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Plasmid Files

pPIC6α C

Pichia pastoris vector for methanol-inducible expression of a secreted protein. For other reading frames, use pPIC6α A or pPIC6α B.

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pPIC6(alpha) C Sequence and MappPIC6(alpha) C.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BglII (1) AlwNI (3338) PciI (2922) MluI (2833) BsrGI (2826) BsrBI (2711) EcoRV (2659) StuI * (2625) KflI (2617) NmeAIII (2603) BmgBI (2598) DraIII (2574) TsoI (2536) PvuI (2404) NruI * (2400) BsgI - Bts α I (2356) MscI (2172) NcoI - StyI (2167) AleI - MslI (76) PflMI (190) Eco53kI (206) SacI (208) DraI - PmeI (413) NsiI (677) BstXI - XcmI (706) MfeI (864) HindIII (872) BstBI (933) BstAPI (995) PsiI (1136) BsrDI (1155) BspDI - ClaI (1208) EcoRI (1213) BsaAI - PmlI (1220) NgoMIV (1229) SfiI (1230) NaeI (1231) BsmBI (1241) Acc65I (1246) KpnI (1250) PspXI (1251) SacII (1260) NotI (1263) XbaI (1276) SalI (1319) AccI (1320) AgeI (1401) BsiWI (1533) BamHI (1682) BtgZI (1741) pPIC6 α C 3651 bp
BglII  (1)
1 site
A G A T C T T C T A G A
AlwNI  (3338)
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  (2922)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
MluI  (2833)
1 site
A C G C G T T G C G C A
BsrGI  (2826)
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  (2711)
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  (2659)
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  (2625)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
KflI  (2617)
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  (2603)
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  (2598)
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  (2574)
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  (2536)
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  (2404)
1 site
C G A T C G G C T A G C
NruI  (2400)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BsgI  (2356)
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  (2356)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
MscI  (2172)
1 site
T G G C C A A C C G G T
NcoI  (2167)
1 site
C C A T G G G G T A C C
StyI  (2167)
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
BstAPI  (995)
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  (1136)
1 site
T T A T A A A A T A T T
BsrDI  (1155)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
BspDI  (1208)
1 site
A T C G A T T A G C T A
ClaI  (1208)
1 site
A T C G A T T A G C T A
EcoRI  (1213)
1 site
G A A T T C C T T A A G
BsaAI  (1220)
1 site
Y A C G T R R T G C A Y
PmlI  (1220)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NgoMIV  (1229)
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  (1230)
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  (1231)
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  (1241)
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  (1246)
1 site
G G T A C C C C A T G G
KpnI  (1250)
1 site
G G T A C C C C A T G G
PspXI  (1251)
1 site
V C T C G A G B B G A G C T C V
SacII  (1260)
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  (1263)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1276)
1 site
T C T A G A A G A T C T
SalI  (1319)
1 site
G T C G A C C A G C T G
AccI  (1320)
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  (1401)
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  (1533)
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  (1682)
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  (1741)
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
2983 .. 3571  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2983 .. 3571  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
TEF1 promoter
1683 .. 2096  =  414 bp
promoter for EF-1α
TEF1 promoter
1683 .. 2096  =  414 bp
promoter for EF-1α
BSD
2169 .. 2567  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
2169 .. 2567  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
α-factor secretion signal
941 .. 1207  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 1:  presequence  
   941 .. 997  =  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
941 .. 1207  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 2:  pro region  
   998 .. 1195  =  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
941 .. 1207  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 3:  
   1196 .. 1207  =  12 bp
   4 amino acids  =  418.4 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
941 .. 1207  =  267 bp
89 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
2661 .. 2908  =  248 bp
transcription terminator for CYC1
CYC1 terminator
2661 .. 2908  =  248 bp
transcription terminator for CYC1
AOX1 terminator
1422 .. 1668  =  247 bp
transcription terminator for AOX1
AOX1 terminator
1422 .. 1668  =  247 bp
transcription terminator for AOX1
EM7 promoter
2103 .. 2150  =  48 bp
synthetic bacterial promoter
EM7 promoter
2103 .. 2150  =  48 bp
synthetic bacterial promoter
Myc
1280 .. 1309  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
1280 .. 1309  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
6xHis
1325 .. 1342  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1325 .. 1342  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
MCS
1207 .. 1281  =  75 bp
multiple cloning site
MCS
1207 .. 1281  =  75 bp
multiple cloning site
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