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Plasmid Files

pTRE3G-IRES

Vector for doxycycline-inducible expression of two genes in cells containing the Tet-On® 3G transactivator protein.

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pTRE3G-IRES Sequence and MappTRE3G-IRES.dna
Map and Sequence File   
Sequence Author:  Clontech
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 EcoRI (4021) AatII (3950) ZraI (3948) ScaI (3508) TatI (3506) FspI (3250) NmeAIII (3176) BsaI (3089) AhdI (3028) AlwNI (2551) PspFI (2443) BseYI (2439) DrdI (2243) BspQI - SapI (2019) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1) BmeT110I (2) KasI (274) NarI (275) SfoI (276) PluTI (278) Eco53kI (292) SacI (294) SalI (383) AccI (384) BglII (399) BspDI * - ClaI * (406) EagI (411) NgoMIV (413) NaeI (415) FseI (417) AvrII (570) BsaAI - PmlI (735) AarI - BfuAI - BspMI (758) DraIII (782) Acc65I (860) KpnI (864) BtgI (953) BmgBI (962) MscI (997) EcoRV (1014) MluI (1018) NdeI (1025) NheI (1030) BmtI (1034) PstI - SbfI (1040) BamHI (1042) BtgZI (1079) BsgI (1485) BsaBI * (1716) MfeI (1804) HpaI (1817) pTRE3G-IRES 4026 bp
EcoRI  (4021)
1 site
G A A T T C C T T A A G
AatII  (3950)
1 site
G A C G T C C T G C A G
ZraI  (3948)
1 site
G A C G T C C T G C A G
ScaI  (3508)
1 site
A G T A C T T C A T G A
TatI  (3506)
1 site
W G T A C W W C A T G W
FspI  (3250)
1 site
T G C G C A A C G C G T
NmeAIII  (3176)
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  (3089)
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  (3028)
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  (2551)
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  (2443)
1 site
C C C A G C G G G T C G
BseYI  (2439)
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  (2243)
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.
BspQI  (2019)
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  (2019)
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.
AvaI  (1)
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  (1)
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.
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
BmeT110I  (2)
1 site
C Y C G R G G R G C Y C
KasI  (274)
1 site
G G C G C C C C G C G G
NarI  (275)
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  (276)
1 site
G G C G C C C C G C G G
PluTI  (278)
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.
Eco53kI  (292)
1 site
G A G C T C C T C G A G
SacI  (294)
1 site
G A G C T C C T C G A G
SalI  (383)
1 site
G T C G A C C A G C T G
AccI  (384)
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.
BglII  (399)
1 site
A G A T C T T C T A G A
BspDI  (406)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (406)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
EagI  (411)
1 site
C G G C C G G C C G G C
NgoMIV  (413)
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  (415)
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.
FseI  (417)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
AvrII  (570)
1 site
C C T A G G G G A T C C
BsaAI  (735)
1 site
Y A C G T R R T G C A Y
PmlI  (735)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (758)
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  (758)
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  (758)
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.
DraIII  (782)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
Acc65I  (860)
1 site
G G T A C C C C A T G G
KpnI  (864)
1 site
G G T A C C C C A T G G
BtgI  (953)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
BmgBI  (962)
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.
MscI  (997)
1 site
T G G C C A A C C G G T
EcoRV  (1014)
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.
MluI  (1018)
1 site
A C G C G T T G C G C A
NdeI  (1025)
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.
NheI  (1030)
1 site
G C T A G C C G A T C G
BmtI  (1034)
1 site
G C T A G C C G A T C G
PstI  (1040)
1 site
C T G C A G G A C G T C
SbfI  (1040)
1 site
C C T G C A G G G G A C G T C C
BamHI  (1042)
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.
BtgZI  (1079)
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.
BsgI  (1485)
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).
BsaBI  (1716)
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.
MfeI  (1804)
1 site
C A A T T G G T T A A C
HpaI  (1817)
1 site
G T T A A C C A A T T G
AmpR
2955 .. 3815  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2955 .. 3746  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2955 .. 3815  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3747 .. 3815  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2955 .. 3815  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2196 .. 2784  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2196 .. 2784  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES2
418 .. 1005  =  588 bp
   Segment 1:  
   418 .. 993  =  576 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
418 .. 1005  =  588 bp
   Segment 2:  ATG  
   994 .. 996  =  3 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
418 .. 1005  =  588 bp
   Segment 3:  
   997 .. 1005  =  9 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES2
418 .. 1005  =  588 bp
3 segments
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
TRE3G promoter
4 .. 382  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On 3G
TRE3G promoter
4 .. 382  =  379 bp
3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On 3G
SV40 poly(A) signal
1818 .. 1939  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1818 .. 1939  =  122 bp
SV40 polyadenylation signal
AmpR promoter
3816 .. 3920  =  105 bp
AmpR promoter
3816 .. 3920  =  105 bp
small t intron
1178 .. 1243  =  66 bp
simian virus 40 (SV40) small t antigen intron
small t intron
1178 .. 1243  =  66 bp
simian virus 40 (SV40) small t antigen intron
MCS 2
1012 .. 1047  =  36 bp
multiple cloning site 2
MCS 2
1012 .. 1047  =  36 bp
multiple cloning site 2
MCS 1
383 .. 416  =  34 bp
multiple cloning site 1
MCS 1
383 .. 416  =  34 bp
multiple cloning site 1
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
84 .. 102  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
84 .. 102  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
120 .. 138  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
120 .. 138  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
156 .. 174  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
156 .. 174  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
192 .. 210  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
192 .. 210  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
228 .. 246  =  19 bp
bacterial operator O2 for the tetR and tetA genes
tet operator
228 .. 246  =  19 bp
bacterial operator O2 for the tetR and tetA genes
ATG
994 .. 996  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
ATG
994 .. 996  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon for translation from IRES2
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