pF3K WG (BYDV)

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

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) T7 promoter 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 - BmeT110I - BsoBI (660) 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
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

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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.
BmeT110I  (660)
1 site
C Y C G R G G R G C Y C
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.
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
ORF:  233 .. 568  =  336 bp
ORF:  111 amino acids  =  12.5 kDa
ORF:  1235 .. 2029  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  1407 .. 1793  =  387 bp
ORF:  128 amino acids  =  14.7 kDa
ORF:  3150 .. 3497  =  348 bp
ORF:  115 amino acids  =  12.4 kDa
ORF:  3259 .. 3585  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  35 .. 289  =  255 bp
ORF:  84 amino acids  =  9.5 kDa
ORF:  1544 .. 1798  =  255 bp
ORF:  84 amino acids  =  9.8 kDa
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