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Plasmid Files

pFA6a-3HA-His3MX6

Plasmid with a HIS3MX6 marker for adding a C-terminal triple-HA tag.

 
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 BsiWI (25) HindIII (19) EcoO109I (3814) SspI (3642) XmnI (3437) ScaI (3318) PvuI (3208) NmeAIII (2986) BmrI (2878) BanI (2786) AlwNI (2361) PspFI (2253) BseYI (2249) SalI (37) F2 (42 .. 61) AvaI - BsoBI - TspMI - XmaI (48) BmeT110I (49) SmaI (50) PacI (58) BbvCI (167) AscI (172) BstZ17I (250) BglII (380) BstEII (410) BmgBI (463) BseRI (574) MluI (627) NcoI - StyI (767) BtgZI (810) SphI (895) BclI * (1026) BsaBI (1028) NgoMIV (1096) NaeI (1098) XbaI (1147) AfeI (1335) PmeI (1659) Eco53kI (1666) BanII - SacI (1668) EcoRI (1670) R1 (1656 .. 1675) EcoRV (1684) SfiI (1707) SacII (1714) HpaI (1766) BspQI - SapI (1829) pFA6a-3HA-His3MX6 4092 bp
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.
HindIII  (19)
1 site
A A G C T T T T C G A A
EcoO109I  (3814)
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  (3642)
1 site
A A T A T T T T A T A A
XmnI  (3437)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (3318)
1 site
A G T A C T T C A T G A
PvuI  (3208)
1 site
C G A T C G G C T A G C
NmeAIII  (2986)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BmrI  (2878)
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  (2786)
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  (2361)
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.
PspFI  (2253)
1 site
C C C A G C G G G T C G
BseYI  (2249)
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.
SalI  (37)
1 site
G T C G A C C A G C T G
AvaI  (48)
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  (48)
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  (48)
1 site
C C C G G G G G G C C C
XmaI  (48)
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  (49)
1 site
C Y C G R G G R G C Y C
SmaI  (50)
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  (58)
1 site
T T A A T T A A A A T T A A T T
BbvCI  (167)
1 site
C C T C A G C G G A G T C G
AscI  (172)
1 site
G G C G C G C C C C G C G C G G
BstZ17I  (250)
1 site
G T A T A C C A T A T G
BglII  (380)
1 site
A G A T C T T C T A G A
BstEII  (410)
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  (463)
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.
BseRI  (574)
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  (627)
1 site
A C G C G T T G C G C A
NcoI  (767)
1 site
C C A T G G G G T A C C
StyI  (767)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BtgZI  (810)
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.
SphI  (895)
1 site
G C A T G C C G T A C G
BclI  (1026)
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  (1028)
1 site
G A T N N N N A T C C T A N N N N T A G
NgoMIV  (1096)
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  (1098)
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  (1147)
1 site
T C T A G A A G A T C T
AfeI  (1335)
1 site
A G C G C T T C G C G A
PmeI  (1659)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1666)
1 site
G A G C T C C T C G A G
BanII  (1668)
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  (1668)
1 site
G A G C T C C T C G A G
EcoRI  (1670)
1 site
G A A T T C C T T A A G
EcoRV  (1684)
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.
SfiI  (1707)
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  (1714)
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  (1766)
1 site
G T T A A C C A A T T G
BspQI  (1829)
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  (1829)
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.
F2
20-mer  /  50% GC
1 binding site
42 .. 61  =  20 annealed bases
Tm  =  53°C
Forward primer for C-terminal tagging. 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
1656 .. 1675  =  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.
HIS3MX6
425 .. 1625  =  1201 bp
HIS3MX6
425 .. 1625  =  1201 bp
AmpR
2765 .. 3625  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2765 .. 3556  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2765 .. 3625  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3557 .. 3625  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2765 .. 3625  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2006 .. 2594  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2006 .. 2594  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ADH1 terminator
191 .. 378  =  188 bp
ADH1 terminator
191 .. 378  =  188 bp
AmpR promoter
3626 .. 3730  =  105 bp
AmpR promoter
3626 .. 3730  =  105 bp
3xHA
69 .. 158  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
3xHA
69 .. 158  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
T7 promoter
1730 .. 1748  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1730 .. 1748  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4076 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4076 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
S. pombe his5
769 .. 1422  =  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
769 .. 1422  =  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
425 .. 767  =  343 bp
Ashbya gossypii TEF promoter
TEF promoter
425 .. 767  =  343 bp
Ashbya gossypii TEF promoter
TEF terminator
1428 .. 1625  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1428 .. 1625  =  198 bp
Ashbya gossypii TEF terminator
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