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Plasmid Files

pF3K WG (BYDV)

Flexi® vector with a kanamycin resistance marker, for cell-free translation in a wheat germ extract.

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pF3K WG (BYDV) Sequence and MappF3K WG (BYDV).dna
Map and Sequence File   
Sequence Author:  Promega
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 AseI (20) AclI (3524) BsaI (3388) ScaI (3201) TatI (3199) MluI (3117) BspEI (2904) BspHI (2860) ApaLI (2454) PciI (2140) AgeI (2072) BstBI (2045) RsrII (1879) NaeI (1865) SP6 promoter T7 promoter BYDV 5' UTR BbvCI - Bpu10I (174) AsiSI - PvuI - SgfI (206) BtgI - NcoI (209) ZraI (367) AatII (369) BpmI (407) DraI - PmeI (573) EcoRI (578) Eco53kI (586) SacI (588) Acc65I (590) KpnI (594) BbsI (606) BamHI (624) AvaI - BsoBI (660) BmeT110I (661) XbaI (703) SnaBI (711) NruI (717) SalI (721) AccI (722) SbfI (731) BlpI (796) EcoO109I (823) BstAPI (1061) XcmI (1179) BclI * (1204) EagI (1269) MscI (1445) FspI (1465) PflFI - Tth111I (1481) BsrDI (1596) NgoMIV (1863) pF3K WG (BYDV) 3684 bp
AseI  (20)
1 site
A T T A A T T A A T T A
AclI  (3524)
1 site
A A C G T T T T G C A A
BsaI  (3388)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
ScaI  (3201)
1 site
A G T A C T T C A T G A
TatI  (3199)
1 site
W G T A C W W C A T G W
MluI  (3117)
1 site
A C G C G T T G C G C A
BspEI  (2904)
1 site
T C C G G A A G G C C T
BspHI  (2860)
1 site
T C A T G A A G T A C T
ApaLI  (2454)
1 site
G T G C A C C A C G T G
PciI  (2140)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2072)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BstBI  (2045)
1 site
T T C G A A A A G C T T
RsrII  (1879)
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.
NaeI  (1865)
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.
BbvCI  (174)
1 site
C C T C A G C G G A G T C G
Bpu10I  (174)
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.
AsiSI  (206)
1 site
G C G A T C G C C G C T A G C G
PvuI  (206)
1 site
C G A T C G G C T A G C
SgfI  (206)
1 site
G C G A T C G C C G C T A G C G
BtgI  (209)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (209)
1 site
C C A T G G G G T A C C
ZraI  (367)
1 site
G A C G T C C T G C A G
AatII  (369)
1 site
G A C G T C C T G C A G
BpmI  (407)
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.
DraI  (573)
1 site
T T T A A A A A A T T T
PmeI  (573)
1 site
G T T T A A A C C A A A T T T G
EcoRI  (578)
1 site
G A A T T C C T T A A G
Eco53kI  (586)
1 site
G A G C T C C T C G A G
SacI  (588)
1 site
G A G C T C C T C G A G
Acc65I  (590)
1 site
G G T A C C C C A T G G
KpnI  (594)
1 site
G G T A C C C C A T G G
BbsI  (606)
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.
BamHI  (624)
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  (660)
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  (660)
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.
BmeT110I  (661)
1 site
C Y C G R G G R G C Y C
XbaI  (703)
1 site
T C T A G A A G A T C T
SnaBI  (711)
1 site
T A C G T A A T G C A T
NruI  (717)
1 site
T C G C G A A G C G C T
SalI  (721)
1 site
G T C G A C C A G C T G
AccI  (722)
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.
SbfI  (731)
1 site
C C T G C A G G G G A C G T C C
BlpI  (796)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
EcoO109I  (823)
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.
BstAPI  (1061)
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.
XcmI  (1179)
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.
BclI  (1204)
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.
EagI  (1269)
1 site
C G G C C G G C C G G C
MscI  (1445)
1 site
T G G C C A A C C G G T
FspI  (1465)
1 site
T G C G C A A C G C G T
PflFI  (1481)
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  (1481)
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.
BsrDI  (1596)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
NgoMIV  (1863)
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.
NeoR/KanR
1235 .. 2029  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1235 .. 2029  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
2201 .. 2789  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2201 .. 2789  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
barnase
233 .. 568  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
233 .. 568  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
cer region
2905 .. 3188  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
2905 .. 3188  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
BYDV 5' UTR
65 .. 201  =  137 bp
barley yellow dwarf virus 5' untranslated region;
required for function of the cap-independent 3'
translation enhancer (Guo et al., 2001)
BYDV 5' UTR
65 .. 201  =  137 bp
barley yellow dwarf virus 5' untranslated region;
required for function of the cap-independent 3'
translation enhancer (Guo et al., 2001)
BYDV 3' TE
596 .. 702  =  107 bp
cap-independent 3' translation enhancer from barley
yellow dwarf virus (Wang et al., 1997)
BYDV 3' TE
596 .. 702  =  107 bp
cap-independent 3' translation enhancer from barley
yellow dwarf virus (Wang et al., 1997)
rrnB T1 terminator
3418 .. 3504  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3418 .. 3504  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
T7 terminator
807 .. 854  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
807 .. 854  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
rrnB T2 terminator
3596 .. 3623  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
3596 .. 3623  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
SP6 promoter
23 .. 41  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
23 .. 41  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
T7 promoter
46 .. 63  =  18 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
46 .. 63  =  18 bp
promoter for bacteriophage T7 RNA polymerase
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