pESC-HIS

Yeast episomal vector with a HIS3 marker, for galactose-regulated expression and tagging of up to two genes.

Sequence Author: Agilent Technologies

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MfeI (6588) XcmI (6587) BmgBI (6376) XbaI (6275) SnaBI (5938) ScaI (4858) BpmI (4448) BsaI (4439) BmrI (4418) AhdI (4378) AlwNI (3901) PspFI (3793) BseYI (3789) BspQI - SapI (3369) PfoI (46) BsaBI * (561) NdeI (694) MscI (711) BsmI (716) BsiWI (916) BclI * (1052) AleI (1063) PstI (1187) BssHII (1312) DraIII (1647) NgoMIV (1748) NaeI (1750) PacI (2260) Eco53kI (2266) SacI (2268) BspDI - ClaI (2300) SpeI (2305) EagI - NotI (2312) EcoRI (2334) AgeI (2629) BstAPI (2881) BamHI (3012) PspOMI (3036) TspMI - XmaI (3039) ApaI (3040) SmaI - SrfI (3041) SalI (3046) PaeR7I - PspXI - XhoI (3082) BtgI (3099) SacII (3102) PpuMI (3159) BsrGI (3233) MluI (3240) pESC-HIS 6706 bp
MfeI  (6588)
1 site
C A A T T G G T T A A C
XcmI  (6587)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BmgBI  (6376)
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.
XbaI  (6275)
1 site
T C T A G A A G A T C T
SnaBI  (5938)
1 site
T A C G T A A T G C A T
ScaI  (4858)
1 site
A G T A C T T C A T G A
BpmI  (4448)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BsaI  (4439)
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.
BmrI  (4418)
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.
AhdI  (4378)
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  (3901)
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.
PspFI  (3793)
1 site
C C C A G C G G G T C G
BseYI  (3789)
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.
BspQI  (3369)
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  (3369)
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.
PfoI  (46)
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.
BsaBI  (561)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
NdeI  (694)
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.
MscI  (711)
1 site
T G G C C A A C C G G T
BsmI  (716)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BsiWI  (916)
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.
BclI  (1052)
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.
AleI  (1063)
1 site
C A C N N N N G T G G T G N N N N C A C
PstI  (1187)
1 site
C T G C A G G A C G T C
BssHII  (1312)
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.
DraIII  (1647)
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.
NgoMIV  (1748)
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  (1750)
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.
PacI  (2260)
1 site
T T A A T T A A A A T T A A T T
Eco53kI  (2266)
1 site
G A G C T C C T C G A G
SacI  (2268)
1 site
G A G C T C C T C G A G
BspDI  (2300)
1 site
A T C G A T T A G C T A
ClaI  (2300)
1 site
A T C G A T T A G C T A
SpeI  (2305)
1 site
A C T A G T T G A T C A
EagI  (2312)
1 site
C G G C C G G C C G G C
NotI  (2312)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (2334)
1 site
G A A T T C C T T A A G
AgeI  (2629)
1 site
A C C G G T T G G C C A
BstAPI  (2881)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BamHI  (3012)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
PspOMI  (3036)
1 site
G G G C C C C C C G G G
TspMI  (3039)
1 site
C C C G G G G G G C C C
XmaI  (3039)
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.
ApaI  (3040)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SmaI  (3041)
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  (3041)
1 site
G C C C G G G C C G G G C C C G
SalI  (3046)
1 site
G T C G A C C A G C T G
PaeR7I  (3082)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (3082)
1 site
V C T C G A G B B G A G C T C V
XhoI  (3082)
1 site
C T C G A G G A G C T C
BtgI  (3099)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SacII  (3102)
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.
PpuMI  (3159)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BsrGI  (3233)
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.
MluI  (3240)
1 site
A C G C G T T G C G C A
2μ ori
5297 .. 6639  =  1343 bp
yeast 2μ plasmid origin of replication
2μ ori
5297 .. 6639  =  1343 bp
yeast 2μ plasmid origin of replication
AmpR
4305 .. 5165  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   4305 .. 5096  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4305 .. 5165  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   5097 .. 5165  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4305 .. 5165  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GAL1,10 promoter
2344 .. 3008  =  665 bp
divergent inducible promoter, regulated by Gal4
GAL1,10 promoter
2344 .. 3008  =  665 bp
divergent inducible promoter, regulated by Gal4
HIS3
504 .. 1163  =  660 bp
219 amino acids  =  23.8 kDa
Product: imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis
yeast auxotrophic marker
HIS3
504 .. 1163  =  660 bp
219 amino acids  =  23.8 kDa
Product: imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis
yeast auxotrophic marker
ori
3546 .. 4134  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3546 .. 4134  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1423 .. 1878  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1423 .. 1878  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
CYC1 terminator
3114 .. 3303  =  190 bp
transcription terminator for CYC1
CYC1 terminator
3114 .. 3303  =  190 bp
transcription terminator for CYC1
HIS3 promoter
317 .. 503  =  187 bp
HIS3 promoter
317 .. 503  =  187 bp
ADH1 terminator
1961 .. 2126  =  166 bp
transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene