  =  56 bp
rabbit β-globin polyadenylation signal
β-globin poly(A) signal
3773 .. 3828  =  56 bp
rabbit β-globin polyadenylation signal
HSV TK poly(A) signal
1315 .. 1363  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
1315 .. 1363  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
105 .. 240  =  136 bp
SV40 origin of replication
SV40 ori
105 .. 240  =  136 bp
SV40 origin of replication
ORF:  289 .. 1083  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  461 .. 847  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  1988 .. 2983  =  996 bp
ORF:  331 amino acids  =  38.0 kDa
ORF:  5435 .. 5701  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1104 .. 1463  =  360 bp
ORF:  119 amino acids  =  13.7 kDa
ORF:  4146 .. 4445  =  300 bp
ORF:  99 amino acids  =  11.4 kDa
ORF:  1163 .. 1402  =  240 bp
ORF:  79 amino acids  =  9.0 kDa
ORF:  598 .. 1134  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  2047 .. 2295  =  249 bp
ORF:  82 amino acids  =  9.4 kDa
ORF:  5305 .. 6165  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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