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Plasmid Files

pFLDα

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

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pFLD(alpha) Sequence and MappFLD(alpha).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BglII (4689) MluI (4595) BsrGI (4588) PpuMI (4514) EcoRV (4421) StuI (4387) DraIII (4336) FseI (4259) 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) AanI - AanI - PsiI (817) PaeR7I - XhoI (865) MfeI (889) BsaAI - PmlI (899) SfiI (909) Acc65I (925) KpnI (929) SacII (933) PspOMI (949) ApaI (953) MfeI (954) 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.
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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
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.
AanI  (817)
1 site
T T A T A A A A T A T T
AanI  (817)
1 site
T T A T A A A A T A T T
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
MfeI  (889)
2 sites
C A A T T G G T T A A 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

PmlI gradually loses activity when stored at -20°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.
MfeI  (954)
2 sites
C A A T T G G T T A A 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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
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
   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
   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
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™
α-factor secretion signal
622 .. 885  =  264 bp
88 amino acids  =  9.3 kDa
   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
   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
   Segment 3:  
   877 .. 885  =  9 bp
   3 amino acids  =  347.4 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
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.
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
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
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