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pPink-LC

Pichia pastoris integrating vector for low-copy expression in the PichiaPink™ system.

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pPink-LC Sequence and MappPink-LC.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 PluTI (7570) SfoI (7568) NarI (7567) KasI (7566) NdeI (7515) AatII (7266) ZraI (7264) AhdI (6344) AflII (4841) PshAI (4802) BmgBI (4523) SpeI (4388) EcoNI (4256) PsiI (4063) BglII (1) Eco53kI (206) SacI (208) PmeI (413) XcmI (706) EcoRI (941) RsrII (948) SphI (958) StuI * (962) Acc65I (966) KpnI (970) NgoMIV (973) NaeI (975) FseI (977) SwaI (983) EcoRV (1011) BsrGI (1180) MluI (1187) BamHI (1268) NotI - SacII (1275) BstEII (1313) NheI (1427) BmtI (1431) BbsI (2096) DraIII (2381) BsaAI (3097) BsgI (3204) AscI - BssHII (3257) NruI (3343) BsaBI (3448) pPink-LC 7732 bp
PluTI  (7570)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (7568)
1 site
G G C G C C C C G C G G
NarI  (7567)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (7566)
1 site
G G C G C C C C G C G G
NdeI  (7515)
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.
AatII  (7266)
1 site
G A C G T C C T G C A G
ZraI  (7264)
1 site
G A C G T C C T G C A G
AhdI  (6344)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AflII  (4841)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
PshAI  (4802)
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.
BmgBI  (4523)
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.
SpeI  (4388)
1 site
A C T A G T T G A T C A
EcoNI  (4256)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PsiI  (4063)
1 site
T T A T A A A A T A T T
BglII  (1)
1 site
A G A T C T T C T A G A
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
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.
EcoRI  (941)
1 site
G A A T T C C T T A A G
RsrII  (948)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
SphI  (958)
1 site
G C A T G C C G T A C G
StuI  (962)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
Acc65I  (966)
1 site
G G T A C C C C A T G G
KpnI  (970)
1 site
G G T A C C C C A T G G
NgoMIV  (973)
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.
NaeI  (975)
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.
FseI  (977)
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.
SwaI  (983)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
EcoRV  (1011)
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.
BsrGI  (1180)
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.
MluI  (1187)
1 site
A C G C G T T G C G C A
BamHI  (1268)
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.
NotI  (1275)
1 site
G C G G C C G C C G C C G G C G
SacII  (1275)
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.
BstEII  (1313)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
NheI  (1427)
1 site
G C T A G C C G A T C G
BmtI  (1431)
1 site
G C T A G C C G A T C G
BbsI  (2096)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
DraIII  (2381)
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.
BsaAI  (3097)
1 site
Y A C G T R R T G C A Y
BsgI  (3204)
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).
AscI  (3257)
1 site
G G C G C G C C C C G C G C G G
BssHII  (3257)
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.
NruI  (3343)
1 site
T C G C G A A G C G C T
BsaBI  (3448)
1 site
G A T N N N N A T C C T A N N N N T A G
PpADE2
1364 .. 3055  =  1692 bp
563 amino acids  =  61.4 kDa
Product: phosphoribosylaminoimidazole carboxylase
from Pichia pastoris
essential for the biosynthesis of purine nucleotides,
including adenine
PpADE2
1364 .. 3055  =  1692 bp
563 amino acids  =  61.4 kDa
Product: phosphoribosylaminoimidazole carboxylase
from Pichia pastoris
essential for the biosynthesis of purine nucleotides,
including adenine
PpTRP2
3432 .. 5048  =  1617 bp
538 amino acids  =  60.8 kDa
Product: anthranilate synthase from Pichia pastoris
catalyzes the initial step of tryptophan biosynthesis
PpTRP2
3432 .. 5048  =  1617 bp
538 amino acids  =  60.8 kDa
Product: anthranilate synthase from Pichia pastoris
catalyzes the initial step of tryptophan biosynthesis
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AOX1 promoter
2 .. 940  =  939 bp
inducible promoter, regulated by methanol
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6271 .. 7062  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7063 .. 7131  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
5512 .. 6100  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5512 .. 6100  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CYC1 terminator
1013 .. 1262  =  250 bp
transcription terminator for CYC1
CYC1 terminator
1013 .. 1262  =  250 bp
transcription terminator for CYC1
PpTRP2 promoter
3282 .. 3431  =  150 bp
promoter for Pichia pastoris TRP2
PpTRP2 promoter
3282 .. 3431  =  150 bp
promoter for Pichia pastoris TRP2
AmpR promoter
7132 .. 7236  =  105 bp
AmpR promoter
7132 .. 7236  =  105 bp
PpADE2 LC promoter
1282 .. 1363  =  82 bp
strong 82-bp promoter fragment that requires only
low-copy integration for efficient expression
PpADE2 LC promoter
1282 .. 1363  =  82 bp
strong 82-bp promoter fragment that requires only
low-copy integration for efficient expression
MCS
941 .. 987  =  47 bp
multiple cloning site
MCS
941 .. 987  =  47 bp
multiple cloning site
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