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Plasmid Files

pKLAC2

Integrative expression vector for the yeast Kluyveromyces lactis. For an alternative MCS, use pKLAC1.

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pKLAC2 Sequence and MappKLAC2.dna
Map and Sequence File   
Sequence Author:  New England Biolabs
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 HindIII (1) SnaBI (8814) BstXI (7539) SacII (7478) AanI (7211) AhdI (5934) SacII (4651) PaeR7I - XhoI (257) NdeI (291) NcoI (297) NotI (304) EcoRV (313) SalI - SgrDI (317) BamHI (323) EcoRI (329) SbfI (340) StuI (356) XcmI (515) BsrGI (1010) BglII (1701) BssHII (1748) PshAI (2229) EcoO109I - PpuMI (2537) KasI (2614) NarI * (2615) SfoI (2616) BbeI (2618) AleI (3132) BstEII (3146) BspEI * (3236) AgeI (3257) TspMI - XmaI (3360) SmaI (3362) NruI (3383) Bsu36I (3404) PfoI * (3456) XbaI * (3538) AanI (3681) BmgBI (4259) BstXI (4326) PflFI - Tth111I (4369) pKLAC2 9107 bp
HindIII  (1)
1 site
A A G C T T T T C G A A
SnaBI  (8814)
1 site
T A C G T A A T G C A T
BstXI  (7539)
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  (7478)
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  (7211)
2 sites
T T A T A A A A T A T T
AhdI  (5934)
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  (4651)
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
NdeI  (291)
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.
NcoI  (297)
1 site
C C A T G G G G T A C C
NotI  (304)
1 site
G C G G C C G C C G C C G G C G
EcoRV  (313)
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.
SalI  (317)
1 site
G T C G A C C A G C T G
SgrDI  (317)
1 site
C G T C G A C G G C A G C T G C
BamHI  (323)
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.
EcoRI  (329)
1 site
G A A T T C C T T A A G
SbfI  (340)
1 site
C C T G C A G G G G A C G T C C
StuI  (356)
1 site
A G G C C T T C C G G A
XcmI  (515)
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  (1010)
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.
BglII  (1701)
1 site
A G A T C T T C T A G A
BssHII  (1748)
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.
PshAI  (2229)
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  (2537)
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  (2537)
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  (2614)
1 site
G G C G C C C C G C G G
NarI  (2615)
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  (2616)
1 site
G G C G C C C C G C G G
BbeI  (2618)
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.
AleI  (3132)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (3146)
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.
BspEI  (3236)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
AgeI  (3257)
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  (3360)
1 site
C C C G G G G G G C C C
XmaI  (3360)
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  (3362)
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  (3383)
1 site
T C G C G A A G C G C T
Bsu36I  (3404)
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  (3456)
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  (3538)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
AanI  (3681)
2 sites
T T A T A A A A T A T T
BmgBI  (4259)
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  (4326)
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  (4369)
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  (4369)
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
1713 .. 3359  =  1647 bp
548 amino acids  =  60.2 kDa
Product: acetamidase from Aspergillus nidulans
confers ability to process acetamide for selection in
fungal cells
amdS
1713 .. 3359  =  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')
7479 .. 9007  =  1529 bp
3' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 promoter (3')
7479 .. 9007  =  1529 bp
3' portion of a modified Kluyveromyces lactis LAC4
promoter
AmpR
5861 .. 6721  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5861 .. 6652  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5861 .. 6721  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6653 .. 6721  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5861 .. 6721  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ADH1 promoter
990 .. 1692  =  703 bp
promoter for alcohol dehydrogenase 1
ADH1 promoter
990 .. 1692  =  703 bp
promoter for alcohol dehydrogenase 1
LAC4 promoter (5')
3995 .. 4651  =  657 bp
5' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 promoter (5')
3995 .. 4651  =  657 bp
5' portion of a modified Kluyveromyces lactis LAC4
promoter
LAC4 terminator
371 .. 989  =  619 bp
transcription terminator for Kluyveromyces lactis
LAC4
LAC4 terminator
371 .. 989  =  619 bp
transcription terminator for Kluyveromyces lactis
LAC4
ori
5102 .. 5690  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5102 .. 5690  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6853 .. 7308  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6853 .. 7308  =  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
6722 .. 6826  =  105 bp
AmpR promoter
6722 .. 6826  =  105 bp
MCS
257 .. 359  =  103 bp
multiple cloning site
MCS
257 .. 359  =  103 bp
multiple cloning site
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