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Plasmid Files

pTRE-Cycle1

Bidirectional expression vector for cycling the level of a protein by alternating expression and rapid degradation.

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pTRE-Cycle1 Sequence and MappTRE-Cycle1.dna
Map and Sequence File   
Sequence Author:  Clontech
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 EcoRI (3109) AgeI (3103) NdeI (3098) SacII (3094) ApaI (3089) EcoO109I (3086) PspOMI (3085) BglII (3079) Bsu36I (3073) PstI (3070) XbaI (3060) AatII (2868) ZraI (2866) SspI (2750) EarI (2741) XmnI (2545) ScaI (2426) TatI (2424) PvuI (2316) FspI (2168) AseI (2118) NmeAIII (2094) BsaI (2007) AhdI (1946) PaeR7I - PspXI - XhoI (1) Acc65I (335) KpnI (339) NcoI - StyI (350) AarI - BfuAI - BspMI (351) BsmBI (377) BsgI (379) BbsI (639) BamHI (676) PvuII (690) MluI (694) NheI (700) BmtI (704) EagI - NotI (707) BspDI - ClaI (715) HindIII (720) SalI (726) AccI (727) EcoRV (734) PciI (1053) DrdI (1161) HaeII (1301) AlwNI (1469) pTRE-Cycle1 3188 bp
EcoRI  (3109)
1 site
G A A T T C C T T A A G
AgeI  (3103)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
NdeI  (3098)
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  (3094)
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.
ApaI  (3089)
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  (3086)
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  (3085)
1 site
G G G C C C C C C G G G
BglII  (3079)
1 site
A G A T C T T C T A G A
Bsu36I  (3073)
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  (3070)
1 site
C T G C A G G A C G T C
XbaI  (3060)
1 site
T C T A G A A G A T C T
AatII  (2868)
1 site
G A C G T C C T G C A G
ZraI  (2866)
1 site
G A C G T C C T G C A G
SspI  (2750)
1 site
A A T A T T T T A T A A
EarI  (2741)
1 site
C T C T T C N G A G A A G N N N N

Efficient cleavage requires at least two copies of the EarI
recognition sequence.
Sticky ends from different EarI sites may not be compatible.
XmnI  (2545)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2426)
1 site
A G T A C T T C A T G A
TatI  (2424)
1 site
W G T A C W W C A T G W
PvuI  (2316)
1 site
C G A T C G G C T A G C
FspI  (2168)
1 site
T G C G C A A C G C G T
AseI  (2118)
1 site
A T T A A T T A A T T A
NmeAIII  (2094)
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  (2007)
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  (1946)
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
NcoI  (350)
1 site
C C A T G G G G T A C C
StyI  (350)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AarI  (351)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BfuAI  (351)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (351)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsmBI  (377)
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.
BsgI  (379)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BbsI  (639)
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.
BamHI  (676)
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  (690)
1 site
C A G C T G G T C G A C
MluI  (694)
1 site
A C G C G T T G C G C A
NheI  (700)
1 site
G C T A G C C G A T C G
BmtI  (704)
1 site
G C T A G C C G A T C G
EagI  (707)
1 site
C G G C C G G C C G G C
NotI  (707)
1 site
G C G G C C G C C G C C G G C G
BspDI  (715)
1 site
A T C G A T T A G C T A
ClaI  (715)
1 site
A T C G A T T A G C T A
HindIII  (720)
1 site
A A G C T T T T C G A A
SalI  (726)
1 site
G T C G A C C A G C T G
AccI  (727)
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  (734)
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  (1053)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (1161)
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  (1301)
1 site
R G C G C Y Y C G C G R
AlwNI  (1469)
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
1873 .. 2733  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   1873 .. 2664  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1873 .. 2733  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   2665 .. 2733  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1873 .. 2733  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1114 .. 1702  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1114 .. 1702  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
bidirectional TRE promoter
3119 .. 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
3119 .. 318  =  388 bp
Tet-responsive bidirectional promoter PTight-BI,
consisting of seven tet operator sequences flanked
on each side by the minimal CMV promoter
DD
352 .. 675  =  324 bp
108 amino acids  =  11.9 kDa
   Segment 1:  
   352 .. 354  =  3 bp
   1 amino acid  =  149.2 Da
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
DD
352 .. 675  =  324 bp
108 amino acids  =  11.9 kDa
   Segment 2:  
   355 .. 675  =  321 bp
   107 amino acids  =  11.8 kDa
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
DD
352 .. 675  =  324 bp
108 amino acids  =  11.9 kDa
2 segments
Product: destabilization domain that can be
stabilized by Shield1 in the ProteoTuner™ system
L106P mutant of FKBP12
AmpR promoter
2734 .. 2838  =  105 bp
AmpR promoter
2734 .. 2838  =  105 bp
SV40 poly(A) signal
851 .. 932  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
851 .. 932  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2871 .. 2952  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2871 .. 2952  =  82 bp
SV40 polyadenylation signal
MCS 1
676 .. 737  =  62 bp
multiple cloning site 1
MCS 1
676 .. 737  =  62 bp
multiple cloning site 1
MCS 2
3060 .. 3114  =  55 bp
multiple cloning site 2
MCS 2
3060 .. 3114  =  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
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