pET161-DEST

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

Sequence Author: Thermo Fisher (Invitrogen)

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BglII (1) SgrAI (7440) SphI (7292) EcoNI (7227) BstEII (6577) ApaI (6556) PspOMI (6552) HpaI (6257) PshAI (5918) NruI (5660) FspAI (5178) PflFI - Tth111I (4413) BsaAI (4407) BspQI - SapI (4272) PciI (4155) T7 promoter lac operator XbaI (67) NdeI (107) NheI (112) BmtI (116) NotI (280) lac UV5 promoter EcoRI (600) PasI (832) NcoI (901) BbvCI (1325) TspMI - XmaI (1469) SmaI - SrfI (1471) BmgBI (1505) SalI (1728) BstBI (1866) AgeI (1914) 6xHis BlpI (1997) BspDI - ClaI (2241) ZraI (2346) AatII (2348) PvuI (2898) AhdI (3267) pET161-DEST 7480 bp
BglII  (1)
1 site
A G A T C T T C T A G A
SgrAI  (7440)
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  (7292)
1 site
G C A T G C C G T A C G
EcoNI  (7227)
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  (6577)
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  (6556)
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  (6552)
1 site
G G G C C C C C C G G G
HpaI  (6257)
1 site
G T T A A C C A A T T G
PshAI  (5918)
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  (5660)
1 site
T C G C G A A G C G C T
FspAI  (5178)
1 site
R T G C G C A Y Y A C G C G T R
PflFI  (4413)
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  (4413)
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  (4407)
1 site
Y A C G T R R T G C A Y
BspQI  (4272)
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  (4272)
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  (4155)
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.
NheI  (112)
1 site
G C T A G C C G A T C G
BmtI  (116)
1 site
G C T A G C C G A T C G
NotI  (280)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (600)
1 site
G A A T T C C T T A A G
PasI  (832)
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  (901)
1 site
C C A T G G G G T A C C
BbvCI  (1325)
1 site
C C T C A G C G G A G T C G
TspMI  (1469)
1 site
C C C G G G G G G C C C
XmaI  (1469)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (1471)
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  (1471)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1505)
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  (1728)
1 site
G T C G A C C A G C T G
BstBI  (1866)
1 site
T T C G A A A A G C T T
AgeI  (1914)
1 site
A C C G G T T G G C C A
BlpI  (1997)
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  (2241)
1 site
A T C G A T T A G C T A
ClaI  (2241)
1 site
A T C G A T T A G C T A
ZraI  (2346)
1 site
G A C G T C C T G C A G
AatII  (2348)
1 site
G A C G T C C T G C A G
PvuI  (2898)
1 site
C G A T C G G C T A G C
AhdI  (3267)
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
6032 .. 7114  =  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
6032 .. 7114  =  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
2480 .. 3340  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   2480 .. 2548  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2480 .. 3340  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   2549 .. 3340  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2480 .. 3340  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
387 .. 1046  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
387 .. 1046  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3511 .. 4099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3511 .. 4099  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ccdB
1388 .. 1693  =  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
1388 .. 1693  =  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
4529 .. 4720  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
4529 .. 4720  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
attR1
154 .. 278  =  125 bp
recombination site for the Gateway® LR reaction
attR1
154 .. 278  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1734 .. 1858  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1734 .. 1858  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2375 .. 2479  =  105 bp
AmpR promoter
2375 .. 2479  =  105 bp
lacI promoter
7115 .. 7192  =  78 bp
lacI promoter
7115 .. 7192  =  78 bp
tetracysteine tag
1887 .. 1904  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
tetracysteine tag
1887 .. 1904  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
6xHis
1920 .. 1937  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1920 .. 1937  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
T7 terminator
2008 .. 2055  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
2008 .. 2055  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 tag (gene 10 leader)
109 .. 141  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene 10
promotes efficient translation in E. coli
T7 tag (gene 10 leader)
109 .. 141  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene 10
promotes efficient translation in E. coli
lac UV5 promoter
303 .. 333  =  31 bp
3 segments
   Segment 1:  -35  
   303 .. 308  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
303 .. 333  =  31 bp
3 segments
   Segment 2:  
   309 .. 326  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
303 .. 333  =  31 bp
3 segments
   Segment 3:  -10  
   327 .. 333  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
303 .. 333  =  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).
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
94 .. 101  =  8 bp
ribosome binding site
RBS
94 .. 101  =  8 bp
ribosome binding site
ORF:  5554 .. 6249  =  696 bp
ORF:  231 amino acids  =  25.4 kDa
ORF:  7153 .. 7416  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1388 .. 1693  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  2480 .. 3340  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  387 .. 1046  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  4752 .. 5120  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  7290 .. 49  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  4529 .. 4753  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  5117 .. 5455  =  339 bp
ORF:  112 amino acids  =  12.6 kDa
ORF:  6032 .. 6991  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  7361 .. 108  =  228 bp
ORF:  75 amino acids  =  7.9 kDa
ORF:  2944 .. 3210  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  5359 .. 5976  =  618 bp
ORF:  205 amino acids  =  21.8 kDa
ORF:  6012 .. 6275  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  6864 .. 7364  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
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Individual Sequences & Maps

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