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Plasmid Files

pFA6a-hphMX6

Plasmid for yeast gene deletion using the hphMX6 selectable marker conferring hygromycin resistance.

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pFA6a-hphMX6 Sequence and MappFA6a-hphMX6.dna
Map and Sequence File   
Sequence Author:  EUROSCARF
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 PvuII (13) PfoI (3934) EcoO109I (3877) SspI (3705) XmnI (3500) BmrI (2941) AlwNI (2424) HindIII (17) BsiWI (23) SalI (35) AccI (36) F1 (40 .. 59) BamHI (41) AvaI - BsoBI - TspMI - XmaI (46) BmeT110I (47) SmaI (48) PacI (56) AscI - BssHII (61) BglII (68) BstEII (98) BstXI (115) BmgBI (151) Bpu10I (160) BseRI (262) MluI (315) PshAI (487) EcoRI (703) AsiSI (824) RsrII (868) BtgZI (1132) BsmI (1675) PmeI (1722) Eco53kI (1729) BanII - SacI (1731) EcoRI (1733) R1 (1719 .. 1738) BspDI - ClaI (1740) EcoRV (1747) SpeI (1757) SfiI (1770) HpaI (1829) BspQI - SapI (1892) PciI (2008) pFA6a-hphMX6 4157 bp
PvuII  (13)
1 site
C A G C T G G T C G A C
PfoI  (3934)
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  (3877)
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  (3705)
1 site
A A T A T T T T A T A A
XmnI  (3500)
1 site
G A A N N N N T T C C T T N N N N A A G
BmrI  (2941)
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  (2424)
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.
AvaI  (46)
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  (46)
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  (46)
1 site
C C C G G G G G G C C C
XmaI  (46)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
BmeT110I  (47)
1 site
C Y C G R G G R G C Y C
SmaI  (48)
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  (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
BssHII  (61)
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.
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.
BmgBI  (151)
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  (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.
BseRI  (262)
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.
MluI  (315)
1 site
A C G C G T T G C G C A
PshAI  (487)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
EcoRI  (703)
2 sites
G A A T T C C T T A A G
AsiSI  (824)
1 site
G C G A T C G C C G C T A G C G
RsrII  (868)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BtgZI  (1132)
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.
BsmI  (1675)
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  (1722)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1729)
1 site
G A G C T C C T C G A G
BanII  (1731)
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  (1731)
1 site
G A G C T C C T C G A G
EcoRI  (1733)
2 sites
G A A T T C C T T A A G
BspDI  (1740)
1 site
A T C G A T T A G C T A
ClaI  (1740)
1 site
A T C G A T T A G C T A
EcoRV  (1747)
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  (1757)
1 site
A C T A G T T G A T C A
SfiI  (1770)
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.
HpaI  (1829)
1 site
G T T A A C C A A T T G
BspQI  (1892)
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  (1892)
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  (2008)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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
1719 .. 1738  =  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.
hphMX6
113 .. 1688  =  1576 bp
yeast selectable marker conferring hygromycin
resistance
hphMX6
113 .. 1688  =  1576 bp
yeast selectable marker conferring hygromycin
resistance
AmpR
2828 .. 3688  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2828 .. 3619  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2828 .. 3688  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3620 .. 3688  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2828 .. 3688  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2069 .. 2657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2069 .. 2657  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
AmpR promoter
3689 .. 3793  =  105 bp
AmpR promoter
3689 .. 3793  =  105 bp
T7 promoter
1793 .. 1811  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1793 .. 1811  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4139 .. 4157  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4139 .. 4157  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
HygR
457 .. 1485  =  1029 bp
342 amino acids  =  38.1 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
457 .. 1485  =  1029 bp
342 amino acids  =  38.1 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
1491 .. 1688  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1491 .. 1688  =  198 bp
Ashbya gossypii TEF terminator
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