Resources
Plasmid Files

pGAPZα C

Pichia pastoris vector for constitutive expression of a secreted protein. For other reading frames, use pGAPZα A or pGAPZα B.

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pGAPZ(alpha) C Sequence and MappGAPZ(alpha) C.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 NsiI (3145) AlwNI (2835) BciVI (2622) BssS α I (2592) PciI (2419) MluI (2330) BsrGI (2323) AclI (2289) EcoRV (2156) StuI * (2122) KflI (2114) DraIII (2071) FseI (1994) SexAI * (1881) SgrAI (1832) SmaI (1808) TspMI - XmaI (1806) AatII (1764) ZraI (1762) BssHII - MauBI (1754) MscI (1721) NcoI (1716) AseI (1660) BglII (1) TsoI (61) BfuAI - BspMI (96) AvrII (190) BspQI - SapI (251) PasI (301) PshAI (351) BspHI (355) DraI (439) MfeI (469) BstBI (485) BstAPI (547) PsiI (688) BspDI - ClaI (760) EcoRI (765) BsaAI - PmlI (772) SfiI (782) Acc65I (798) KpnI (802) PspXI (803) SacII (812) NotI (815) XbaI (828) SalI (871) AccI (872) AgeI (952) BsmI (993) BsiWI (1084) BclI * (1100) PvuII (1118) BamHI (1233) BtgZI (1292) pGAPZ α C 3152 bp
NsiI  (3145)
1 site
A T G C A T T A C G T A
AlwNI  (2835)
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.
BciVI  (2622)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
BssSαI  (2592)
1 site
C A C G A G G T G C T C
PciI  (2419)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
MluI  (2330)
1 site
A C G C G T T G C G C A
BsrGI  (2323)
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.
AclI  (2289)
1 site
A A C G T T T T G C A A
EcoRV  (2156)
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  (2122)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
KflI  (2114)
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.
DraIII  (2071)
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  (1994)
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  (1881)
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  (1832)
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  (1808)
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  (1806)
1 site
C C C G G G G G G C C C
XmaI  (1806)
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.
AatII  (1764)
1 site
G A C G T C C T G C A G
ZraI  (1762)
1 site
G A C G T C C T G C A G
BssHII  (1754)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
MauBI  (1754)
1 site
C G C G C G C G G C G C G C G C
MscI  (1721)
1 site
T G G C C A A C C G G T
NcoI  (1716)
1 site
C C A T G G G G T A C C
AseI  (1660)
1 site
A T T A A T T A A T T A
BglII  (1)
1 site
A G A T C T T C T A G A
TsoI  (61)
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).
BfuAI  (96)
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  (96)
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.
AvrII  (190)
1 site
C C T A G G G G A T C C
BspQI  (251)
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  (251)
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.
PasI  (301)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PshAI  (351)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BspHI  (355)
1 site
T C A T G A A G T A C T
DraI  (439)
1 site
T T T A A A A A A T T T
MfeI  (469)
1 site
C A A T T G G T T A A C
BstBI  (485)
1 site
T T C G A A A A G C T T
BstAPI  (547)
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  (688)
1 site
T T A T A A A A T A T T
BspDI  (760)
1 site
A T C G A T T A G C T A
ClaI  (760)
1 site
A T C G A T T A G C T A
EcoRI  (765)
1 site
G A A T T C C T T A A G
BsaAI  (772)
1 site
Y A C G T R R T G C A Y
PmlI  (772)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SfiI  (782)
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  (798)
1 site
G G T A C C C C A T G G
KpnI  (802)
1 site
G G T A C C C C A T G G
PspXI  (803)
1 site
V C T C G A G B B G A G C T C V
SacII  (812)
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  (815)
1 site
G C G G C C G C C G C C G G C G
XbaI  (828)
1 site
T C T A G A A G A T C T
SalI  (871)
1 site
G T C G A C C A G C T G
AccI  (872)
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  (952)
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.
BsmI  (993)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BsiWI  (1084)
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  (1100)
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  (1118)
1 site
C A G C T G G T C G A C
BamHI  (1233)
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  (1292)
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.
ori
2480 .. 3068  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2480 .. 3068  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
GAP promoter
7 .. 483  =  477 bp
promoter for Pichia pastoris
glyceraldehyde-3-phosphate dehydrogenase
GAP promoter
7 .. 483  =  477 bp
promoter for Pichia pastoris
glyceraldehyde-3-phosphate dehydrogenase
TEF1 promoter
1234 .. 1645  =  412 bp
promoter for EF-1α
TEF1 promoter
1234 .. 1645  =  412 bp
promoter for EF-1α
BleoR
1718 .. 2092  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
BleoR
1718 .. 2092  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and
Zeocin™
α-factor secretion signal
493 .. 759  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 1:  presequence  
   493 .. 549  =  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
493 .. 759  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 2:  pro region  
   550 .. 747  =  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
493 .. 759  =  267 bp
89 amino acids  =  9.3 kDa
   Segment 3:  
   748 .. 759  =  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
493 .. 759  =  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
2158 .. 2405  =  248 bp
transcription terminator for CYC1
CYC1 terminator
2158 .. 2405  =  248 bp
transcription terminator for CYC1
AOX1 terminator
973 .. 1219  =  247 bp
transcription terminator for AOX1
AOX1 terminator
973 .. 1219  =  247 bp
transcription terminator for AOX1
EM7 promoter
1652 .. 1699  =  48 bp
synthetic bacterial promoter
EM7 promoter
1652 .. 1699  =  48 bp
synthetic bacterial promoter
Myc
832 .. 861  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
Myc
832 .. 861  =  30 bp
10 amino acids  =  1.2 kDa
Product: Myc (human c-Myc oncogene) epitope tag
6xHis
877 .. 894  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
877 .. 894  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
MCS
759 .. 833  =  75 bp
multiple cloning site
MCS
759 .. 833  =  75 bp
multiple cloning site
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