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Plasmid Files

pEU-His-FV

Flexi® Vector with the barnase gene, for in vitro transcription and translation of an N-terminally 6xHis-tagged protein.

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pEU-His-FV Sequence and MappEU-His-FV.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 AfeI (24) PfoI (4692) XmnI (4258) FspI (3881) BsaI (3720) AhdI (3659) AlwNI (3182) PciI (2766) BspQI - SapI (2650) lac operator SgrAI (106) NgoMIV (115) NaeI (117) BsrGI (309) ATG AsiSI - SgfI (549) Bpu10I (997) BspEI (1220) PasI - PflMI * (1456) MscI (1491) PmeI (1675) Eco53kI (1688) SacI (1690) Acc65I (1692) KpnI (1696) XbaI (1707) SalI (1713) AccI (1714) HincII (1715) PstI - SbfI (1723) BlpI (1788) SpeI (1809) PshAI (1839) NsiI (2147) PmlI (2186) BsiWI - SnaBI (2193) BssHII (2250) BstBI (2312) PspOMI (2338) ApaI (2342) HindIII (2392) SacII (2403) pEU-His-FV 4966 bp
AfeI  (24)
1 site
A G C G C T T C G C G A
PfoI  (4692)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
XmnI  (4258)
1 site
G A A N N N N T T C C T T N N N N A A G
FspI  (3881)
1 site
T G C G C A A C G C G T
BsaI  (3720)
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.
AhdI  (3659)
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.
AlwNI  (3182)
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.
PciI  (2766)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2650)
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  (2650)
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.
SgrAI  (106)
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.
NgoMIV  (115)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (117)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
BsrGI  (309)
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.
AsiSI  (549)
1 site
G C G A T C G C C G C T A G C G
SgfI  (549)
1 site
G C G A T C G C C G C T A G C G
Bpu10I  (997)
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.
BspEI  (1220)
1 site
T C C G G A A G G C C T
PasI  (1456)
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  (1456)
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.
MscI  (1491)
1 site
T G G C C A A C C G G T
PmeI  (1675)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1688)
1 site
G A G C T C C T C G A G
SacI  (1690)
1 site
G A G C T C C T C G A G
Acc65I  (1692)
1 site
G G T A C C C C A T G G
KpnI  (1696)
1 site
G G T A C C C C A T G G
XbaI  (1707)
1 site
T C T A G A A G A T C T
SalI  (1713)
1 site
G T C G A C C A G C T G
AccI  (1714)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
HincII  (1715)
1 site
G T Y R A C C A R Y T G
PstI  (1723)
1 site
C T G C A G G A C G T C
SbfI  (1723)
1 site
C C T G C A G G G G A C G T C C
BlpI  (1788)
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.
SpeI  (1809)
1 site
A C T A G T T G A T C A
PshAI  (1839)
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.
NsiI  (2147)
1 site
A T G C A T T A C G T A
PmlI  (2186)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BsiWI  (2193)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SnaBI  (2193)
1 site
T A C G T A A T G C A T
BssHII  (2250)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BstBI  (2312)
1 site
T T C G A A A A G C T T
PspOMI  (2338)
1 site
G G G C C C C C C G G G
ApaI  (2342)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HindIII  (2392)
1 site
A A G C T T T T C G A A
SacII  (2403)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
AmpR
3586 .. 4446  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3586 .. 4377  =  792 bp
   263 amino acids  =  29.0 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3586 .. 4446  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4378 .. 4446  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3586 .. 4446  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
1011 .. 1670  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1011 .. 1670  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
2827 .. 3415  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2827 .. 3415  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
576 .. 911  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
576 .. 911  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
AmpR promoter
4447 .. 4551  =  105 bp
AmpR promoter
4447 .. 4551  =  105 bp
lac UV5 promoter
927 .. 957  =  31 bp
   Segment 1:  -35  
   927 .. 932  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
927 .. 957  =  31 bp
   Segment 2:  
   933 .. 950  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
927 .. 957  =  31 bp
   Segment 3:  -10  
   951 .. 957  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
927 .. 957  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac promoter
2473 .. 2503  =  31 bp
   Segment 3:  -10  
   2473 .. 2479  =  7 bp
promoter for the E. coli lac operon
lac promoter
2473 .. 2503  =  31 bp
   Segment 2:  
   2480 .. 2497  =  18 bp
promoter for the E. coli lac operon
lac promoter
2473 .. 2503  =  31 bp
   Segment 1:  -35  
   2498 .. 2503  =  6 bp
promoter for the E. coli lac operon
lac promoter
2473 .. 2503  =  31 bp
3 segments
promoter for the E. coli lac operon
SP6 promoter
373 .. 391  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
373 .. 391  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
6xHis
527 .. 544  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
527 .. 544  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
M13 rev
2425 .. 2441  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
2425 .. 2441  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
2449 .. 2465  =  17 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
2449 .. 2465  =  17 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).
ATG
521 .. 523  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
521 .. 523  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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