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Plasmid Files

pETDuet™-1

Bacterial vector for the co-expression of two genes.

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pETDuet-1 Sequence and MappETDuet-1.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 EcoRI (112) BamHI (106) NcoI (69) RBS XbaI (30) lac operator T7 promoter BspDI * - ClaI * (5383) pET Upstream Primer (5358 .. 5373) SgrAI (5339) SphI (5191) EcoNI (5126) BstAPI (4982) MluI (4658) BclI * (4644) BstEII (4476) ApaI (4455) PspOMI (4451) HpaI (4156) PpuMI (3789) Bpu10I (3689) PflFI - Tth111I (3052) BstZ17I (3027) BspQI - SapI (2911) PciI (2794) Eco53kI (120) SacI (122) BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) SalI (137) HindIII (143) NotI (150) AflII (163) DuetUP2 Primer (189 .. 208) BsrGI (190) DuetDOWN1 Primer (189 .. 208) T7 promoter RBS NdeI (298) BglII (305) MfeI (311) EcoRV (319) FseI (328) AsiSI (337) ZraI (344) AatII (346) Acc65I (348) KpnI (352) AvaI - BsoBI - PaeR7I - PspXI - XhoI (354) BmeT110I (355) PacI (429) AvrII (433) BlpI (451) T7 Terminator Primer (448 .. 466) T7 terminator DraIII (779) PsiI (904) SspI (984) AhdI (1189) BsaI (1250) BglI (1309) FspI (1411) ScaI (1669) AlwNI (2385) pETDuet™-1 5420 bp
EcoRI  (112)
1 site
G A A T T C C T T A A G
BamHI  (106)
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.
NcoI  (69)
1 site
C C A T G G G G T A C C
XbaI  (30)
1 site
T C T A G A A G A T C T
BspDI  (5383)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (5383)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (5339)
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.
SphI  (5191)
1 site
G C A T G C C G T A C G
EcoNI  (5126)
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.
BstAPI  (4982)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (4658)
1 site
A C G C G T T G C G C A
BclI  (4644)
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.
BstEII  (4476)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
ApaI  (4455)
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  (4451)
1 site
G G G C C C C C C G G G
HpaI  (4156)
1 site
G T T A A C C A A T T G
PpuMI  (3789)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
Bpu10I  (3689)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
PflFI  (3052)
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  (3052)
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.
BstZ17I  (3027)
1 site
G T A T A C C A T A T G
BspQI  (2911)
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  (2911)
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.
PciI  (2794)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
Eco53kI  (120)
1 site
G A G C T C C T C G A G
SacI  (122)
1 site
G A G C T C C T C G A G
BfuAI  (124)
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  (124)
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.
AscI  (125)
1 site
G G C G C G C C C C G C G C G G
PstI  (135)
1 site
C T G C A G G A C G T C
SbfI  (135)
1 site
C C T G C A G G G G A C G T C C
SalI  (137)
1 site
G T C G A C C A G C T G
HindIII  (143)
1 site
A A G C T T T T C G A A
NotI  (150)
1 site
G C G G C C G C C G C C G G C G
AflII  (163)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
BsrGI  (190)
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.
NdeI  (298)
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.
BglII  (305)
1 site
A G A T C T T C T A G A
MfeI  (311)
1 site
C A A T T G G T T A A C
EcoRV  (319)
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.
FseI  (328)
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.
AsiSI  (337)
1 site
G C G A T C G C C G C T A G C G
ZraI  (344)
1 site
G A C G T C C T G C A G
AatII  (346)
1 site
G A C G T C C T G C A G
Acc65I  (348)
1 site
G G T A C C C C A T G G
KpnI  (352)
1 site
G G T A C C C C A T G G
AvaI  (354)
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  (354)
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  (354)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (354)
1 site
V C T C G A G B B G A G C T C V
XhoI  (354)
1 site
C T C G A G G A G C T C
BmeT110I  (355)
1 site
C Y C G R G G R G C Y C
PacI  (429)
1 site
T T A A T T A A A A T T A A T T
AvrII  (433)
1 site
C C T A G G G G A T C C
BlpI  (451)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
DraIII  (779)
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.
PsiI  (904)
1 site
T T A T A A A A T A T T
SspI  (984)
1 site
A A T A T T T T A T A A
AhdI  (1189)
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.
BsaI  (1250)
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.
BglI  (1309)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
FspI  (1411)
1 site
T G C G C A A C G C G T
ScaI  (1669)
1 site
A G T A C T T C A T G A
AlwNI  (2385)
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.
pET Upstream Primer
16-mer  /  63% GC
1 binding site
5358 .. 5373  =  16 annealed bases
Tm  =  57°C
DuetUP2 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  56°C
DuetDOWN1 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  56°C
T7 Terminator Primer
19-mer  /  53% GC
1 binding site
448 .. 466  =  19 annealed bases
Tm  =  56°C
lacI
3931 .. 5013  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
3931 .. 5013  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
AmpR
1116 .. 1976  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1116 .. 1907  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1116 .. 1976  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1908 .. 1976  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1116 .. 1976  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2150 .. 2738  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2150 .. 2738  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
546 .. 1001  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
546 .. 1001  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
3168 .. 3359  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3168 .. 3359  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
MCS-2
297 .. 438  =  142 bp
multiple cloning site #2
MCS-2
297 .. 438  =  142 bp
multiple cloning site #2
MCS-1
69 .. 168  =  100 bp
multiple cloning site #1
MCS-1
69 .. 168  =  100 bp
multiple cloning site #1
AmpR promoter
1977 .. 2069  =  93 bp
AmpR promoter
1977 .. 2069  =  93 bp
lacI promoter
5014 .. 5091  =  78 bp
lacI promoter
5014 .. 5091  =  78 bp
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
3 .. 27  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
3 .. 27  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
233 .. 257  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
233 .. 257  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5404 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5404 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
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