pFA6a-His3MX6-PGAL1-GFP

Plasmid with a HIS3MX6 marker for swapping in the GAL1 promoter and adding a GFP tag.
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HomePlasmidsYeast PlasmidspFA6a-His3MX6-PGAL1-GFP
HindIII (19) PfoI (5027) EcoO109I (4970) AatII (4916) ZraI (4914) SspI (4798) XmnI (4593) ScaI (4474) PvuI (4364) BmrI (4034) BanI (3942) BspQI - SapI (2985) SacII (2870) SfiI (2863) EcoRV (2840) F4 (2812 .. 2831) EcoRI (2826) SacI (2824) Eco53kI (2822) BsiWI (25) SalI (37) AscI (251) R5 (261 .. 280) BstBI (350) MfeI (410) PmlI (649) BsrGI (694) SnaBI (780) MscI (803) PacI (977) BstAPI (1128) AgeI (1373) BglII (1536) BstEII (1566) BmgBI (1619) Bpu10I (1628) MluI (1783) SphI (2051) BclI * (2182) BsaBI (2184) NgoMIV (2252) NaeI (2254) XbaI (2303) AfeI (2491) PmeI (2815) pFA6a-His3MX6-PGAL1-GFP 5248 bp
HindIII  (19)
1 site		 A A G C T T T T C G A A
PfoI  (5027)
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  (4970)
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  (4916)
1 site		 G A C G T C C T G C A G
ZraI  (4914)
1 site		 G A C G T C C T G C A G
SspI  (4798)
1 site		 A A T A T T T T A T A A
XmnI  (4593)
1 site		 G A A N N N N T T C C T T N N N N A A G
ScaI  (4474)
1 site		 A G T A C T T C A T G A
PvuI  (4364)
1 site		 C G A T C G G C T A G C
BmrI  (4034)
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  (3942)
1 site		 G G Y R C C C C R Y G G
Sticky ends from different BanI sites may not be compatible.
BspQI  (2985)
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  (2985)
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  (2870)
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.
SfiI  (2863)
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.
EcoRV  (2840)
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.
EcoRI  (2826)
1 site		 G A A T T C C T T A A G
SacI  (2824)
1 site		 G A G C T C C T C G A G
Eco53kI  (2822)
1 site		 G A G C T C C T C G A 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
AscI  (251)
1 site		 G G C G C G C C C C G C G C G G
BstBI  (350)
1 site		 T T C G A A A A G C T T
MfeI  (410)
1 site		 C A A T T G G T T A A C
PmlI  (649)
1 site		 C A C G T G G T G C A C
BsrGI  (694)
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.
SnaBI  (780)
1 site		 T A C G T A A T G C A T
MscI  (803)
1 site		 T G G C C A A C C G G T
PacI  (977)
1 site		 T T A A T T A A A A T T A A T T
BstAPI  (1128)
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  (1373)
1 site		 A C C G G T T G G C C A
BglII  (1536)
1 site		 A G A T C T T C T A G A
BstEII  (1566)
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  (1619)
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  (1628)
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  (1783)
1 site		 A C G C G T T G C G C A
SphI  (2051)
1 site		 G C A T G C C G T A C G
BclI  (2182)
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  (2184)
1 site		 G A T N N N N A T C C T A N N N N T A G
NgoMIV  (2252)
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  (2254)
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  (2303)
1 site		 T C T A G A A G A T C T
AfeI  (2491)
1 site		 A G C G C T T C G C G A
PmeI  (2815)
1 site		 G T T T A A A C C A A A T T T G
F4
20-mer  /  40% GC
1 binding site
2812 .. 2831  =  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.
R5
20-mer  /  35% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  50°C
Reverse primer for promoter swapping and adding an N-terminal GFP tag. A gene-specific sequence should be added at the 5' end of the primer.
HIS3MX6
1581 .. 2781  =  1201 bp
yeast selectable marker encoding the S. pombe his5 gene, which corresponds to S. cerevisiae HIS3
HIS3MX6
1581 .. 2781  =  1201 bp
yeast selectable marker encoding the S. pombe his5 gene, which corresponds to S. cerevisiae HIS3
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3921 .. 4712  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   4713 .. 4781  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3921 .. 4781  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GFP(S65T)
258 .. 970  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
GFP(S65T)
258 .. 970  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
start codon
984 .. 986  =  3 bp
1 amino acid  =  149.2 Da
start codon
984 .. 986  =  3 bp
1 amino acid  =  149.2 Da
ori
3162 .. 3750  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
3162 .. 3750  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
GAL1 promoter
1006 .. 1447  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
1006 .. 1447  =  442 bp
inducible promoter, regulated by Gal4
ADH1 terminator
50 .. 237  =  188 bp
ADH1 terminator
50 .. 237  =  188 bp
AmpR promoter
4782 .. 4886  =  105 bp
AmpR promoter
4782 .. 4886  =  105 bp
T7 promoter
2886 .. 2904  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2886 .. 2904  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
5232 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
5232 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
S. pombe his5
1925 .. 2578  =  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
1925 .. 2578  =  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
1581 .. 1923  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
1581 .. 1923  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
2584 .. 2781  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
2584 .. 2781  =  198 bp
Ashbya gossypii TEF terminator
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
UAS
1330 .. 1447  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
ORF:  1522 .. 1791  =  270 bp
ORF:  89 amino acids  =  10.2 kDa
ORF:  4051 .. 4317  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1925 .. 2578  =  654 bp
ORF:  217 amino acids  =  23.6 kDa
ORF:  258 .. 986  =  729 bp
ORF:  242 amino acids  =  27.4 kDa
ORF:  3921 .. 4781  =  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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