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Plasmid Files

pEU-HSBC

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

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pEU-HSBC Sequence and MappEU-HSBC.dna
Map and Sequence File   
Sequence Author:  Center for Eukaryotic Structural Genomics
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 PfoI (6062) AatII (5951) ZraI (5949) XmnI (5628) FspI (5251) BsaI (5090) AhdI (5029) AlwNI (4552) BspQI - SapI (4020) lac operator ApaI (3712) PspOMI (3708) BstBI (3682) BssHII (3620) BsiWI (3563) PmlI (3556) NsiI (3517) PshAI (3209) SpeI (3179) AfeI (24) SgrAI (106) NgoMIV (115) NaeI (117) ATG AsiSI - SgfI (549) BstZ17I (661) StuI * (1031) BsgI (1355) PsiI (1430) EcoRV (2233) BspEI (2590) PasI - PflMI * (2826) PmeI (3045) Eco53kI (3058) SacI (3060) AvaI - BsoBI - TspMI - XmaI (3066) BmeT110I (3067) SmaI (3068) XbaI (3077) SalI (3083) PstI - SbfI (3093) BlpI (3158) pEU-HSBC 6336 bp
PfoI  (6062)
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.
AatII  (5951)
1 site
G A C G T C C T G C A G
ZraI  (5949)
1 site
G A C G T C C T G C A G
XmnI  (5628)
1 site
G A A N N N N T T C C T T N N N N A A G
FspI  (5251)
1 site
T G C G C A A C G C G T
BsaI  (5090)
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  (5029)
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  (4552)
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.
BspQI  (4020)
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  (4020)
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.
ApaI  (3712)
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  (3708)
1 site
G G G C C C C C C G G G
BstBI  (3682)
1 site
T T C G A A A A G C T T
BssHII  (3620)
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.
BsiWI  (3563)
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.
PmlI  (3556)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NsiI  (3517)
1 site
A T G C A T T A C G T A
PshAI  (3209)
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.
SpeI  (3179)
1 site
A C T A G T T G A T C A
AfeI  (24)
1 site
A G C G C T T C G C G A
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.
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
BstZ17I  (661)
1 site
G T A T A C C A T A T G
StuI  (1031)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BsgI  (1355)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PsiI  (1430)
1 site
T T A T A A A A T A T T
EcoRV  (2233)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to
delete a base after cleavage.
BspEI  (2590)
1 site
T C C G G A A G G C C T
PasI  (2826)
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  (2826)
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.
PmeI  (3045)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (3058)
1 site
G A G C T C C T C G A G
SacI  (3060)
1 site
G A G C T C C T C G A G
AvaI  (3066)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (3066)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (3066)
1 site
C C C G G G G G G C C C
XmaI  (3066)
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.
BmeT110I  (3067)
1 site
C Y C G R G G R G C Y C
SmaI  (3068)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
XbaI  (3077)
1 site
T C T A G A A G A T C T
SalI  (3083)
1 site
G T C G A C C A G C T G
PstI  (3093)
1 site
C T G C A G G A C G T C
SbfI  (3093)
1 site
C C T G C A G G G G A C G T C C
BlpI  (3158)
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.
SacB
790 .. 2211  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 1:  signal peptide  
   790 .. 876  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
790 .. 2211  =  1422 bp
473 amino acids  =  53.0 kDa
   Segment 2:  
   877 .. 2211  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
SacB
790 .. 2211  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders
bacterial growth sensitive to sucrose
negative selection marker
AmpR
4956 .. 5816  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4956 .. 5747  =  792 bp
   263 amino acids  =  29.0 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4956 .. 5816  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5748 .. 5816  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4956 .. 5816  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
2381 .. 3040  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
2381 .. 3040  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4197 .. 4785  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4197 .. 4785  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
5817 .. 5921  =  105 bp
AmpR promoter
5817 .. 5921  =  105 bp
lac UV5 promoter
2297 .. 2327  =  31 bp
   Segment 1:  -35  
   2297 .. 2302  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2297 .. 2327  =  31 bp
   Segment 2:  
   2303 .. 2320  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2297 .. 2327  =  31 bp
   Segment 3:  -10  
   2321 .. 2327  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
2297 .. 2327  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
lac promoter
3843 .. 3873  =  31 bp
   Segment 3:  -10  
   3843 .. 3849  =  7 bp
promoter for the E. coli lac operon
lac promoter
3843 .. 3873  =  31 bp
   Segment 2:  
   3850 .. 3867  =  18 bp
promoter for the E. coli lac operon
lac promoter
3843 .. 3873  =  31 bp
   Segment 1:  -35  
   3868 .. 3873  =  6 bp
promoter for the E. coli lac operon
lac promoter
3843 .. 3873  =  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
3795 .. 3811  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3795 .. 3811  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
3819 .. 3835  =  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
3819 .. 3835  =  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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