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Plasmid Files

pYM44

Plasmid with a HIS3MX6 marker that provides a template for PCR to fuse yeast enhanced GFP (yeGFP) to the C-terminus of a protein of interest.

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pYM44 Sequence and MappYM44.dna
Map and Sequence File   
Sequence Author:  EUROSCARF
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 HindIII (19) NotI (6) PfoI (4528) EcoO109I (4471) AatII (4417) ZraI (4415) SspI (4299) XmnI (4094) PvuI (3865) NmeAIII (3643) BmrI (3535) BanI (3443) PspFI (2910) BseYI (2906) BspQI - SapI (2486) SacII (2371) NotI (2365) BsiWI (25) SalI (37) AarI - BamHI (43) BspEI * (46) MscI (252) MfeI (641) PflMI (777) BsrGI (788) BstZ17I (879) I-SceI (1025) BglII (1037) BstEII (1067) BmgBI (1120) Bpu10I (1129) BseRI (1231) MluI (1284) BtgZI (1467) SphI (1552) BclI * (1683) BsaBI (1685) NgoMIV (1753) NaeI (1755) XbaI (1804) AfeI (1992) PmeI (2316) Eco53kI (2323) BanII - SacI (2325) EcoRI (2327) EcoRV (2341) SfiI (2364) pYM44 4749 bp
HindIII  (19)
1 site
A A G C T T T T C G A A
NotI  (6)
2 sites
G C G G C C G C C G C C G G C G
PfoI  (4528)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
EcoO109I  (4471)
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.
AatII  (4417)
1 site
G A C G T C C T G C A G
ZraI  (4415)
1 site
G A C G T C C T G C A G
SspI  (4299)
1 site
A A T A T T T T A T A A
XmnI  (4094)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (3865)
1 site
C G A T C G G C T A G C
NmeAIII  (3643)
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).
BmrI  (3535)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the
absence of magnesium.
BanI  (3443)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
PspFI  (2910)
1 site
C C C A G C G G G T C G
BseYI  (2906)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
BspQI  (2486)
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  (2486)
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.
SacII  (2371)
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  (2365)
2 sites
G C G G C C G C C G C C G G C G
BsiWI  (25)
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.
SalI  (37)
1 site
G T C G A C C A G C T G
AarI  (43)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
BamHI  (43)
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.
BspEI  (46)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
MscI  (252)
1 site
T G G C C A A C C G G T
MfeI  (641)
1 site
C A A T T G G T T A A C
PflMI  (777)
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.
BsrGI  (788)
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.
BstZ17I  (879)
1 site
G T A T A C C A T A T G
I-SceI  (1025)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its
electrophoretic mobility.
BglII  (1037)
1 site
A G A T C T T C T A G A
BstEII  (1067)
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.
BmgBI  (1120)
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.
Bpu10I  (1129)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BseRI  (1231)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
MluI  (1284)
1 site
A C G C G T T G C G C A
BtgZI  (1467)
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.
SphI  (1552)
1 site
G C A T G C C G T A C G
BclI  (1683)
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.
BsaBI  (1685)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (1753)
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  (1755)
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.
XbaI  (1804)
1 site
T C T A G A A G A T C T
AfeI  (1992)
1 site
A G C G C T T C G C G A
PmeI  (2316)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (2323)
1 site
G A G C T C C T C G A G
BanII  (2325)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (2325)
1 site
G A G C T C C T C G A G
EcoRI  (2327)
1 site
G A A T T C C T T A A G
EcoRV  (2341)
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.
SfiI  (2364)
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.
HIS3MX6
1082 .. 2282  =  1201 bp
HIS3MX6
1082 .. 2282  =  1201 bp
AmpR
3422 .. 4282  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3422 .. 4213  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3422 .. 4282  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4214 .. 4282  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3422 .. 4282  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
yeGFP
85 .. 798  =  714 bp
237 amino acids  =  26.7 kDa
Product: yeast-enhanced green fluorescent protein
codons optimized for translation in S. cerevisiae and
C. albicans
yeGFP
85 .. 798  =  714 bp
237 amino acids  =  26.7 kDa
Product: yeast-enhanced green fluorescent protein
codons optimized for translation in S. cerevisiae and
C. albicans
ori
2663 .. 3251  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2663 .. 3251  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ADH1 terminator
820 .. 1007  =  188 bp
transcription terminator for alcohol dehydrogenase 1
ADH1 terminator
820 .. 1007  =  188 bp
transcription terminator for alcohol dehydrogenase 1
AmpR promoter
4283 .. 4387  =  105 bp
AmpR promoter
4283 .. 4387  =  105 bp
SP6 promoter
4733 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4733 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
S. pombe his5
1426 .. 2079  =  654 bp
217 amino acids  =  23.6 kDa
Product: imidazoleglycerol-phosphate dehydratase,
required for histidine biosynthesis
yeast auxotrophic marker; corresponds to S.
cerevisiae HIS3
S. pombe his5
1426 .. 2079  =  654 bp
217 amino acids  =  23.6 kDa
Product: imidazoleglycerol-phosphate dehydratase,
required for histidine biosynthesis
yeast auxotrophic marker; corresponds to S.
cerevisiae HIS3
TEF promoter
1082 .. 1425  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
1082 .. 1425  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
2085 .. 2282  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
2085 .. 2282  =  198 bp
Ashbya gossypii TEF terminator
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