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Plasmid Files

pPIC9K

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

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pPIC9K Sequence and MappPIC9K.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AatII (9101) ZraI (9099) ScaI (8659) AflIII - PciI (7286) BspQI - SapI (7170) NdeI (7109) BstZ17I (7059) PflFI - Tth111I (7033) BglII (6874) SphI (6036) SgrAI (5880) PspXI (5709) NruI (5654) SmaI (5437) TspMI - XmaI (5435) AsiSI (5313) BbvCI (4644) BglII (1) AleI (76) Eco53kI (206) SacI (208) PmeI (413) BlpI (588) XcmI (706) BamHI (938) PsiI (1144) SnaBI (1218) EcoRI (1222) AvrII (1228) MCS EagI - NotI (1235) AgeI (1300) BtgZI (1754) FspAI (1826) XbaI (2032) Bsu36I (2088) HpaI (2138) SalI (3177) NcoI (3632) BspEI (3844) BsrGI (4298) pPIC9K 9276 bp
AatII  (9101)
1 site
G A C G T C C T G C A G
ZraI  (9099)
1 site
G A C G T C C T G C A G
ScaI  (8659)
1 site
A G T A C T T C A T G A
AflIII  (7286)
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  (7286)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (7170)
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  (7170)
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  (7109)
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  (7059)
1 site
G T A T A C C A T A T G
PflFI  (7033)
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  (7033)
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  (6874)
2 sites
A G A T C T T C T A G A
SphI  (6036)
1 site
G C A T G C C G T A C G
SgrAI  (5880)
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.
PspXI  (5709)
1 site
V C T C G A G B B G A G C T C V
NruI  (5654)
1 site
T C G C G A A G C G C T
SmaI  (5437)
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  (5435)
1 site
C C C G G G G G G C C C
XmaI  (5435)
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  (5313)
1 site
G C G A T C G C C G C T A G C G
BbvCI  (4644)
1 site
C C T C A G C G G A G T 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.
PsiI  (1144)
1 site
T T A T A A A A T A T T
SnaBI  (1218)
1 site
T A C G T A A T G C A T
EcoRI  (1222)
1 site
G A A T T C C T T A A G
AvrII  (1228)
1 site
C C T A G G G G A T C C
EagI  (1235)
1 site
C G G C C G G C C G G C
NotI  (1235)
1 site
G C G G C C G C C G C C G G C G
AgeI  (1300)
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  (1754)
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  (1826)
1 site
R T G C G C A Y Y A C G C G T R
XbaI  (2032)
1 site
T C T A G A A G A T C T
Bsu36I  (2088)
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  (2138)
1 site
G T T A A C C A A T T G
SalI  (3177)
1 site
G T C G A C C A G C T G
NcoI  (3632)
1 site
C C A T G G G G T A C C
BspEI  (3844)
1 site
T C C G G A A G G C C T
BsrGI  (4298)
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
1980 .. 4514  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
PpHIS4
1980 .. 4514  =  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
8106 .. 8966  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   8106 .. 8897  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8106 .. 8966  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   8898 .. 8966  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
8106 .. 8966  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
KanR
4928 .. 5743  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
4928 .. 5743  =  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
6122 .. 6878  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
6122 .. 6878  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
7347 .. 7935  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7347 .. 7935  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
α-factor secretion signal
949 .. 1215  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 1:  presequence  
   949 .. 1005  =  57 bp
   19 amino acids  =  2.0 kDa
Product: N-terminal secretion signal from S.
cerevisiae
alpha-factor
Cleavage by the Kex2 protease occurs after the
dibasic KR sequence. The EA dipeptides are then
removed by dipeptidyl aminopeptidase A.
α-factor secretion signal
949 .. 1215  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 2:  pro region  
   1006 .. 1203  =  198 bp
   66 amino acids  =  7.0 kDa
Product: N-terminal secretion signal from S.
cerevisiae
alpha-factor
Cleavage by the Kex2 protease occurs after the
dibasic KR sequence. The EA dipeptides are then
removed by dipeptidyl aminopeptidase A.
α-factor secretion signal
949 .. 1215  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 3:  
   1204 .. 1215  =  12 bp
   4 amino acids  =  418.4 Da
Product: N-terminal secretion signal from S.
cerevisiae
alpha-factor
Cleavage by the Kex2 protease occurs after the
dibasic KR sequence. The EA dipeptides are then
removed by dipeptidyl aminopeptidase A.
α-factor secretion signal
949 .. 1215  =  267 bp
89 amino acids  =  9.3 kDa
3 segments
Product: N-terminal secretion signal from S.
cerevisiae
alpha-factor
Cleavage by the Kex2 protease occurs after the
dibasic KR sequence. The EA dipeptides are then
removed by dipeptidyl aminopeptidase A.
AOX1 terminator
1321 .. 1567  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1321 .. 1567  =  247 bp
transcription terminator for AOX1
AmpR promoter
8967 .. 9071  =  105 bp
AmpR promoter
8967 .. 9071  =  105 bp
MCS
1216 .. 1241  =  26 bp
multiple cloning site
MCS
1216 .. 1241  =  26 bp
multiple cloning site
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