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Plasmid Files

pPIC3.5K

Pichia pastoris multi-copy integrating vector for methanol-inducible intracellular expression of a protein.

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pPIC3.5K Sequence and MappPIC3.5K.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AatII (8829) ZraI (8827) ScaI (8387) AflIII - PciI (7014) BspQI - SapI (6898) NdeI (6837) BstZ17I (6787) PflFI - Tth111I (6761) BglII (6602) SphI (5764) SgrAI (5608) PaeR7I - PspXI - XhoI (5437) NruI (5382) SmaI (5165) TspMI - XmaI (5163) AsiSI (5041) BglII (1) AleI (76) Eco53kI (206) SacI (208) PmeI (413) BlpI (588) 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) NcoI (3360) BspEI (3572) BsrGI (4026) BbvCI (4372) pPIC3.5K 9004 bp
AatII  (8829)
1 site
G A C G T C C T G C A G
ZraI  (8827)
1 site
G A C G T C C T G C A G
ScaI  (8387)
1 site
A G T A C T T C A T G A
AflIII  (7014)
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  (7014)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (6898)
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  (6898)
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  (6837)
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  (6787)
1 site
G T A T A C C A T A T G
PflFI  (6761)
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  (6761)
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  (6602)
2 sites
A G A T C T T C T A G A
SphI  (5764)
1 site
G C A T G C C G T A C G
SgrAI  (5608)
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.
PaeR7I  (5437)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5437)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5437)
1 site
C T C G A G G A G C T C
NruI  (5382)
1 site
T C G C G A A G C G C T
SmaI  (5165)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (5163)
1 site
C C C G G G G G G C C C
XmaI  (5163)
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.
AsiSI  (5041)
1 site
G C G A T C G C C G C T A G C G
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.
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
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
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.
BbvCI  (4372)
1 site
C C T C A G C G G A G T C G
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
7834 .. 8694  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   7834 .. 8625  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7834 .. 8694  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   8626 .. 8694  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7834 .. 8694  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
KanR
4656 .. 5471  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
4656 .. 5471  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
AOX1 3' fragment
5850 .. 6606  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
5850 .. 6606  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
7075 .. 7663  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7075 .. 7663  =  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
8695 .. 8799  =  105 bp
AmpR promoter
8695 .. 8799  =  105 bp
MCS
938 .. 969  =  32 bp
multiple cloning site
MCS
938 .. 969  =  32 bp
multiple cloning site
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