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Plasmid Files

pAO815

Pichia pastoris vector for creating multimers to drive high levels of methanol-inducible protein expression.

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pAO815 Sequence and MappAO815.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BspDI - ClaI (7635) AatII (7534) ZraI (7532) ScaI (7092) PvuI (6982) PstI (6857) AflIII - PciI (5719) BspQI - SapI (5603) NdeI (5542) BstZ17I (5492) BsaAI (5473) PflFI - Tth111I (5466) BglII (5306) SphI (4468) SgrAI (4312) NaeI (4305) NgoMIV (4303) BfuAI - BspMI (4168) BsrGI (3983) BglII (1) AleI (76) Eco53kI (206) SacI (208) PmeI (413) BlpI (588) NsiI (677) XcmI (706) HindIII (872) EcoRI (943) AgeI (997) BamHI (1278) BtgZI (1439) FspAI (1511) XbaI (1717) Bsu36I (1773) HpaI (1823) SalI (2862) StuI (2947) DraIII (3309) NcoI (3317) BspEI (3529) pAO815 7709 bp
BspDI  (7635)
1 site
A T C G A T T A G C T A
ClaI  (7635)
1 site
A T C G A T T A G C T A
AatII  (7534)
1 site
G A C G T C C T G C A G
ZraI  (7532)
1 site
G A C G T C C T G C A G
ScaI  (7092)
1 site
A G T A C T T C A T G A
PvuI  (6982)
1 site
C G A T C G G C T A G C
PstI  (6857)
1 site
C T G C A G G A C G T C
AflIII  (5719)
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  (5719)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5603)
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  (5603)
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  (5542)
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  (5492)
1 site
G T A T A C C A T A T G
BsaAI  (5473)
1 site
Y A C G T R R T G C A Y
PflFI  (5466)
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  (5466)
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  (5306)
2 sites
A G A T C T T C T A G A
SphI  (4468)
1 site
G C A T G C C G T A C G
SgrAI  (4312)
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.
NaeI  (4305)
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.
NgoMIV  (4303)
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.
BfuAI  (4168)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (4168)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BsrGI  (3983)
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.
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.
NsiI  (677)
1 site
A T G C A T T A C G T A
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.
HindIII  (872)
1 site
A A G C T T T T C G A A
EcoRI  (943)
1 site
G A A T T C C T T A A G
AgeI  (997)
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.
BamHI  (1278)
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.
BtgZI  (1439)
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  (1511)
1 site
R T G C G C A Y Y A C G C G T R
XbaI  (1717)
1 site
T C T A G A A G A T C T
Bsu36I  (1773)
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  (1823)
1 site
G T T A A C C A A T T G
SalI  (2862)
1 site
G T C G A C C A G C T G
StuI  (2947)
1 site
A G G C C T T C C G G A
DraIII  (3309)
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.
NcoI  (3317)
1 site
C C A T G G G G T A C C
BspEI  (3529)
1 site
T C C G G A A G G C C T
PpHIS4
1665 .. 4199  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
PpHIS4
1665 .. 4199  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for
histidine biosynthesis
auxotrophic marker for Pichia pastoris
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AmpR
6539 .. 7399  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6539 .. 7330  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6539 .. 7399  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   7331 .. 7399  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6539 .. 7399  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AOX1 3' fragment
4554 .. 5310  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
4554 .. 5310  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
5780 .. 6368  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5780 .. 6368  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AOX1 terminator
1018 .. 1264  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1018 .. 1264  =  247 bp
transcription terminator for AOX1
AmpR promoter
7400 .. 7504  =  105 bp
AmpR promoter
7400 .. 7504  =  105 bp
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