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Plasmid Files

pKLAC1

Integrative expression vector for the yeast Kluyveromyces lactis. For an alternative MCS, use pKLAC2.

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pKLAC1 Sequence and MappKLAC1.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 HindIII (1) SnaBI (8798) BstXI (7523) SacII (7462) AanI (7195) AhdI (5918) SacII (4635) PaeR7I - XhoI (257) BglII (290) AvrII (296) Acc65I (302) KpnI (306) SalI - SgrDI (308) AscI (315) NotI (324) PacI (335) StuI (340) XcmI (499) BsrGI (994) PshAI (2213) EcoO109I - PpuMI (2521) KasI (2598) NarI * (2599) SfoI (2600) BbeI (2602) Eco53kI (2926) SacI (2928) AleI (3116) BstEII (3130) AgeI (3241) TspMI - XmaI (3344) SmaI (3346) NruI (3367) Bsu36I (3388) PfoI * (3440) XbaI * (3522) AanI (3665) SbfI (3859) BmgBI (4243) BstXI (4310) PflFI - Tth111I (4353) pKLAC1 9091 bp
HindIII  (1)
1 site
A A G C T T T T C G A A
SnaBI  (8798)
1 site
T A C G T A A T G C A T
BstXI  (7523)
2 sites
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.
SacII  (7462)
2 sites
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
AanI  (7195)
2 sites
T T A T A A A A T A T T
AhdI  (5918)
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.
SacII  (4635)
2 sites
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
PaeR7I  (257)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (257)
1 site
C T C G A G G A G C T C
BglII  (290)
1 site
A G A T C T T C T A G A
AvrII  (296)
1 site
C C T A G G G G A T C C
Acc65I  (302)
1 site
G G T A C C C C A T G G
KpnI  (306)
1 site
G G T A C C C C A T G G
SalI  (308)
1 site
G T C G A C C A G C T G
SgrDI  (308)
1 site
C G T C G A C G G C A G C T G C
AscI  (315)
1 site
G G C G C G C C C C G C G C G G
NotI  (324)
1 site
G C G G C C G C C G C C G G C G
PacI  (335)
1 site
T T A A T T A A A A T T A A T T
StuI  (340)
1 site
A G G C C T T C C G G A
XcmI  (499)
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.
BsrGI  (994)
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.
PshAI  (2213)
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.
EcoO109I  (2521)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PpuMI  (2521)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
KasI  (2598)
1 site
G G C G C C C C G C G G
NarI  (2599)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (2600)
1 site
G G C G C C C C G C G G
BbeI  (2602)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the BbeI
recognition sequence.
Eco53kI  (2926)
1 site
G A G C T C C T C G A G
SacI  (2928)
1 site
G A G C T C C T C G A G
AleI  (3116)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (3130)
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.
AgeI  (3241)
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.
TspMI  (3344)
1 site
C C C G G G G G G C C C
XmaI  (3344)
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.
SmaI  (3346)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
NruI  (3367)
1 site
T C G C G A A G C G C T
Bsu36I  (3388)
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.
PfoI  (3440)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
XbaI  (3522)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
AanI  (3665)
2 sites
T T A T A A A A T A T T
SbfI  (3859)
1 site
C C T G C A G G G G A C G T C C
BmgBI  (4243)
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.
BstXI  (4310)
2 sites
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.
PflFI  (4353)
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  (4353)
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.
amdS
1697 .. 3343  =  1647 bp
548 amino acids  =  60.2 kDa
Product: acetamidase from Aspergillus nidulans
confers ability to process acetamide for selection in
fungal cells
amdS
1697 .. 3343  =  1647 bp
548 amino acids  =  60.2 kDa
Product: acetamidase from Aspergillus nidulans
confers ability to process acetamide for selection in
fungal cells
LAC4 promoter (3')
7463 .. 8991  =  1529 bp
3' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 promoter (3')
7463 .. 8991  =  1529 bp
3' portion of a modified Kluyveromyces lactis LAC4
promoter
AmpR
5845 .. 6705  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5845 .. 6636  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5845 .. 6705  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6637 .. 6705  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5845 .. 6705  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ADH1 promoter
974 .. 1676  =  703 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
974 .. 1676  =  703 bp
promoter for alcohol dehydrogenase 1
LAC4 promoter (5')
3979 .. 4635  =  657 bp
5' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 promoter (5')
3979 .. 4635  =  657 bp
5' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 terminator
355 .. 973  =  619 bp
transcription terminator for Kluyveromyces lactis
LAC4
LAC4 terminator
355 .. 973  =  619 bp
transcription terminator for Kluyveromyces lactis
LAC4
ori
5086 .. 5674  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5086 .. 5674  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6837 .. 7292  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6837 .. 7292  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
α-factor secretion signal
14 .. 280  =  267 bp
89 amino acids  =  9.4 kDa
Product: N-terminal secretion signal from
Kluyveromyces lactis 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
14 .. 280  =  267 bp
89 amino acids  =  9.4 kDa
Product: N-terminal secretion signal from
Kluyveromyces lactis alpha-factor
Cleavage by the Kex2 protease occurs after the
dibasic KR sequence. The EA dipeptides are then
removed by dipeptidyl aminopeptidase A.
AmpR promoter
6706 .. 6810  =  105 bp
AmpR promoter
6706 .. 6810  =  105 bp
MCS
257 .. 343  =  87 bp
multiple cloning site
MCS
257 .. 343  =  87 bp
multiple cloning site
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