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Plasmid Files

pFA6a-GFP(S65T)-TRP1

Plasmid with a TRP1 marker for adding a C-terminal GFP tag or truncating a gene.

 
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pFA6a-GFP(S65T)-TRP1.dna
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BsiWI (25) PfoI (4110) EcoO109I (4053) AatII (3999) ZraI (3997) SspI (3881) ScaI (3557) TsoI (3476) PvuI (3447) FspI (3299) NmeAIII (3225) BanI (3025) PciI (2184) PstI (35) SalI (37) F2 (42 .. 61) BamHI (43) AvaI - BsoBI - TspMI - XmaI (48) BmeT110I (49) SmaI (50) PacI (58) NcoI - StyI (225) MscI (230) SnaBI (253) BtgZI (640) BstBI (681) F3 (777 .. 793) AscI - BssHII (778) PsiI (839) BglII (986) BspEI * (1004) BstAPI (1218) XbaI (1223) Bsu36I (1381) BstXI (1437) BsgI (1552) AarI (1572) PmeI (1898) Eco53kI (1905) SacI (1907) EcoRI (1909) R1 (1895 .. 1914) BspDI - ClaI (1916) SpeI (1933) SfiI (1946) SacII (1953) BspQI - SapI (2068) pFA6a-GFP(S65T)-TRP1 4331 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.
PfoI  (4110)
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  (4053)
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.
AatII  (3999)
1 site
G A C G T C C T G C A G
ZraI  (3997)
1 site
G A C G T C C T G C A G
SspI  (3881)
1 site
A A T A T T T T A T A A
ScaI  (3557)
1 site
A G T A C T T C A T G A
TsoI  (3476)
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  (3447)
1 site
C G A T C G G C T A G C
FspI  (3299)
1 site
T G C G C A A C G C G T
NmeAIII  (3225)
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).
BanI  (3025)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
PciI  (2184)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PstI  (35)
1 site
C T G C A G G A C G T C
SalI  (37)
1 site
G T C G A C C A G C T G
BamHI  (43)
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  (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
NcoI  (225)
1 site
C C A T G G G G T A C C
StyI  (225)
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  (230)
1 site
T G G C C A A C C G G T
SnaBI  (253)
1 site
T A C G T A A T G C A T
BtgZI  (640)
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.
BstBI  (681)
1 site
T T C G A A A A G C T T
AscI  (778)
1 site
G G C G C G C C C C G C G C G G
BssHII  (778)
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.
PsiI  (839)
1 site
T T A T A A A A T A T T
BglII  (986)
1 site
A G A T C T T C T A G A
BspEI  (1004)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
BstAPI  (1218)
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.
XbaI  (1223)
1 site
T C T A G A A G A T C T
Bsu36I  (1381)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BstXI  (1437)
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.
BsgI  (1552)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
AarI  (1572)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
PmeI  (1898)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1905)
1 site
G A G C T C C T C G A G
SacI  (1907)
1 site
G A G C T C C T C G A G
EcoRI  (1909)
1 site
G A A T T C C T T A A G
BspDI  (1916)
1 site
A T C G A T T A G C T A
ClaI  (1916)
1 site
A T C G A T T A G C T A
SpeI  (1933)
1 site
A C T A G T T G A T C A
SfiI  (1946)
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  (1953)
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.
BspQI  (2068)
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  (2068)
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.
F3
20-mer  /  55% GC
1 binding site
777 .. 793  =  17 annealed bases
Tm  =  58°C
Forward primer for C-terminal truncation. This primer includes a stop codon and an AscI recognition sequence. A gene-specific sequence should be added at the 5' end of the primer.
R1
20-mer  /  40% GC
1 binding site
1895 .. 1914  =  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.
AmpR
3004 .. 3864  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3004 .. 3795  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3004 .. 3864  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3796 .. 3864  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3004 .. 3864  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GFP(S65T)
64 .. 776  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
GFP(S65T)
64 .. 776  =  713 bp
236 amino acids  =  26.7 kDa
Product: green fluorescent protein, S65T variant
TRP1
1140 .. 1814  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
1140 .. 1814  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2245 .. 2833  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2245 .. 2833  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ADH1 terminator
797 .. 984  =  188 bp
ADH1 terminator
797 .. 984  =  188 bp
TRP1 promoter
992 .. 1139  =  148 bp
TRP1 promoter
992 .. 1139  =  148 bp
AmpR promoter
3865 .. 3969  =  105 bp
AmpR promoter
3865 .. 3969  =  105 bp
T7 promoter
1969 .. 1987  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1969 .. 1987  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4315 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4315 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
ORF:  1390 .. 1671  =  282 bp
ORF:  93 amino acids  =  10.8 kDa
ORF:  3134 .. 3400  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  291 .. 776  =  486 bp
ORF:  161 amino acids  =  18.6 kDa
ORF:  1140 .. 1814  =  675 bp
ORF:  224 amino acids  =  24.1 kDa
ORF:  3004 .. 3864  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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