pPINK(alpha)-HC

Pichia pastoris integrating vector for high-copy expression of a secreted protein in the PichiaPink™ system.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsYeast PlasmidspPINK(alpha)-HC
PluTI (7736) SfoI (7734) NarI (7733) KasI (7732) NdeI (7681) AatII (7432) ZraI (7430) AhdI (6510) AflII (5007) PshAI (4968) BmgBI (4689) SpeI (4554) EcoNI (4422) BspEI (4101) BglII (1) Eco53kI (206) SacI (208) PmeI (413) XcmI (706) AvaI - BsoBI - PaeR7I - XhoI (1184) StuI (1195) Bsu36I (1197) Acc65I (1201) KpnI (1205) NgoMIV (1208) MCS NaeI (1210) FseI (1212) SwaI (1218) EcoRV (1246) BsrGI (1415) MluI (1422) BamHI (1503) NotI - SacII (1510) NheI (1593) BmtI (1597) BbsI (2262) DraIII (2547) BsaAI (3263) BsgI (3370) AscI - BssHII (3423) NruI (3509) BsaBI (3614) pPINK α- HC 7898 bp
PluTI  (7736)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (7734)
1 site		 G G C G C C C C G C G G
NarI  (7733)
1 site		 G G C G C C C C G C G G
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (7732)
1 site		 G G C G C C C C G C G G
NdeI  (7681)
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.
AatII  (7432)
1 site		 G A C G T C C T G C A G
ZraI  (7430)
1 site		 G A C G T C C T G C A G
AhdI  (6510)
1 site		 G A C N N N N N G T C C T G N N N N N C A G
The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AflII  (5007)
1 site		 C T T A A G G A A T T C
PshAI  (4968)
1 site		 G A C N N N N G T C C T G N N N N C A G
PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
BmgBI  (4689)
1 site		 C A C G T C G T G C A G
This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
SpeI  (4554)
1 site		 A C T A G T T G A T C A
EcoNI  (4422)
1 site		 C C T N N N N N A G G G G A N N N N N T C C
The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BspEI  (4101)
1 site		 T C C G G A A G G C C T
BglII  (1)
1 site		 A G A T C T T C T A G A
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
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.
AvaI  (1184)
1 site		 C Y C G R G G R G C Y C
Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1184)
1 site		 C Y C G R G G R G C Y C
Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (1184)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1184)
1 site		 C T C G A G G A G C T C
StuI  (1195)
1 site		 A G G C C T T C C G G A
Bsu36I  (1197)
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.
Acc65I  (1201)
1 site		 G G T A C C C C A T G G
KpnI  (1205)
1 site		 G G T A C C C C A T G G
NgoMIV  (1208)
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.
NaeI  (1210)
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.
FseI  (1212)
1 site		 G G C C G G C C C C G G C C G G
FseI gradually loses activity when stored at -20°C.
SwaI  (1218)
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.
EcoRV  (1246)
1 site		 G A T A T C C T A T A G
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
BsrGI  (1415)
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.
MluI  (1422)
1 site		 A C G C G T T G C G C A
BamHI  (1503)
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.
NotI  (1510)
1 site		 G C G G C C G C C G C C G G C G
SacII  (1510)
1 site		 C C G C G G G G C G C C
Efficient cleavage requires at least two copies of the SacII recognition sequence.
NheI  (1593)
1 site		 G C T A G C C G A T C G
BmtI  (1597)
1 site		 G C T A G C C G A T C G
BbsI  (2262)
1 site		 G A A G A C N N C T T C T G N N ( N ) 4
Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
DraIII  (2547)
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.
BsaAI  (3263)
1 site		 Y A C G T R R T G C A Y
BsgI  (3370)
1 site		 G T G C A G ( N ) 14 N N C A C G T C ( N ) 14
Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AscI  (3423)
1 site		 G G C G C G C C C C G C G C G G
BssHII  (3423)
1 site		 G C G C G C C G C G C G
BssHII is typically used at 50°C, but is 75% active at 37°C.
NruI  (3509)
1 site		 T C G C G A A G C G C T
BsaBI  (3614)
1 site		 G A T N N N N A T C C T A N N N N T A G
PpADE2
1530 .. 3221  =  1692 bp
563 amino acids  =  61.4 kDa
Product: phosphoribosylaminoimidazole carboxylase from Pichia pastoris
essential for the biosynthesis of purine nucleotides, including adenine
PpADE2
1530 .. 3221  =  1692 bp
563 amino acids  =  61.4 kDa
Product: phosphoribosylaminoimidazole carboxylase from Pichia pastoris
essential for the biosynthesis of purine nucleotides, including adenine
PpTRP2
3598 .. 5214  =  1617 bp
538 amino acids  =  60.8 kDa
Product: anthranilate synthase from Pichia pastoris
catalyzes the initial step of tryptophan biosynthesis
PpTRP2
3598 .. 5214  =  1617 bp
538 amino acids  =  60.8 kDa
Product: anthranilate synthase from Pichia pastoris
catalyzes the initial step of tryptophan biosynthesis
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AmpR
6437 .. 7297  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   6437 .. 7228  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6437 .. 7297  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   7229 .. 7297  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6437 .. 7297  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
5678 .. 6266  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5678 .. 6266  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
α-factor secretion signal
941 .. 1195  =  255 bp
85 amino acids  =  8.9 kDa
2 segments
   Segment 1:  presequence  
   941 .. 997  =  57 bp
   19 amino acids  =  2.0 kDa
Product: N-terminal secretion signal from S. cerevisiae alpha-factor (modified)
Cleavage by the Kex2 protease occurs after the dibasic KR sequence.
α-factor secretion signal
941 .. 1195  =  255 bp
85 amino acids  =  8.9 kDa
2 segments
   Segment 2:  pro region  
   998 .. 1195  =  198 bp
   66 amino acids  =  7.0 kDa
Product: N-terminal secretion signal from S. cerevisiae alpha-factor (modified)
Cleavage by the Kex2 protease occurs after the dibasic KR sequence.
α-factor secretion signal
941 .. 1195  =  255 bp
85 amino acids  =  8.9 kDa
2 segments
Product: N-terminal secretion signal from S. cerevisiae alpha-factor (modified)
Cleavage by the Kex2 protease occurs after the dibasic KR sequence.
CYC1 terminator
1248 .. 1497  =  250 bp
transcription terminator for CYC1
CYC1 terminator
1248 .. 1497  =  250 bp
transcription terminator for CYC1
PpTRP2 promoter
3448 .. 3597  =  150 bp
promoter for Pichia pastoris TRP2
PpTRP2 promoter
3448 .. 3597  =  150 bp
promoter for Pichia pastoris TRP2
AmpR promoter
7298 .. 7402  =  105 bp
AmpR promoter
7298 .. 7402  =  105 bp
MCS
1196 .. 1222  =  27 bp
multiple cloning site
MCS
1196 .. 1222  =  27 bp
multiple cloning site
PpADE2 HC promoter
1517 .. 1529  =  13 bp
weak 13-bp promoter fragment that requires high-copy integration for efficient expression
PpADE2 HC promoter
1517 .. 1529  =  13 bp
weak 13-bp promoter fragment that requires high-copy integration for efficient expression
ORF:  3598 .. 5214  =  1617 bp
ORF:  538 amino acids  =  60.8 kDa
ORF:  941 .. 1255  =  315 bp
ORF:  104 amino acids  =  10.9 kDa
ORF:  486 .. 725  =  240 bp
ORF:  79 amino acids  =  9.2 kDa
ORF:  1530 .. 3221  =  1692 bp
ORF:  563 amino acids  =  61.4 kDa
ORF:  6567 .. 6833  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  7683 .. 33  =  249 bp
ORF:  82 amino acids  =  9.8 kDa
ORF:  7643 .. 47  =  303 bp
ORF:  100 amino acids  =  11.8 kDa
ORF:  3789 .. 4034  =  246 bp
ORF:  81 amino acids  =  8.5 kDa
ORF:  6437 .. 7297  =  861 bp
ORF:  286 amino acids  =  31.6 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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