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Plasmid Files

pFA6a-bleMX6

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

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pFA6a-bleMX6 Sequence and MappFA6a-bleMX6.dna
Map and Sequence File   
Sequence Author:  EUROSCARF
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 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) 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

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  (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.
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  =  52°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
   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
   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
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