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Plasmid Files

pPIC6 A

Pichia pastoris vector for methanol-inducible intracellular expression of a protein. For other reading frames, use pPIC6 B or pPIC6 C.

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pPIC6 A.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Invitrogen)
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BglII (1) AlwNI (3069) PciI (2653) MluI (2564) BsrGI (2557) BsrBI (2442) EcoRV (2390) StuI * (2356) KflI (2348) NmeAIII (2334) BmgBI (2329) DraIII (2305) TsoI (2267) PvuI (2135) NruI * (2131) BsgI - Bts α I (2087) MscI (1903) NcoI - StyI (1898) AleI - MslI (76) PflMI (190) Eco53kI (206) SacI (208) DraI - PmeI (413) NsiI (677) BstXI - XcmI (706) MfeI (864) HindIII (872) BstBI (933) EcoRI (943) BsaAI - PmlI (950) NgoMIV (959) SfiI (960) NaeI (961) BsmBI (971) Acc65I (976) KpnI (980) AvaI - BsoBI - PaeR7I - PspXI - XhoI (981) BmeT110I (982) SacII (990) NotI (993) PspOMI (1006) ApaI (1010) SalI (1051) AccI (1052) AgeI (1133) BsiWI (1265) BamHI (1414) BtgZI (1473) pPIC6 A 3382 bp
BglII  (1)
1 site
A G A T C T T C T A G A
AlwNI  (3069)
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.
PciI  (2653)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
MluI  (2564)
1 site
A C G C G T T G C G C A
BsrGI  (2557)
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.
BsrBI  (2442)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures up to 50°C.
EcoRV  (2390)
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  (2356)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
KflI  (2348)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
NmeAIII  (2334)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BmgBI  (2329)
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.
DraIII  (2305)
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.
TsoI  (2267)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (2135)
1 site
C G A T C G G C T A G C
NruI  (2131)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BsgI  (2087)
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).
BtsαI  (2087)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsαI sites may not be compatible.
MscI  (1903)
1 site
T G G C C A A C C G G T
NcoI  (1898)
1 site
C C A T G G G G T A C C
StyI  (1898)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI 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
MslI  (76)
1 site
C A Y N N N N R T G G T R N N N N Y A C
PflMI  (190)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
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
DraI  (413)
1 site
T T T A A A A A A T T T
PmeI  (413)
1 site
G T T T A A A C C A A A T T T G
NsiI  (677)
1 site
A T G C A T T A C G T A
BstXI  (706)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
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.
MfeI  (864)
1 site
C A A T T G G T T A A C
HindIII  (872)
1 site
A A G C T T T T C G A A
BstBI  (933)
1 site
T T C G A A A A G C T T
EcoRI  (943)
1 site
G A A T T C C T T A A G
BsaAI  (950)
1 site
Y A C G T R R T G C A Y
PmlI  (950)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NgoMIV  (959)
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.
SfiI  (960)
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.
NaeI  (961)
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.
BsmBI  (971)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
Acc65I  (976)
1 site
G G T A C C C C A T G G
KpnI  (980)
1 site
G G T A C C C C A T G G
AvaI  (981)
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  (981)
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  (981)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (981)
1 site
V C T C G A G B B G A G C T C V
XhoI  (981)
1 site
C T C G A G G A G C T C
BmeT110I  (982)
1 site
C Y C G R G G R G C Y C
SacII  (990)
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.
NotI  (993)
1 site
G C G G C C G C C G C C G G C G
PspOMI  (1006)
1 site
G G G C C C C C C G G G
ApaI  (1010)
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  (1051)
1 site
G T C G A C C A G C T G
AccI  (1052)
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  (1133)
1 site
A C C G G T T G G C C A
BsiWI  (1265)
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.
BamHI  (1414)
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  (1473)
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.
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
ori
2714 .. 3302  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2714 .. 3302  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
TEF1 promoter
1415 .. 1826  =  412 bp
promoter for EF-1α
TEF1 promoter
1415 .. 1826  =  412 bp
promoter for EF-1α
BSD
1900 .. 2298  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
1900 .. 2298  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CYC1 terminator
2392 .. 2639  =  248 bp
transcription terminator for CYC1
CYC1 terminator
2392 .. 2639  =  248 bp
transcription terminator for CYC1
AOX1 terminator
1154 .. 1400  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1154 .. 1400  =  247 bp
transcription terminator for AOX1
EM7 promoter
1834 .. 1881  =  48 bp
synthetic bacterial promoter
EM7 promoter
1834 .. 1881  =  48 bp
synthetic bacterial promoter
Myc
1012 .. 1041  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1012 .. 1041  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
6xHis
1057 .. 1074  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1057 .. 1074  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
MCS
932 .. 1011  =  80 bp
multiple cloning site
MCS
932 .. 1011  =  80 bp
multiple cloning site
ORF:  1900 .. 2298  =  399 bp
ORF:  132 amino acids  =  13.7 kDa
ORF:  486 .. 725  =  240 bp
ORF:  79 amino acids  =  9.2 kDa
ORF:  1846 .. 2202  =  357 bp
ORF:  118 amino acids  =  12.8 kDa
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