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Plasmid Files

pET160-DEST

Gateway® destination vector for high-level bacterial expression of proteins with an N-terminal tetracysteine (Lumio™) tag.

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pET160-DEST Sequence and MappET160-DEST.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BglII (1) SphI (7249) EcoNI (7184) BclI * (6702) BstEII (6534) ApaI (6513) PspOMI (6509) HpaI (6214) PshAI (5875) NruI (5617) FspAI (5135) PflFI - Tth111I (4370) BsaAI (4364) BspQI - SapI (4229) PciI (4112) T7 promoter XbaI (67) RBS NdeI (107) ATG NsiI (113) 6xHis AleI (128) tetracysteine tag TEV Site NotI (322) lac UV5 promoter EcoRI (642) PasI (874) AgeI (1142) BbvCI (1346) TspMI - XmaI (1490) SmaI - SrfI (1492) BmgBI (1526) SalI (1749) PacI (1889) BlpI (1950) T7 terminator BspDI - ClaI (2198) ZraI (2303) AatII (2305) PvuI (2855) AhdI (3224) pET160-DEST 7437 bp
BglII  (1)
1 site
A G A T C T T C T A G A
SphI  (7249)
1 site
G C A T G C C G T A C G
EcoNI  (7184)
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.
BclI  (6702)
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  (6534)
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  (6513)
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  (6509)
1 site
G G G C C C C C C G G G
HpaI  (6214)
1 site
G T T A A C C A A T T G
PshAI  (5875)
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  (5617)
1 site
T C G C G A A G C G C T
FspAI  (5135)
1 site
R T G C G C A Y Y A C G C G T R
PflFI  (4370)
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  (4370)
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  (4364)
1 site
Y A C G T R R T G C A Y
BspQI  (4229)
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  (4229)
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  (4112)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
XbaI  (67)
1 site
T C T A G A A G A T C T
NdeI  (107)
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.
NsiI  (113)
1 site
A T G C A T T A C G T A
AleI  (128)
1 site
C A C N N N N G T G G T G N N N N C A C
NotI  (322)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (642)
1 site
G A A T T C C T T A A G
PasI  (874)
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.
AgeI  (1142)
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.
BbvCI  (1346)
1 site
C C T C A G C G G A G T C G
TspMI  (1490)
1 site
C C C G G G G G G C C C
XmaI  (1490)
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  (1492)
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  (1492)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1526)
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  (1749)
1 site
G T C G A C C A G C T G
PacI  (1889)
1 site
T T A A T T A A A A T T A A T T
BlpI  (1950)
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  (2198)
1 site
A T C G A T T A G C T A
ClaI  (2198)
1 site
A T C G A T T A G C T A
ZraI  (2303)
1 site
G A C G T C C T G C A G
AatII  (2305)
1 site
G A C G T C C T G C A G
PvuI  (2855)
1 site
C G A T C G G C T A G C
AhdI  (3224)
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
5989 .. 7071  =  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
5989 .. 7071  =  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
2437 .. 3297  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   2437 .. 2505  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2437 .. 3297  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2506 .. 3297  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2437 .. 3297  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
429 .. 1088  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
429 .. 1088  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3468 .. 4056  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3468 .. 4056  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
1409 .. 1714  =  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
1409 .. 1714  =  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
4486 .. 4677  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
4486 .. 4677  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
attR1
196 .. 320  =  125 bp
recombination site for the Gateway® LR reaction
attR1
196 .. 320  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1755 .. 1879  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1755 .. 1879  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2332 .. 2436  =  105 bp
AmpR promoter
2332 .. 2436  =  105 bp
lacI promoter
7072 .. 7149  =  78 bp
lacI promoter
7072 .. 7149  =  78 bp
T7 terminator
1961 .. 2008  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
1961 .. 2008  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac UV5 promoter
345 .. 375  =  31 bp
   Segment 1:  -35  
   345 .. 350  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
345 .. 375  =  31 bp
   Segment 2:  
   351 .. 368  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
345 .. 375  =  31 bp
   Segment 3:  -10  
   369 .. 375  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
345 .. 375  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac operator
40 .. 64  =  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
40 .. 64  =  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).
TEV Site
169 .. 189  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) recognition and
cleavage site
TEV Site
169 .. 189  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) recognition and
cleavage site
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
112 .. 129  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
112 .. 129  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
tetracysteine tag
142 .. 159  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
tetracysteine tag
142 .. 159  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical
labeling reagents
RBS
94 .. 101  =  8 bp
ribosome binding site
RBS
94 .. 101  =  8 bp
ribosome binding site
ATG
109 .. 111  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
109 .. 111  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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