pFLD(alpha)

Pichia pastoris vector for methylamine-inducible expression of a secreted protein.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsYeast PlasmidspFLD(alpha)
BglII (4689) MluI (4595) BsrGI (4588) PpuMI (4514) EcoRV (4421) StuI (4387) DraIII (4336) FseI (4259) CsiI - SexAI * (4146) SgrAI (4097) SmaI (4073) TspMI - XmaI (4071) MauBI (4019) MscI (3986) NcoI - StyI (3981) HaeII (3265) AlwNI (3096) AhdI (2617) BmrI (2577) FspI (2394) SphI (5) BbvCI (49) NsiI (284) NdeI (405) BspDI - ClaI (492) BstAPI (676) PsiI (817) PaeR7I - XhoI (865) BsaAI - PmlI (899) SfiI (909) Acc65I (925) KpnI (929) SacII (933) PspOMI (949) ApaI (953) SalI (1009) AccI (1010) 6xHis AgeI (1091) BsiWI (1223) BclI * (1239) PvuII (1257) BamHI (1372) AscI (1379) SspI (1812) XmnI (2017) PvuI (2248) pFLD α 4694 bp
BglII  (4689)
1 site		 A G A T C T T C T A G A
MluI  (4595)
1 site		 A C G C G T T G C G C A
BsrGI  (4588)
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.
PpuMI  (4514)
1 site		 R G G W C C Y Y C C W G G R
Sticky ends from different PpuMI sites may not be compatible.
EcoRV  (4421)
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.
StuI  (4387)
1 site		 A G G C C T T C C G G A
DraIII  (4336)
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.
FseI  (4259)
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.
CsiI  (4146)
1 site		 A C C W G G T T G G W C C A
Sticky ends from different CsiI sites may not be compatible.
SexAI  (4146)
1 site		 A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
SgrAI  (4097)
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.
SmaI  (4073)
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  (4071)
1 site		 C C C G G G G G G C C C
XmaI  (4071)
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.
MauBI  (4019)
1 site		 C G C G C G C G G C G C G C G C
MscI  (3986)
1 site		 T G G C C A A C C G G T
NcoI  (3981)
1 site		 C C A T G G G G T A C C
StyI  (3981)
1 site		 C C W W G G G G W W C C
Sticky ends from different StyI sites may not be compatible.
HaeII  (3265)
1 site		 R G C G C Y Y C G C G R
AlwNI  (3096)
1 site		 C A G N N N C T G G T C N N N G A C
Sticky ends from different AlwNI sites may not be compatible.
AhdI  (2617)
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.
BmrI  (2577)
1 site		 A C T G G G ( N ) 4 N T G A C C C ( N ) 4
The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
FspI  (2394)
1 site		 T G C G C A A C G C G T
SphI  (5)
1 site		 G C A T G C C G T A C G
BbvCI  (49)
1 site		 C C T C A G C G G A G T C G
NsiI  (284)
1 site		 A T G C A T T A C G T A
NdeI  (405)
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.
BspDI  (492)
1 site		 A T C G A T T A G C T A
ClaI  (492)
1 site		 A T C G A T T A G C T A
BstAPI  (676)
1 site		 G C A N N N N N T G C C G T N N N N N A C G
Sticky ends from different BstAPI sites may not be compatible.
PsiI  (817)
1 site		 T T A T A A A A T A T T
PaeR7I  (865)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (865)
1 site		 C T C G A G G A G C T C
BsaAI  (899)
1 site		 Y A C G T R R T G C A Y
PmlI  (899)
1 site		 C A C G T G G T G C A C
SfiI  (909)
1 site		 G G C C N N N N N G G C C C C G G N N N N N C C G G
Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
Acc65I  (925)
1 site		 G G T A C C C C A T G G
KpnI  (929)
1 site		 G G T A C C C C A T G G
SacII  (933)
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.
PspOMI  (949)
1 site		 G G G C C C C C C G G G
ApaI  (953)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
SalI  (1009)
1 site		 G T C G A C C A G C T G
AccI  (1010)
1 site		 G T M K A C C A K M T G
Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
AgeI  (1091)
1 site		 A C C G G T T G G C C A
BsiWI  (1223)
1 site		 C G T A C G G C A T G C
BsiWI is typically used at 55°C, but is 50% active at 37°C.
BclI  (1239)
1 site		 T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
PvuII  (1257)
1 site		 C A G C T G G T C G A C
BamHI  (1372)
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.
AscI  (1379)
1 site		 G G C G C G C C C C G C G C G G
SspI  (1812)
1 site		 A A T A T T T T A T A A
XmnI  (2017)
1 site		 G A A N N N N T T C C T T N N N N A A G
PvuI  (2248)
1 site		 C G A T C G G C T A G C
AmpR
1830 .. 2690  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1830 .. 1898  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1830 .. 2690  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1899 .. 2690  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1830 .. 2690  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
FLD1 promoter
1 .. 597  =  597 bp
methanol- and methylamine-inducible promoter for Pichia pastoris formaldehyde dehydrogenase
FLD1 promoter
1 .. 597  =  597 bp
methanol- and methylamine-inducible promoter for Pichia pastoris formaldehyde dehydrogenase
ori
2861 .. 3449  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2861 .. 3449  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
α-factor secretion signal
622 .. 885  =  264 bp
88 amino acids  =  9.3 kDa
3 segments
   Segment 1:  presequence  
   622 .. 678  =  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
622 .. 885  =  264 bp
88 amino acids  =  9.3 kDa
3 segments
   Segment 2:  pro region  
   679 .. 876  =  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
622 .. 885  =  264 bp
88 amino acids  =  9.3 kDa
3 segments
   Segment 3:  
   877 .. 885  =  9 bp
   3 amino acids  =  347.3 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
622 .. 885  =  264 bp
88 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.
V5 tag
964 .. 1005  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
964 .. 1005  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
1015 .. 1032  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1015 .. 1032  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEF1 promoter
3504 .. 3910  =  407 bp
promoter for EF-1α
TEF1 promoter
3504 .. 3910  =  407 bp
promoter for EF-1α
BleoR
3983 .. 4357  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
3983 .. 4357  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
CYC1 terminator
4423 .. 4670  =  248 bp
transcription terminator for CYC1
CYC1 terminator
4423 .. 4670  =  248 bp
transcription terminator for CYC1
AOX1 terminator
1112 .. 1358  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1112 .. 1358  =  247 bp
transcription terminator for AOX1
AmpR promoter
1725 .. 1829  =  105 bp
AmpR promoter
1725 .. 1829  =  105 bp
MCS
889 .. 959  =  71 bp
multiple cloning site
MCS
889 .. 959  =  71 bp
multiple cloning site
EM7 promoter
3917 .. 3964  =  48 bp
synthetic bacterial promoter
EM7 promoter
3917 .. 3964  =  48 bp
synthetic bacterial promoter
ORF:  622 .. 1035  =  414 bp
ORF:  137 amino acids  =  14.6 kDa
ORF:  3983 .. 4357  =  375 bp
ORF:  124 amino acids  =  13.8 kDa
ORF:  1830 .. 2690  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2294 .. 2560  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3929 .. 4426  =  498 bp
ORF:  165 amino acids  =  17.6 kDa
ORF:  4662 .. 231  =  264 bp
ORF:  87 amino acids  =  10.0 kDa
ORF:  808 .. 1053  =  246 bp
ORF:  81 amino acids  =  9.2 kDa
ORF:  3124 .. 3513  =  390 bp
ORF:  129 amino acids  =  14.4 kDa
ORF:  3976 .. 4263  =  288 bp
ORF:  95 amino acids  =  10.1 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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