pEXP4-DEST

Gateway® destination vector for cell-free expression and C-terminal tagging. See also pEXP2-DEST.

Sequence Author: Thermo Fisher (Invitrogen)

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BspQI - SapI (4352) NspI (4239) AflIII - PciI (4235) DrdI (4133) PspFI (3936) BseYI (3932) AhdI (3350) BmrI (3310) FspI (3127) PvuI (2981) XmnI (2750) DraIII (2521) FseI (2444) CsiI - SexAI * (2331) SgrAI (2282) AatII (2214) XbaI (58) NotI (224) PvuII (444) ApoI - EcoRI (544) PasI - PflMI * (776) SspI (856) BglII (991) BstZ17I (1079) BbvCI (1269) SrfI (1415) BstXI (1532) BfuAI - BspMI (1659) PstI (1670) SalI (1672) BclI * (1802) BstBI (1810) HindIII (1813) tetracysteine tag AgeI (1858) BlpI (1941) EcoO109I (1968) SfiI (2168) MauBI (2204) ZraI (2212) pEXP4-DEST 4415 bp
BspQI  (4352)
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  (4352)
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.
NspI  (4239)
1 site
R C A T G Y Y G T A C R
AflIII  (4235)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (4235)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (4133)
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.
PspFI  (3936)
1 site
C C C A G C G G G T C G
BseYI  (3932)
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.
AhdI  (3350)
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.
BmrI  (3310)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
FspI  (3127)
1 site
T G C G C A A C G C G T
PvuI  (2981)
1 site
C G A T C G G C T A G C
XmnI  (2750)
1 site
G A A N N N N T T C C T T N N N N A A G
DraIII  (2521)
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.
FseI  (2444)
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.
CsiI  (2331)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2331)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
SgrAI  (2282)
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.
AatII  (2214)
1 site
G A C G T C C T G C A G
XbaI  (58)
1 site
T C T A G A A G A T C T
NotI  (224)
1 site
G C G G C C G C C G C C G G C G
PvuII  (444)
1 site
C A G C T G G T C G A C
ApoI  (544)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (544)
1 site
G A A T T C C T T A A G
PasI  (776)
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.
PflMI  (776)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
SspI  (856)
1 site
A A T A T T T T A T A A
BglII  (991)
1 site
A G A T C T T C T A G A
BstZ17I  (1079)
1 site
G T A T A C C A T A T G
BbvCI  (1269)
1 site
C C T C A G C G G A G T C G
SrfI  (1415)
1 site
G C C C G G G C C G G G C C C G
BstXI  (1532)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BfuAI  (1659)
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  (1659)
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.
PstI  (1670)
1 site
C T G C A G G A C G T C
SalI  (1672)
1 site
G T C G A C C A G C T G
BclI  (1802)
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.
BstBI  (1810)
1 site
T T C G A A A A G C T T
HindIII  (1813)
1 site
A A G C T T T T C G A A
AgeI  (1858)
1 site
A C C G G T T G G C C A
BlpI  (1941)
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.
EcoO109I  (1968)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
SfiI  (2168)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
MauBI  (2204)
1 site
C G C G C G C G G C G C G C G C
ZraI  (2212)
1 site
G A C G T C C T G C A G
AmpR
2563 .. 3423  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   2563 .. 2631  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2563 .. 3423  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   2632 .. 3423  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2563 .. 3423  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
331 .. 990  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
331 .. 990  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3594 .. 4179  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3594 .. 4179  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
BleoR
2168 .. 2542  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
2168 .. 2542  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
ccdB
1332 .. 1637  =  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
1332 .. 1637  =  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.
attR1
98 .. 222  =  125 bp
recombination site for the Gateway® LR reaction
attR1
98 .. 222  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1678 .. 1802  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1678 .. 1802  =  125 bp
recombination site for the Gateway® LR reaction
tetracysteine tag
1831 .. 1848  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
tetracysteine tag
1831 .. 1848  =  18 bp
6 amino acids  =  584.8 Da
Product: tetracysteine peptide that binds biarsenical labeling reagents
6xHis
1864 .. 1881  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1864 .. 1881  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
T7 terminator
1952 .. 1999  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1952 .. 1999  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac UV5 promoter
247 .. 277  =  31 bp
3 segments
   Segment 1:  -35  
   247 .. 252  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
247 .. 277  =  31 bp
3 segments
   Segment 2:  
   253 .. 270  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
247 .. 277  =  31 bp
3 segments
   Segment 3:  -10  
   271 .. 277  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
247 .. 277  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
T7 promoter
20 .. 38  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
20 .. 38  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  331 .. 990  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  2563 .. 3423  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2168 .. 2542  =  375 bp
ORF:  124 amino acids  =  13.8 kDa
ORF:  1332 .. 1637  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  3027 .. 3293  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2101 .. 2577  =  477 bp
ORF:  158 amino acids  =  16.9 kDa
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