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Plasmid Files

pTRE-Cycle3

Vector for expressing ZsGreen1 while cycling the level of a second protein by alternating expression and rapid degradation.

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pTRE-Cycle3 Sequence and MappTRE-Cycle3.dna
Map and Sequence File   
Sequence Author:  Clontech
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 EcoRI (3793) BclI * (3699) SgrAI (3694) PflFI - Tth111I (3550) NaeI (3519) NgoMIV (3517) PluTI (3482) SfoI (3480) NarI (3479) KasI (3478) BseRI (3427) BmgBI (3294) BsrGI (3231) BtgZI (3128) ApaI (3089) EcoO109I (3086) PspOMI (3085) BglII (3079) PstI (3070) XbaI (3060) AatII (2868) ZraI (2866) SspI (2750) EarI (2741) XmnI (2545) ScaI (2426) PvuI (2316) FspI (2168) AseI (2118) BsaI (2007) PaeR7I - PspXI - XhoI (1) Acc65I (335) KpnI (339) AarI (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) EcoRV (734) PciI (1053) DrdI (1161) AlwNI (1469) pTRE-Cycle3 3872 bp
EcoRI  (3793)
1 site
G A A T T C C T T A A G
BclI  (3699)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
SgrAI  (3694)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
PflFI  (3550)
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  (3550)
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.
NaeI  (3519)
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.
NgoMIV  (3517)
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.
PluTI  (3482)
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.
SfoI  (3480)
1 site
G G C G C C C C G C G G
NarI  (3479)
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.
KasI  (3478)
1 site
G G C G C C C C G C G G
BseRI  (3427)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
BmgBI  (3294)
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.
BsrGI  (3231)
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.
BtgZI  (3128)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
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
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
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
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.
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
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.
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
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.
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
ZsGreen1
3091 .. 3786  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
3091 .. 3786  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
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
3803 .. 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
3803 .. 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
676 .. 737  =  62 bp
multiple cloning site
MCS
676 .. 737  =  62 bp
multiple cloning site
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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