pFA6a-His3MX6-PGAL1

Plasmid for swapping in the GAL1 promoter using the HIS3MX6 selectable marker.
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HomePlasmidsYeast PlasmidspFA6a-His3MX6-PGAL1
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) BspQI - SapI (2066) BsiWI (25) SalI (37) BamHI (43) AvaI - BsoBI - TspMI - XmaI (48) 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) pFA6a-His3MX6-PGAL1 4329 bp
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.
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.
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
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
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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
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
Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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
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  =  53°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
2 segments
   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
2 segments
   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
ORF:  1006 .. 1659  =  654 bp
ORF:  217 amino acids  =  23.6 kDa
ORF:  603 .. 872  =  270 bp
ORF:  89 amino acids  =  10.2 kDa
ORF:  3132 .. 3398  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3002 .. 3862  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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Individual Sequences & Maps

bleMX6
pFA6a-kanMX6
pPIC6(alpha) B
pYES2.1-E
hphMX6
pFA6a-kanMX6-PGAL1
pPIC6(alpha) C
pYES2 CT
kanMX
pFA6a-kanMX6-PGAL1-3HA
pPIC9
pYES2 NT A
kanMX2
pFA6a-kanMX6-PGAL1-GFP
pPIC9K
pYES2 NT B
kanMX4
pFA6a-kanMX6-PGAL1-GST
pPICZ A
pYES2 NT C
kanMX6
pFA6a-natMX6
pPICZ B
pYES3 CT
natMX6
pFA6a-TRP1
pPICZ C
pYES6 CT
pAbAi
pFA6a-TRP1-PGAL1
pPICZ(alpha) A
pYM25
pAD-GAL4
pFA6a-TRP1-PGAL1-3HA
pPICZ(alpha) B
pYM26
pAG25
pFA6a-TRP1-PGAL1-GFP
pPICZ(alpha) C
pYM44
pAG29
pFA6a-TRP1-PGAL1-GST
pPink-HC
YCp50
pAG32
pFLD
pPink-LC
YCp50-poly
pAO815
pFLD(alpha)
pPINK(alpha)-HC
YCplac111
patMX4
pGAD-C1
pRS303
YCplac22
pAUR101
pGAD-C2
pRS304
YCplac33
pAUR112
pGAD-C3
pRS305
YEp13
pAUR123
pGADT7 AD
pRS306
YEp24
pAUR224
pGADT7-Rec
pRS313
YEp351
pBridge
pGADT7-Rec (linearized)
pRS314
YEp352
pDH25
pGAPZ A
pRS315
YEp353
pEG202
pGAPZ B
pRS316
YEp354
pESC-HIS
pGAPZ C
pRS403
YEp355
pESC-LEU
pGAPZ(alpha) A
pRS404
YEp356
pESC-TRP
pGAPZ(alpha) B
pRS405
YEp356R
pESC-URA
pGAPZ(alpha) C
pRS406
YEp357
pFA6-kanMX4
pGBD-C1
pRS413
YEp357R
pFA6a
pGBD-C2
pRS414
YEp358
pFA6a-12Pk-kanMX6
pGBD-C3
pRS415
YEp358R
pFA6a-13Myc-His3MX6
pGBDU-C1
pRS416
YEp363
pFA6a-13Myc-kanMX6
pGBDU-C2
pRS423
YEp364
pFA6a-13Myc-TRP1
pGBDU-C3
pRS424
YEp365
pFA6a-3HA-His3MX6
pGBKT7
pRS425
YEp366
pFA6a-3HA-kanMX6
pGilda
pRS426
YEp366R
pFA6a-3HA-TRP1
pHIL-D2
pSpot5
YEp367
pFA6a-5FLAG-kanMX6
pHIL-S1
pSpot6
YEp367R
pFA6a-bleMX6
pJG4-5
pSpot7
YEp368
pFA6a-GFP(S65T)-His3MX6
pKLAC1
pSpot8
YEp368R
pFA6a-GFP(S65T)-kanMX6
pKLAC2
pTEF1 Zeo
YEplac112
pFA6a-GFP(S65T)-TRP1
pOAD
pUCATPH
YEplac181
pFA6a-GST-His3MX6
pOBD2
pY-secSUMOstar
YEplac195
pFA6a-GST-kanMX6
pP-secSUMO3
pY-SUMOstar
YIp351
pFA6a-GST-TRP1
pPIC3
pYC2-E
YIp352
pFA6a-His3MX6
pPIC3.5
pYC2 CT
YIp5
pFA6a-His3MX6-PGAL1
pPIC3.5K
pYC2 NT A
YIplac128
pFA6a-His3MX6-PGAL1-3HA
pPIC6 A
pYC2 NT B
YIplac204
pFA6a-His3MX6-PGAL1-GFP
pPIC6 B
pYC2 NT C
YIplac211
pFA6a-His3MX6-PGAL1-GST
pPIC6 C
pYC6 CT
YRp7
pFA6a-hphMX6
pPIC6(alpha) A
pYES2
alpha-factor secretion signal

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