pPIC9K

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

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsYeast PlasmidspPIC9K
AatII (9101) ZraI (9099) ScaI (8659) AflIII - PciI (7286) BspQI - SapI (7170) NdeI (7109) BstZ17I (7059) PflFI - Tth111I (7033) SphI (6036) SgrAI (5880) PspXI (5709) NruI (5654) SmaI (5437) TspMI - XmaI (5435) AsiSI (5313) BbvCI (4644) 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.
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
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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
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
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
2 segments
   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
2 segments
   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
3 segments
   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
3 segments
   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
3 segments
   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
ORF:  949 .. 1248  =  300 bp
ORF:  99 amino acids  =  10.5 kDa
ORF:  8236 .. 8502  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  486 .. 725  =  240 bp
ORF:  79 amino acids  =  9.2 kDa
ORF:  1650 .. 1955  =  306 bp
ORF:  101 amino acids  =  10.8 kDa
ORF:  5961 .. 6398  =  438 bp
ORF:  145 amino acids  =  15.4 kDa
ORF:  1980 .. 4514  =  2535 bp
ORF:  844 amino acids  =  92.2 kDa
ORF:  5805 .. 6035  =  231 bp
ORF:  76 amino acids  =  7.7 kDa
ORF:  8106 .. 8966  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  4928 .. 5743  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  5909 .. 6631  =  723 bp
ORF:  240 amino acids  =  26.3 kDa
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Individual Sequences & Maps

bleMX6
pFA6a-kanMX6
pPIC6(alpha) B
pYES2.1-E
hphMX6
pFA6a-kanMX6-PGAL1
pPIC6(alpha) C
pYES2 CT
kanMX
pFA6a-kanMX6-PGAL1-3HA
pPIC9
pYES2 NT A
kanMX2
pFA6a-kanMX6-PGAL1-GFP
pPIC9K
pYES2 NT B
kanMX4
pFA6a-kanMX6-PGAL1-GST
pPICZ A
pYES2 NT C
kanMX6
pFA6a-natMX6
pPICZ B
pYES3 CT
natMX6
pFA6a-TRP1
pPICZ C
pYES6 CT
pAbAi
pFA6a-TRP1-PGAL1
pPICZ(alpha) A
pYM25
pAD-GAL4
pFA6a-TRP1-PGAL1-3HA
pPICZ(alpha) B
pYM26
pAG25
pFA6a-TRP1-PGAL1-GFP
pPICZ(alpha) C
pYM44
pAG29
pFA6a-TRP1-PGAL1-GST
pPink-HC
YCp50
pAG32
pFLD
pPink-LC
YCp50-poly
pAO815
pFLD(alpha)
pPINK(alpha)-HC
YCplac111
patMX4
pGAD-C1
pRS303
YCplac22
pAUR101
pGAD-C2
pRS304
YCplac33
pAUR112
pGAD-C3
pRS305
YEp13
pAUR123
pGADT7 AD
pRS306
YEp24
pAUR224
pGADT7-Rec
pRS313
YEp351
pBridge
pGADT7-Rec (linearized)
pRS314
YEp352
pDH25
pGAPZ A
pRS315
YEp353
pEG202
pGAPZ B
pRS316
YEp354
pESC-HIS
pGAPZ C
pRS403
YEp355
pESC-LEU
pGAPZ(alpha) A
pRS404
YEp356
pESC-TRP
pGAPZ(alpha) B
pRS405
YEp356R
pESC-URA
pGAPZ(alpha) C
pRS406
YEp357
pFA6-kanMX4
pGBD-C1
pRS413
YEp357R
pFA6a
pGBD-C2
pRS414
YEp358
pFA6a-12Pk-kanMX6
pGBD-C3
pRS415
YEp358R
pFA6a-13Myc-His3MX6
pGBDU-C1
pRS416
YEp363
pFA6a-13Myc-kanMX6
pGBDU-C2
pRS423
YEp364
pFA6a-13Myc-TRP1
pGBDU-C3
pRS424
YEp365
pFA6a-3HA-His3MX6
pGBKT7
pRS425
YEp366
pFA6a-3HA-kanMX6
pGilda
pRS426
YEp366R
pFA6a-3HA-TRP1
pHIL-D2
pSpot5
YEp367
pFA6a-5FLAG-kanMX6
pHIL-S1
pSpot6
YEp367R
pFA6a-bleMX6
pJG4-5
pSpot7
YEp368
pFA6a-GFP(S65T)-His3MX6
pKLAC1
pSpot8
YEp368R
pFA6a-GFP(S65T)-kanMX6
pKLAC2
pTEF1 Zeo
YEplac112
pFA6a-GFP(S65T)-TRP1
pOAD
pUCATPH
YEplac181
pFA6a-GST-His3MX6
pOBD2
pY-secSUMOstar
YEplac195
pFA6a-GST-kanMX6
pP-secSUMO3
pY-SUMOstar
YIp351
pFA6a-GST-TRP1
pPIC3
pYC2-E
YIp352
pFA6a-His3MX6
pPIC3.5
pYC2 CT
YIp5
pFA6a-His3MX6-PGAL1
pPIC3.5K
pYC2 NT A
YIplac128
pFA6a-His3MX6-PGAL1-3HA
pPIC6 A
pYC2 NT B
YIplac204
pFA6a-His3MX6-PGAL1-GFP
pPIC6 B
pYC2 NT C
YIplac211
pFA6a-His3MX6-PGAL1-GST
pPIC6 C
pYC6 CT
YRp7
pFA6a-hphMX6
pPIC6(alpha) A
pYES2
alpha-factor secretion signal

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