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Plasmid Files

pFA6a-His3MX6-PGAL1

Plasmid for swapping in the GAL1 promoter using the HIS3MX6 selectable marker.

 
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 BsiWI (25) HindIII (19) NdeI (4246) PfoI (4108) EcoO109I (4051) AatII (3997) ZraI (3995) SspI (3879) XmnI (3674) ScaI (3555) PvuI (3445) BmrI (3115) BanI (3023) AlwNI (2598) PspFI (2490) BseYI (2486) SalI (37) BamHI (43) AvaI - BsoBI - TspMI - XmaI (48) BmeT110I (49) SmaI (50) PacI (58) R2 (65 .. 84) BstAPI (209) AgeI (454) BglII (617) BstEII (647) BmgBI (700) Bpu10I (709) MluI (864) NcoI - StyI (1004) PsiI (1117) SphI (1132) BclI * (1263) BsaBI (1265) NgoMIV (1333) NaeI (1335) XbaI (1384) AfeI (1572) BssHII (1599) PmeI (1896) Eco53kI (1903) SacI (1905) EcoRI (1907) F4 (1893 .. 1912) EcoRV (1921) SfiI (1944) SacII (1951) HpaI (2003) BspQI - SapI (2066) pFA6a-His3MX6-PGAL1 4329 bp
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.
HindIII  (19)
1 site
A A G C T T T T C G A A
NdeI  (4246)
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.
PfoI  (4108)
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  (4051)
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  (3997)
1 site
G A C G T C C T G C A G
ZraI  (3995)
1 site
G A C G T C C T G C A G
SspI  (3879)
1 site
A A T A T T T T A T A A
XmnI  (3674)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3555)
1 site
A G T A C T T C A T G A
PvuI  (3445)
1 site
C G A T C G G C T A G C
BmrI  (3115)
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  (3023)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
AlwNI  (2598)
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.
PspFI  (2490)
1 site
C C C A G C G G G T C G
BseYI  (2486)
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.
SalI  (37)
1 site
G T C G A C C A G C T G
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.
AvaI  (48)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (48)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (48)
1 site
C C C G G G G G G C C C
XmaI  (48)
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.
BmeT110I  (49)
1 site
C Y C G R G G R G C Y C
SmaI  (50)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
PacI  (58)
1 site
T T A A T T A A A A T T A A T T
BstAPI  (209)
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.
AgeI  (454)
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.
BglII  (617)
1 site
A G A T C T T C T A G A
BstEII  (647)
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  (700)
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  (709)
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.
MluI  (864)
1 site
A C G C G T T G C G C A
NcoI  (1004)
1 site
C C A T G G G G T A C C
StyI  (1004)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PsiI  (1117)
1 site
T T A T A A A A T A T T
SphI  (1132)
1 site
G C A T G C C G T A C G
BclI  (1263)
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  (1265)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (1333)
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  (1335)
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  (1384)
1 site
T C T A G A A G A T C T
AfeI  (1572)
1 site
A G C G C T T C G C G A
BssHII  (1599)
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.
PmeI  (1896)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1903)
1 site
G A G C T C C T C G A G
SacI  (1905)
1 site
G A G C T C C T C G A G
EcoRI  (1907)
1 site
G A A T T C C T T A A G
EcoRV  (1921)
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  (1944)
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.
SacII  (1951)
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.
HpaI  (2003)
1 site
G T T A A C C A A T T G
BspQI  (2066)
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  (2066)
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.
R2
20-mer  /  40% GC
1 binding site
65 .. 84  =  20 annealed bases
Tm  =  53°C
Reverse primer for promoter swapping with the
GAL1 promoter. This primer encodes the reverse
complement of a start codon. A gene-specific
sequence should be added at the 5' end of the
primer.
F4
20-mer  /  40% GC
1 binding site
1893 .. 1912  =  20 annealed bases
Tm  =  52°C
Forward primer for promoter swapping. This primer
includes an EcoRI recognition sequence. A
gene-specific sequence should be added at the 5'
end of the primer.
HIS3MX6
662 .. 1862  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
HIS3MX6
662 .. 1862  =  1201 bp
yeast selectable marker encoding the S. pombe his5
gene, which corresponds to S. cerevisiae HIS3
AmpR
3002 .. 3862  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3002 .. 3793  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3002 .. 3862  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3794 .. 3862  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3002 .. 3862  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2243 .. 2831  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2243 .. 2831  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
GAL1 promoter
87 .. 528  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
87 .. 528  =  442 bp
inducible promoter, regulated by Gal4
AmpR promoter
3863 .. 3967  =  105 bp
AmpR promoter
3863 .. 3967  =  105 bp
T7 promoter
1967 .. 1985  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1967 .. 1985  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4313 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4313 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
S. pombe his5
1006 .. 1659  =  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
1006 .. 1659  =  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
662 .. 1004  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
662 .. 1004  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
1665 .. 1862  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1665 .. 1862  =  198 bp
Ashbya gossypii TEF terminator
UAS
411 .. 528  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
UAS
411 .. 528  =  118 bp
upstream activating sequence mediating
Gal4-dependent induction
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