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Plasmid Files

pFA6a-13Myc-TRP1

Plasmid with a TRP1 marker for adding a C-terminal 13xMyc tag.

 
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 PvuII (15) NdeI (4069) PfoI (3931) EcoO109I (3874) AatII (3820) ZraI (3818) SspI (3702) ScaI (3378) TsoI (3297) PvuI (3268) FspI (3120) NmeAIII (3046) BpmI (2968) BanI (2846) AlwNI (2421) BsiWI (25) PstI (35) SalI (37) F2 (42 .. 61) BamHI (43) AvaI - BsoBI - TspMI - XmaI (48) BmeT110I (49) SmaI (50) PacI (58) PsiI (660) BglII (807) BsrGI (812) BspEI * (825) TRP1 promoter PmlI (920) BstAPI (1039) XbaI (1044) MfeI (1100) Bsu36I (1202) BstXI (1258) BsgI (1373) AarI (1393) PmeI (1719) Eco53kI (1726) SacI (1728) R1 (1716 .. 1735) BspDI - ClaI (1737) SfiI (1767) BtgI (1771) SacII (1774) HpaI (1826) BspQI - SapI (1889) AflIII - PciI (2005) pFA6a-13Myc-TRP1 4152 bp
PvuII  (15)
1 site
C A G C T G G T C G A C
NdeI  (4069)
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.
PfoI  (3931)
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  (3874)
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  (3820)
1 site
G A C G T C C T G C A G
ZraI  (3818)
1 site
G A C G T C C T G C A G
SspI  (3702)
1 site
A A T A T T T T A T A A
ScaI  (3378)
1 site
A G T A C T T C A T G A
TsoI  (3297)
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  (3268)
1 site
C G A T C G G C T A G C
FspI  (3120)
1 site
T G C G C A A C G C G T
NmeAIII  (3046)
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).
BpmI  (2968)
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.
BanI  (2846)
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  (2421)
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.
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.
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
PsiI  (660)
1 site
T T A T A A A A T A T T
BglII  (807)
1 site
A G A T C T T C T A G A
BsrGI  (812)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BspEI  (825)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
PmlI  (920)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstAPI  (1039)
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  (1044)
1 site
T C T A G A A G A T C T
MfeI  (1100)
1 site
C A A T T G G T T A A C
Bsu36I  (1202)
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  (1258)
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  (1373)
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  (1393)
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  (1719)
1 site
G T T T A A A C C A A A T T T G
Eco53kI  (1726)
1 site
G A G C T C C T C G A G
SacI  (1728)
1 site
G A G C T C C T C G A G
BspDI  (1737)
1 site
A T C G A T T A G C T A
ClaI  (1737)
1 site
A T C G A T T A G C T A
SfiI  (1767)
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.
BtgI  (1771)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SacII  (1774)
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  (1826)
1 site
G T T A A C C A A T T G
BspQI  (1889)
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  (1889)
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.
AflIII  (2005)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2005)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
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
1716 .. 1735  =  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
2825 .. 3685  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2825 .. 3616  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2825 .. 3685  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3617 .. 3685  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2825 .. 3685  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TRP1
961 .. 1635  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
TRP1
961 .. 1635  =  675 bp
224 amino acids  =  24.1 kDa
Product: phosphoribosylanthranilate isomerase,
required for tryptophan biosynthesis
yeast auxotrophic marker
ori
2066 .. 2654  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2066 .. 2654  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
13xMyc
66 .. 563  =  498 bp
166 amino acids  =  18.6 kDa
Product: 13 tandem Myc epitope tags
13xMyc
66 .. 563  =  498 bp
166 amino acids  =  18.6 kDa
Product: 13 tandem Myc epitope tags
ADH1 terminator
618 .. 805  =  188 bp
ADH1 terminator
618 .. 805  =  188 bp
TRP1 promoter
813 .. 960  =  148 bp
TRP1 promoter
813 .. 960  =  148 bp
AmpR promoter
3686 .. 3790  =  105 bp
AmpR promoter
3686 .. 3790  =  105 bp
T7 promoter
1790 .. 1808  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1790 .. 1808  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SP6 promoter
4136 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4136 .. 2  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
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