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Plasmid Files

pPIC3.5

Pichia pastoris HIS4 vector for methanol-inducible intracellular expression of a protein. The MCS contains no start codons.

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pPIC3.5 Sequence and MappPIC3.5.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AatII (7576) ZraI (7574) ScaI (7134) PvuI (7024) PstI (6899) AflIII - PciI (5761) BspQI - SapI (5645) NdeI (5584) BstZ17I (5534) PflFI - Tth111I (5508) BglII (5349) SphI (4511) SgrAI (4355) NaeI (4348) NgoMIV (4346) BfuAI - BspMI (4211) BsrGI (4026) BglII (1) AleI (76) Eco53kI (206) SacI (208) PmeI (413) BlpI (588) NsiI (677) XcmI (706) BamHI (938) SnaBI (946) EcoRI (950) AvrII (956) EagI - NotI (963) AgeI (1028) BtgZI (1482) FspAI (1554) XbaI (1760) Bsu36I (1816) HpaI (1866) SalI (2905) StuI (2990) DraIII (3352) NcoI (3360) BspEI (3572) pPIC3.5 7751 bp
AatII  (7576)
1 site
G A C G T C C T G C A G
ZraI  (7574)
1 site
G A C G T C C T G C A G
ScaI  (7134)
1 site
A G T A C T T C A T G A
PvuI  (7024)
1 site
C G A T C G G C T A G C
PstI  (6899)
1 site
C T G C A G G A C G T C
AflIII  (5761)
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  (5761)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5645)
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  (5645)
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  (5584)
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  (5534)
1 site
G T A T A C C A T A T G
PflFI  (5508)
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  (5508)
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  (5349)
2 sites
A G A T C T T C T A G A
SphI  (4511)
1 site
G C A T G C C G T A C G
SgrAI  (4355)
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.
NaeI  (4348)
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.
NgoMIV  (4346)
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.
BfuAI  (4211)
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  (4211)
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.
BsrGI  (4026)
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.
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  (413)
1 site
G T T T A A A C C A A A T T T G
BlpI  (588)
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  (677)
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.
BamHI  (938)
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.
SnaBI  (946)
1 site
T A C G T A A T G C A T
EcoRI  (950)
1 site
G A A T T C C T T A A G
AvrII  (956)
1 site
C C T A G G G G A T C C
EagI  (963)
1 site
C G G C C G G C C G G C
NotI  (963)
1 site
G C G G C C G C C G C C G G C G
AgeI  (1028)
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.
BtgZI  (1482)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
FspAI  (1554)
1 site
R T G C G C A Y Y A C G C G T R
XbaI  (1760)
1 site
T C T A G A A G A T C T
Bsu36I  (1816)
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  (1866)
1 site
G T T A A C C A A T T G
SalI  (2905)
1 site
G T C G A C C A G C T G
StuI  (2990)
1 site
A G G C C T T C C G G A
DraIII  (3352)
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.
NcoI  (3360)
1 site
C C A T G G G G T A C C
BspEI  (3572)
1 site
T C C G G A A G G C C T
PpHIS4
1708 .. 4242  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
PpHIS4
1708 .. 4242  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
AOX1 promoter
2 .. 937  =  936 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 937  =  936 bp
inducible promoter, regulated by methanol
AmpR
6581 .. 7441  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6581 .. 7372  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6581 .. 7441  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7373 .. 7441  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6581 .. 7441  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AOX1 3' fragment
4597 .. 5353  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
4597 .. 5353  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
5822 .. 6410  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5822 .. 6410  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AOX1 terminator
1049 .. 1295  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1049 .. 1295  =  247 bp
transcription terminator for AOX1
AmpR promoter
7442 .. 7546  =  105 bp
AmpR promoter
7442 .. 7546  =  105 bp
MCS
938 .. 969  =  32 bp
multiple cloning site
MCS
938 .. 969  =  32 bp
multiple cloning site
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