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pFA6a-natMX6

Plasmid for yeast gene deletion using the natMX6 selectable marker conferring nourseothricin resistance.

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pFA6a-natMX6.dna
Map and Sequence File:    Download    Open   
Sequence Author:  EUROSCARF
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PvuII (13) NdeI (3619) AatII (3370) ZraI (3368) SspI (3252) XmnI (3047) TsoI (2847) PvuI (2818) BmrI (2488) AlwNI (1971) PspFI (1863) HindIII (17) SalI (35) AccI (36) F1 (40 .. 59) BamHI (41) PacI (56) AscI (61) BglII (68) BstXI (115) BmgBI (151) Bpu10I (160) BseRI (262) NcoI (455) AgeI (485) XcmI (528) PpuMI (539) BbsI (552) PflFI - Tth111I (556) PshAI (700) NruI (844) MreI (856) KasI (868) NarI (869) SfoI (870) PluTI (872) SphI (1020) BstAPI (1022) BsmI (1222) PmeI (1269) Eco53kI (1276) SacI (1278) ApoI - EcoRI (1280) R1 (1266 .. 1285) BspDI - ClaI (1287) EcoRV (1294) SpeI (1304) SfiI (1317) SacII (1324) HpaI (1376) BspQI - SapI (1439) PciI (1555) BseYI (1859) pFA6a-natMX6 3704 bp
PvuII  (13)
1 site
C A G C T G G T C G A C
NdeI  (3619)
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.
AatII  (3370)
1 site
G A C G T C C T G C A G
ZraI  (3368)
1 site
G A C G T C C T G C A G
SspI  (3252)
1 site
A A T A T T T T A T A A
XmnI  (3047)
1 site
G A A N N N N T T C C T T N N N N A A G
TsoI  (2847)
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  (2818)
1 site
C G A T C G G C T A G C
BmrI  (2488)
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  (1971)
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  (1863)
1 site
C C C A G C G G G T C G
HindIII  (17)
1 site
A A G C T T T T C G A A
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
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.
NcoI  (455)
1 site
C C A T G G G G T A C C
AgeI  (485)
1 site
A C C G G T T G G C C A
XcmI  (528)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
PpuMI  (539)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BbsI  (552)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
PflFI  (556)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (556)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PshAI  (700)
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.
NruI  (844)
1 site
T C G C G A A G C G C T
MreI  (856)
1 site
C G C C G G C G G C G G C C G C
KasI  (868)
1 site
G G C G C C C C G C G G
NarI  (869)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (870)
1 site
G G C G C C C C G C G G
PluTI  (872)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SphI  (1020)
1 site
G C A T G C C G T A C G
BstAPI  (1022)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BsmI  (1222)
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  (1269)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1276)
1 site
G A G C T C C T C G A G
SacI  (1278)
1 site
G A G C T C C T C G A G
ApoI  (1280)
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  (1280)
1 site
G A A T T C C T T A A G
BspDI  (1287)
1 site
A T C G A T T A G C T A
ClaI  (1287)
1 site
A T C G A T T A G C T A
EcoRV  (1294)
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  (1304)
1 site
A C T A G T T G A T C A
SfiI  (1317)
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  (1324)
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  (1376)
1 site
G T T A A C C A A T T G
BspQI  (1439)
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  (1439)
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  (1555)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1859)
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.
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
1266 .. 1285  =  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.
natMX6
113 .. 1235  =  1123 bp
yeast selectable marker conferring nourseothricin resistance
natMX6
113 .. 1235  =  1123 bp
yeast selectable marker conferring nourseothricin resistance
AmpR
2375 .. 3235  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2375 .. 3166  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2375 .. 3235  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3167 .. 3235  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2375 .. 3235  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1616 .. 2204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1616 .. 2204  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3236 .. 3340  =  105 bp
AmpR promoter
3236 .. 3340  =  105 bp
T7 promoter
1340 .. 1358  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1340 .. 1358  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
3686 .. 3704  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
3686 .. 3704  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
NrsR
457 .. 1032  =  576 bp
191 amino acids  =  20.6 kDa
Product: nourseothricin acetyltransferase
confers resistance to nourseothricin
NrsR
457 .. 1032  =  576 bp
191 amino acids  =  20.6 kDa
Product: nourseothricin acetyltransferase
confers resistance to nourseothricin
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF promoter
113 .. 456  =  344 bp
Ashbya gossypii TEF promoter
TEF terminator
1038 .. 1235  =  198 bp
Ashbya gossypii TEF terminator
TEF terminator
1038 .. 1235  =  198 bp
Ashbya gossypii TEF terminator
ORF:  457 .. 1032  =  576 bp
ORF:  191 amino acids  =  20.6 kDa
ORF:  2505 .. 2771  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3698 .. 227  =  234 bp
ORF:  77 amino acids  =  8.3 kDa
ORF:  483 .. 788  =  306 bp
ORF:  101 amino acids  =  10.5 kDa
ORF:  2375 .. 3235  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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