pFA6a-bleMX6

Plasmid for yeast gene deletion using the bleMX6 selectable marker conferring phleomycin resistance.

Sequence Author: EUROSCARF

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HomePlasmidsYeast PlasmidspFA6a-bleMX6
PvuII (13) NdeI (3418) EcoO109I (3223) SspI (3051) XmnI (2846) TsoI (2646) PvuI (2617) BpmI (2317) BmrI (2287) AlwNI (1770) HindIII (17) BsiWI (23) SalI (35) AccI (36) F1 (40 .. 59) BamHI (41) PacI (56) AscI (61) BglII (68) BstEII (98) BstXI (115) Bpu10I (160) MluI (315) NcoI - StyI (455) MscI (460) MauBI (493) SgrAI (571) CsiI - SexAI * (620) FseI (733) DraIII (810) BclI * (829) BsmI (1021) PmeI (1068) Eco53kI (1075) BanII - SacI (1077) ApoI - EcoRI (1079) R1 (1065 .. 1084) BspDI - ClaI (1086) EcoRV (1093) SpeI (1103) SfiI (1116) SacII (1123) HpaI (1175) BspQI - SapI (1238) PciI (1354) BseYI (1658) PspFI (1662) pFA6a-bleMX6 3503 bp
PvuII  (13)
1 site		 C A G C T G G T C G A C
NdeI  (3418)
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.
EcoO109I  (3223)
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  (3051)
1 site		 A A T A T T T T A T A A
XmnI  (2846)
1 site		 G A A N N N N T T C C T T N N N N A A G
TsoI  (2646)
1 site		 T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9
Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (2617)
1 site		 C G A T C G G C T A G C
BpmI  (2317)
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.
BmrI  (2287)
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.
AlwNI  (1770)
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.
HindIII  (17)
1 site		 A A G C T T T T C G A A
BsiWI  (23)
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  (35)
1 site		 G T C G A C C A G C T G
AccI  (36)
1 site		 G T M K A C C A K M T G
Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BamHI  (41)
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  (56)
1 site		 T T A A T T A A A A T T A A T T
AscI  (61)
1 site		 G G C G C G C C C C G C G C G G
BglII  (68)
1 site		 A G A T C T T C T A G A
BstEII  (98)
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.
BstXI  (115)
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.
Bpu10I  (160)
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  (315)
1 site		 A C G C G T T G C G C A
NcoI  (455)
1 site		 C C A T G G G G T A C C
StyI  (455)
1 site		 C C W W G G G G W W C C
Sticky ends from different StyI sites may not be compatible.
MscI  (460)
1 site		 T G G C C A A C C G G T
MauBI  (493)
1 site		 C G C G C G C G G C G C G C G C
SgrAI  (571)
1 site		 C R C C G G Y G G Y G G C C R C
Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
CsiI  (620)
1 site		 A C C W G G T T G G W C C A
Sticky ends from different CsiI sites may not be compatible.
SexAI  (620)
1 site		 A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
FseI  (733)
1 site		 G G C C G G C C C C G G C C G G
FseI gradually loses activity when stored at -20°C.
DraIII  (810)
1 site		 C A C N N N G T G G T G N N N C A C
Sticky ends from different DraIII sites may not be compatible.
BclI  (829)
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.
BsmI  (1021)
1 site		 G A A T G C N C T T A C G N
Sticky ends from different BsmI sites may not be compatible.
PmeI  (1068)
1 site		 G T T T A A A C C A A A T T T G
Eco53kI  (1075)
1 site		 G A G C T C C T C G A G
BanII  (1077)
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  (1077)
1 site		 G A G C T C C T C G A G
ApoI  (1079)
1 site		 R A A T T Y Y T T A A R
ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (1079)
1 site		 G A A T T C C T T A A G
BspDI  (1086)
1 site		 A T C G A T T A G C T A
ClaI  (1086)
1 site		 A T C G A T T A G C T A
EcoRV  (1093)
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.
SpeI  (1103)
1 site		 A C T A G T T G A T C A
SfiI  (1116)
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  (1123)
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  (1175)
1 site		 G T T A A C C A A T T G
BspQI  (1238)
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  (1238)
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.
PciI  (1354)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BseYI  (1658)
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.
PspFI  (1662)
1 site		 C C C A G C G G G T C G
F1
20-mer  /  50% GC
1 binding site
40 .. 59  =  20 annealed bases
Tm  =  54°C
Forward primer for gene deletion. This primer includes a BamHI recognition sequence. A gene-specific sequence should be added at the 5' end of the primer.
R1
20-mer  /  40% GC
1 binding site
1065 .. 1084  =  20 annealed bases
Tm  =  53°C
Reverse primer for gene deletion or C-terminal tagging. This primer includes an EcoRI recognition sequence. A gene-specific sequence should be added at the 5' end of the primer.
bleMX6
113 .. 1034  =  922 bp
yeast selectable marker conferring phleomycin resistance
bleMX6
113 .. 1034  =  922 bp
yeast selectable marker conferring phleomycin resistance
AmpR
2174 .. 3034  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   2174 .. 2965  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2174 .. 3034  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   2966 .. 3034  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2174 .. 3034  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1415 .. 2003  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1415 .. 2003  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3035 .. 3139  =  105 bp
AmpR promoter
3035 .. 3139  =  105 bp
T7 promoter
1139 .. 1157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1139 .. 1157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
3485 .. 3503  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3485 .. 3503  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
BleoR
457 .. 831  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
BleoR
457 .. 831  =  375 bp
124 amino acids  =  13.8 kDa
Product: antibiotic-binding protein
confers resistance to bleomycin, phleomycin, and Zeocin™
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
837 .. 1034  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
837 .. 1034  =  198 bp
Ashbya gossypii TEF terminator
ORF:  457 .. 831  =  375 bp
ORF:  124 amino acids  =  13.8 kDa
ORF:  2304 .. 2570  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3497 .. 227  =  234 bp
ORF:  77 amino acids  =  8.3 kDa
ORF:  435 .. 737  =  303 bp
ORF:  100 amino acids  =  10.8 kDa
ORF:  2174 .. 3034  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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Download pFA6a-bleMX6.dna file

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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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