pPIC3

Pichia pastoris HIS4 vector for methanol-inducible intracellular expression of a protein.

Sequence Author: Thermo Fisher (Invitrogen)

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AatII (7589) ZraI (7587) ScaI (7147) PvuI (7037) PstI (6912) AflIII - PciI (5774) BspQI - SapI (5658) NdeI (5597) BstZ17I (5547) PflFI - Tth111I (5521) SphI (4524) SgrAI (4368) NaeI (4361) NgoMIV (4359) BfuAI - BspMI (4224) AleI (76) Eco53kI (206) SacI (208) PmeI (413) BlpI (588) NsiI (677) XcmI (706) BamHI (938) SnaBI (959) EcoRI (963) AvrII (969) EagI - NotI (976) AgeI (1041) BtgZI (1495) FspAI (1567) XbaI (1773) Bsu36I (1829) HpaI (1879) SalI (2918) StuI (3003) DraIII (3365) BspEI (3585) BsrGI (4039) pPIC3 7764 bp
AatII  (7589)
1 site
G A C G T C C T G C A G
ZraI  (7587)
1 site
G A C G T C C T G C A G
ScaI  (7147)
1 site
A G T A C T T C A T G A
PvuI  (7037)
1 site
C G A T C G G C T A G C
PstI  (6912)
1 site
C T G C A G G A C G T C
AflIII  (5774)
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  (5774)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (5658)
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  (5658)
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  (5597)
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  (5547)
1 site
G T A T A C C A T A T G
PflFI  (5521)
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  (5521)
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  (4524)
1 site
G C A T G C C G T A C G
SgrAI  (4368)
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  (4361)
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  (4359)
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  (4224)
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  (4224)
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.
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.
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.
SnaBI  (959)
1 site
T A C G T A A T G C A T
EcoRI  (963)
1 site
G A A T T C C T T A A G
AvrII  (969)
1 site
C C T A G G G G A T C C
EagI  (976)
1 site
C G G C C G G C C G G C
NotI  (976)
1 site
G C G G C C G C C G C C G G C G
AgeI  (1041)
1 site
A C C G G T T G G C C A
BtgZI  (1495)
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  (1567)
1 site
R T G C G C A Y Y A C G C G T R
XbaI  (1773)
1 site
T C T A G A A G A T C T
Bsu36I  (1829)
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  (1879)
1 site
G T T A A C C A A T T G
SalI  (2918)
1 site
G T C G A C C A G C T G
StuI  (3003)
1 site
A G G C C T T C C G G A
DraIII  (3365)
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.
BspEI  (3585)
1 site
T C C G G A A G G C C T
BsrGI  (4039)
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
1721 .. 4255  =  2535 bp
844 amino acids  =  92.2 kDa
Product: multifunctional enzyme, required for histidine biosynthesis
auxotrophic marker for Pichia pastoris
PpHIS4
1721 .. 4255  =  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
6594 .. 7454  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   6594 .. 7385  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6594 .. 7454  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   7386 .. 7454  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6594 .. 7454  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AOX1 3' fragment
4610 .. 5366  =  757 bp
region downstream of Pichia pastoris AOX1 gene
AOX1 3' fragment
4610 .. 5366  =  757 bp
region downstream of Pichia pastoris AOX1 gene
ori
5835 .. 6423  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5835 .. 6423  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AOX1 terminator
1062 .. 1308  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1062 .. 1308  =  247 bp
transcription terminator for AOX1
AmpR promoter
7455 .. 7559  =  105 bp
AmpR promoter
7455 .. 7559  =  105 bp
MCS
938 .. 982  =  45 bp
multiple cloning site
MCS
938 .. 982  =  45 bp
multiple cloning site
ATG
949 .. 951  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon that should be utilized or removed when inserting a gene
ATG
949 .. 951  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon that should be utilized or removed when inserting a gene
ORF:  6724 .. 6990  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1391 .. 1696  =  306 bp
ORF:  101 amino acids  =  10.8 kDa
ORF:  486 .. 725  =  240 bp
ORF:  79 amino acids  =  9.2 kDa
ORF:  4449 .. 4886  =  438 bp
ORF:  145 amino acids  =  15.4 kDa
ORF:  4209 .. 4523  =  315 bp
ORF:  104 amino acids  =  11.0 kDa
ORF:  6594 .. 7454  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  1721 .. 4255  =  2535 bp
ORF:  844 amino acids  =  92.2 kDa
ORF:  4397 .. 5119  =  723 bp
ORF:  240 amino acids  =  26.3 kDa
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