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Plasmid Files

pHIL-S1

Pichia pastoris HIS4 vector for methanol-inducible expression of a secreted protein.

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pHIL-S1 Sequence and MappHIL-S1.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AatII (8057) ZraI (8055) ScaI (7615) PvuI (7505) PstI (7380) SwaI (7020) AanI - AanI - PsiI (6923) AflIII - PciI (5805) BspQI - SapI (5689) NdeI (5628) BstZ17I (5578) PflFI - Tth111I (5552) BglII (5393) SphI (4555) SgrAI (4399) BfuAI - BspMI (4255) BglII (1) AleI (76) Eco53kI (206) SacI (208) PmeI (411) BlpI (586) NsiI (676) XcmI (706) PaeR7I - XhoI (1006) EcoRI (1011) TspMI - XmaI (1017) SmaI (1019) BamHI (1021) AgeI (1072) FspAI (1598) XbaI (1804) Bsu36I (1860) HpaI (1910) SalI (2949) StuI (3034) NcoI (3404) BspEI (3616) BsrGI (4070) pHIL-S1 8260 bp
AatII  (8057)
1 site
G A C G T C C T G C A G
ZraI  (8055)
1 site
G A C G T C C T G C A G
ScaI  (7615)
1 site
A G T A C T T C A T G A
PvuI  (7505)
1 site
C G A T C G G C T A G C
PstI  (7380)
1 site
C T G C A G G A C G T C
SwaI  (7020)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AanI  (6923)
1 site
T T A T A A A A T A T T
AanI  (6923)
1 site
T T A T A A A A T A T T
PsiI  (6923)
1 site
T T A T A A A A T A T T
AflIII  (5805)
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  (5805)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5689)
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  (5689)
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.
NdeI  (5628)
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.
BstZ17I  (5578)
1 site
G T A T A C C A T A T G
PflFI  (5552)
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  (5552)
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.
BglII  (5393)
2 sites
A G A T C T T C T A G A
SphI  (4555)
1 site
G C A T G C C G T A C G
SgrAI  (4399)
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.
BfuAI  (4255)
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  (4255)
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.
BglII  (1)
2 sites
A G A T C T T C T A G A
AleI  (76)
1 site
C A C N N N N G T G G T G N N N N C A C
Eco53kI  (206)
1 site
G A G C T C C T C G A G
SacI  (208)
1 site
G A G C T C C T C G A G
PmeI  (411)
1 site
G T T T A A A C C A A A T T T G
BlpI  (586)
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.
NsiI  (676)
1 site
A T G C A T T A C G T A
XcmI  (706)
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.
PaeR7I  (1006)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1006)
1 site
C T C G A G G A G C T C
EcoRI  (1011)
1 site
G A A T T C C T T A A G
TspMI  (1017)
1 site
C C C G G G G G G C C C
XmaI  (1017)
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.
SmaI  (1019)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (1021)
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.
AgeI  (1072)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
FspAI  (1598)
1 site
R T G C G C A Y Y A C G C G T R
XbaI  (1804)
1 site
T C T A G A A G A T C T
Bsu36I  (1860)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
HpaI  (1910)
1 site
G T T A A C C A A T T G
SalI  (2949)
1 site
G T C G A C C A G C T G
StuI  (3034)
1 site
A G G C C T T C C G G A
NcoI  (3404)
1 site
C C A T G G G G T A C C
BspEI  (3616)
1 site
T C C G G A A G G C C T
BsrGI  (4070)
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.
PpHIS4
1752 .. 4286  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
PpHIS4
1752 .. 4286  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
AOX1 promoter
2 .. 941  =  940 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 941  =  940 bp
inducible promoter, regulated by methanol
AmpR
7062 .. 7922  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   7062 .. 7853  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7062 .. 7922  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7854 .. 7922  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7062 .. 7922  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AOX1 3' fragment
4641 .. 5397  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
4641 .. 5397  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
5866 .. 6454  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5866 .. 6454  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6565 .. 7020  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6565 .. 7020  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
AOX1 terminator
1093 .. 1339  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1093 .. 1339  =  247 bp
transcription terminator for AOX1
AmpR promoter
7923 .. 8027  =  105 bp
AmpR promoter
7923 .. 8027  =  105 bp
PHO1 signal sequence
942 .. 1007  =  66 bp
22 amino acids  =  2.4 kDa
Product: signal sequence from a secreted acid
phosphatase of Pichia pastoris
synthetic gene fragment
PHO1 signal sequence
942 .. 1007  =  66 bp
22 amino acids  =  2.4 kDa
Product: signal sequence from a secreted acid
phosphatase of Pichia pastoris
synthetic gene fragment
MCS
1006 .. 1026  =  21 bp
multiple cloning site
MCS
1006 .. 1026  =  21 bp
multiple cloning site
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