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Plasmid Files

pET-DEST42

Gateway® destination vector for high-level T7-regulated bacterial expression and C-terminal tagging.

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pET-DEST42 Sequence and MappET-DEST42.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 EcoNI (44) BstEII (6834) ApaI (6813) PspOMI (6809) HpaI (6514) PshAI (6175) NruI (5917) FspAI (5435) PflFI - Tth111I (4670) BsaAI (4664) BspQI - SapI (4529) PciI (4412) SphI (109) SgrAI (257) BglII (298) T7 promoter XbaI (364) NotI (528) lac UV5 promoter EcoRI (848) PasI (1080) NcoI (1149) BbvCI (1573) TspMI - XmaI (1717) SmaI - SrfI (1719) BmgBI (1753) SalI (1976) BstBI (2114) BseRI (2138) AgeI (2171) BlpI (2254) T7 terminator BspDI - ClaI (2498) ZraI (2603) AatII (2605) PvuI (3155) AhdI (3524) pET-DEST42 7440 bp
EcoNI  (44)
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.
BstEII  (6834)
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  (6813)
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  (6809)
1 site
G G G C C C C C C G G G
HpaI  (6514)
1 site
G T T A A C C A A T T G
PshAI  (6175)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
NruI  (5917)
1 site
T C G C G A A G C G C T
FspAI  (5435)
1 site
R T G C G C A Y Y A C G C G T R
PflFI  (4670)
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  (4670)
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.
BsaAI  (4664)
1 site
Y A C G T R R T G C A Y
BspQI  (4529)
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  (4529)
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  (4412)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SphI  (109)
1 site
G C A T G C C G T A C G
SgrAI  (257)
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.
BglII  (298)
1 site
A G A T C T T C T A G A
XbaI  (364)
1 site
T C T A G A A G A T C T
NotI  (528)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (848)
1 site
G A A T T C C T T A A G
PasI  (1080)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
NcoI  (1149)
1 site
C C A T G G G G T A C C
BbvCI  (1573)
1 site
C C T C A G C G G A G T C G
TspMI  (1717)
1 site
C C C G G G G G G C C C
XmaI  (1717)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (1719)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (1719)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1753)
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.
SalI  (1976)
1 site
G T C G A C C A G C T G
BstBI  (2114)
1 site
T T C G A A A A G C T T
BseRI  (2138)
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.
AgeI  (2171)
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.
BlpI  (2254)
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.
BspDI  (2498)
1 site
A T C G A T T A G C T A
ClaI  (2498)
1 site
A T C G A T T A G C T A
ZraI  (2603)
1 site
G A C G T C C T G C A G
AatII  (2605)
1 site
G A C G T C C T G C A G
PvuI  (3155)
1 site
C G A T C G G C T A G C
AhdI  (3524)
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.
lacI
6289 .. 7371  =  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
6289 .. 7371  =  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
2737 .. 3597  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   2737 .. 2805  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2737 .. 3597  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2806 .. 3597  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2737 .. 3597  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
635 .. 1294  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
635 .. 1294  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3768 .. 4356  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3768 .. 4356  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
1636 .. 1941  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
1636 .. 1941  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
rop
4786 .. 4977  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
4786 .. 4977  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
attR1
402 .. 526  =  125 bp
recombination site for the Gateway® LR reaction
attR1
402 .. 526  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1982 .. 2106  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1982 .. 2106  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2632 .. 2736  =  105 bp
AmpR promoter
2632 .. 2736  =  105 bp
lacI promoter
7372 .. 9  =  78 bp
lacI promoter
7372 .. 9  =  78 bp
T7 terminator
2265 .. 2312  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
2265 .. 2312  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
V5 tag
2126 .. 2167  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
2126 .. 2167  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
lac UV5 promoter
551 .. 581  =  31 bp
   Segment 1:  -35  
   551 .. 556  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
551 .. 581  =  31 bp
   Segment 2:  
   557 .. 574  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
551 .. 581  =  31 bp
   Segment 3:  -10  
   575 .. 581  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
551 .. 581  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
337 .. 361  =  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
337 .. 361  =  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
318 .. 336  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
318 .. 336  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2177 .. 2194  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2177 .. 2194  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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