pTRE-Tight-BI

Vector for co-expressing two genes with the Tet-On® Advanced or Tet-Off® Advanced system.

Sequence Author: Clontech (TaKaRa)

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EcoRI (2777) AgeI (2771) NdeI (2766) SacII (2762) BtgI (2759) ApaI (2757) MCS 2 EcoO109I (2754) PspOMI (2753) BglII (2747) Bsu36I (2741) PstI (2738) XbaI (2728) AatII (2536) ZraI (2534) SspI (2418) EarI (2409) XmnI (2213) ScaI (2094) TatI (2092) PvuI (1984) FspI (1836) AseI (1786) NmeAIII (1762) BsaI (1675) BmrI (1654) AhdI (1614) PaeR7I - PspXI - XhoI (1) Acc65I (335) KpnI - TspMI - XmaI (339) SmaI (341) BamHI (344) PvuII (358) MluI (362) NheI (368) BmtI (372) EagI - NotI (375) BspDI - ClaI (383) HindIII (388) SalI (394) AccI (395) EcoRV (402) PciI (721) NspI (725) DrdI (829) HaeII (969) BseYI (1025) PspFI (1029) AlwNI (1137) pTRE-Tight-BI 2856 bp
EcoRI  (2777)
1 site
G A A T T C C T T A A G
AgeI  (2771)
1 site
A C C G G T T G G C C A
NdeI  (2766)
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.
SacII  (2762)
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.
BtgI  (2759)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
ApaI  (2757)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoO109I  (2754)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PspOMI  (2753)
1 site
G G G C C C C C C G G G
BglII  (2747)
1 site
A G A T C T T C T A G A
Bsu36I  (2741)
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.
PstI  (2738)
1 site
C T G C A G G A C G T C
XbaI  (2728)
1 site
T C T A G A A G A T C T
AatII  (2536)
1 site
G A C G T C C T G C A G
ZraI  (2534)
1 site
G A C G T C C T G C A G
SspI  (2418)
1 site
A A T A T T T T A T A A
EarI  (2409)
1 site
C T C T T C N G A G A A G N N N N

Cleavage may be enhanced when more than one copy of the EarI recognition sequence is present.
Sticky ends from different EarI sites may not be compatible.
XmnI  (2213)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2094)
1 site
A G T A C T T C A T G A
TatI  (2092)
1 site
W G T A C W W C A T G W
PvuI  (1984)
1 site
C G A T C G G C T A G C
FspI  (1836)
1 site
T G C G C A A C G C G T
AseI  (1786)
1 site
A T T A A T T A A T T A
NmeAIII  (1762)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BsaI  (1675)
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.
BmrI  (1654)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
AhdI  (1614)
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.
PaeR7I  (1)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1)
1 site
C T C G A G G A G C T C
Acc65I  (335)
1 site
G G T A C C C C A T G G
KpnI  (339)
1 site
G G T A C C C C A T G G
TspMI  (339)
1 site
C C C G G G G G G C C C
XmaI  (339)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (341)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (344)
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.
PvuII  (358)
1 site
C A G C T G G T C G A C
MluI  (362)
1 site
A C G C G T T G C G C A
NheI  (368)
1 site
G C T A G C C G A T C G
BmtI  (372)
1 site
G C T A G C C G A T C G
EagI  (375)
1 site
C G G C C G G C C G G C
NotI  (375)
1 site
G C G G C C G C C G C C G G C G
BspDI  (383)
1 site
A T C G A T T A G C T A
ClaI  (383)
1 site
A T C G A T T A G C T A
HindIII  (388)
1 site
A A G C T T T T C G A A
SalI  (394)
1 site
G T C G A C C A G C T G
AccI  (395)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
EcoRV  (402)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
PciI  (721)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (725)
1 site
R C A T G Y Y G T A C R
DrdI  (829)
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.
HaeII  (969)
1 site
R G C G C Y Y C G C G R
BseYI  (1025)
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.
PspFI  (1029)
1 site
C C C A G C G G G T C G
AlwNI  (1137)
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.
AmpR
1541 .. 2401  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   1541 .. 2332  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1541 .. 2401  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   2333 .. 2401  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1541 .. 2401  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
782 .. 1370  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
782 .. 1370  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
bidirectional TRE promoter
2787 .. 318  =  388 bp
Tet-responsive bidirectional promoter PTight-BI, consisting of seven tet operator sequences flanked on each side by the minimal CMV promoter
bidirectional TRE promoter
2787 .. 318  =  388 bp
Tet-responsive bidirectional promoter PTight-BI, consisting of seven tet operator sequences flanked on each side by the minimal CMV promoter
AmpR promoter
2402 .. 2506  =  105 bp
AmpR promoter
2402 .. 2506  =  105 bp
SV40 poly(A) signal
519 .. 600  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
519 .. 600  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2539 .. 2620  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2539 .. 2620  =  82 bp
SV40 polyadenylation signal
MCS 1
335 .. 405  =  71 bp
multiple cloning site 1
MCS 1
335 .. 405  =  71 bp
multiple cloning site 1
MCS 2
2728 .. 2782  =  55 bp
multiple cloning site 2
MCS 2
2728 .. 2782  =  55 bp
multiple cloning site 2
tet operator
12 .. 30  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
12 .. 30  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
48 .. 66  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
48 .. 66  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
83 .. 101  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
83 .. 101  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
119 .. 137  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
119 .. 137  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
155 .. 173  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
155 .. 173  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
190 .. 208  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
190 .. 208  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
226 .. 244  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
226 .. 244  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ORF:  70 .. 354  =  285 bp
ORF:  94 amino acids  =  11.0 kDa
ORF:  2711 .. 202  =  348 bp
ORF:  115 amino acids  =  13.2 kDa
ORF:  1671 .. 1937  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  161 .. 418  =  258 bp
ORF:  85 amino acids  =  9.8 kDa
ORF:  1541 .. 2401  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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