pFA6a-TRP1-PGAL1-3HA

Plasmid with a TRP1 marker for swapping in the GAL1 promoter and adding a triple-HA tag.
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HomePlasmidsYeast PlasmidspFA6a-TRP1-PGAL1-3HA
PvuII (15) EcoO109I (3997) SspI (3825) ScaI (3501) PvuI (3391) FspI (3243) BpmI (3091) BanI (2969) AlwNI (2544) AflIII - PciI (2128) BsiWI (25) PstI (35) SalI (37) PsiI (194) AscI - BssHII (251) BbvCI - Bpu10I (257) R3 (261 .. 280) BamHI (297) PacI (371) BtgZI (541) AgeI (767) BseRI (805) BglII (930) BsrGI (935) BspEI * (948) PmlI (1043) XbaI (1167) MfeI (1223) Bsu36I (1325) BstXI (1381) BsgI (1496) PaqCI (1516) PmeI (1842) Eco53kI (1849) SacI (1851) EcoRI (1853) F4 (1839 .. 1858) BspDI - ClaI (1860) SpeI (1877) SfiI (1890) BtgI (1894) SacII (1897) HpaI (1949) BspQI - SapI (2012) pFA6a-TRP1-PGAL1-3HA 4275 bp
PvuII  (15)
1 site		 C A G C T G G T C G A C
EcoO109I  (3997)
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.
SspI  (3825)
1 site		 A A T A T T T T A T A A
ScaI  (3501)
1 site		 A G T A C T T C A T G A
PvuI  (3391)
1 site		 C G A T C G G C T A G C
FspI  (3243)
1 site		 T G C G C A A C G C G T
BpmI  (3091)
1 site		 C T G G A G ( N ) 14 N N G A C C T C ( N ) 14
Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BanI  (2969)
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  (2544)
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.
AflIII  (2128)
1 site		 A C R Y G T T G Y R C A
Sticky ends from different AflIII sites may not be compatible.
PciI  (2128)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
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.
PstI  (35)
1 site		 C T G C A G G A C G T C
SalI  (37)
1 site		 G T C G A C C A G C T G
PsiI  (194)
1 site		 T T A T A A A A T A T T
AscI  (251)
1 site		 G G C G C G C C C C G C G C G G
BssHII  (251)
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.
BbvCI  (257)
1 site		 C C T C A G C G G A G T C G
Bpu10I  (257)
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.
BamHI  (297)
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.
PacI  (371)
1 site		 T T A A T T A A A A T T A A T T
BtgZI  (541)
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.
AgeI  (767)
1 site		 A C C G G T T G G C C A
BseRI  (805)
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.
BglII  (930)
1 site		 A G A T C T T C T A G A
BsrGI  (935)
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.
BspEI  (948)
1 site		 T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (1043)
1 site		 C A C G T G G T G C A C
XbaI  (1167)
1 site		 T C T A G A A G A T C T
MfeI  (1223)
1 site		 C A A T T G G T T A A C
Bsu36I  (1325)
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.
BstXI  (1381)
1 site		 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.
BsgI  (1496)
1 site		 G T G C A G ( N ) 14 N N C A C G T C ( N ) 14
Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PaqCI  (1516)
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 PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PmeI  (1842)
1 site		 G T T T A A A C C A A A T T T G
Eco53kI  (1849)
1 site		 G A G C T C C T C G A G
SacI  (1851)
1 site		 G A G C T C C T C G A G
EcoRI  (1853)
1 site		 G A A T T C C T T A A G
BspDI  (1860)
1 site		 A T C G A T T A G C T A
ClaI  (1860)
1 site		 A T C G A T T A G C T A
SpeI  (1877)
1 site		 A C T A G T T G A T C A
SfiI  (1890)
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.
BtgI  (1894)
1 site		 C C R Y G G G G Y R C C
Sticky ends from different BtgI sites may not be compatible.
SacII  (1897)
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  (1949)
1 site		 G T T A A C C A A T T G
BspQI  (2012)
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  (2012)
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.
R3
20-mer  /  55% GC
1 binding site
261 .. 280  =  20 annealed bases
Tm  =  57°C
Reverse primer for promoter swapping and adding an N-terminal 3xHA tag. A gene-specific sequence should be added at the 5' end of the primer.
F4
20-mer  /  40% GC
1 binding site
1839 .. 1858  =  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.
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2948 .. 3739  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3740 .. 3808  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2948 .. 3808  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TRP1
1084 .. 1758  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
1084 .. 1758  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2189 .. 2777  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2189 .. 2777  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
GAL1 promoter
400 .. 841  =  442 bp
inducible promoter, regulated by Gal4
GAL1 promoter
400 .. 841  =  442 bp
inducible promoter, regulated by Gal4
ADH1 terminator
50 .. 237  =  188 bp
ADH1 terminator
50 .. 237  =  188 bp
TRP1 promoter
936 .. 1083  =  148 bp
TRP1 promoter
936 .. 1083  =  148 bp
3xHA
270 .. 359  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
3xHA
270 .. 359  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
start codon
378 .. 380  =  3 bp
1 amino acid  =  149.2 Da
AmpR promoter
3809 .. 3913  =  105 bp
AmpR promoter
3809 .. 3913  =  105 bp
T7 promoter
1913 .. 1931  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1913 .. 1931  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4259 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4259 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
UAS
724 .. 841  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
UAS
724 .. 841  =  118 bp
upstream activating sequence mediating Gal4-dependent induction
ORF:  1084 .. 1758  =  675 bp
ORF:  224 amino acids  =  24.1 kDa
ORF:  1334 .. 1615  =  282 bp
ORF:  93 amino acids  =  10.8 kDa
ORF:  3078 .. 3344  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2948 .. 3808  =  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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