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pFGC5941

Agrobacterium binary vector for expressing dsRNA, with two multiple cloning sites separated by an intron.

 
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pFGC5941.dna
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BclI * (11,169) EcoRV (10,289) BstZ17I (9225) PluTI (9030) SfoI (9028) NarI (9027) KasI (9026) EcoNI (8554) BsiWI (8125) Acc65I (407) KpnI (411) FspAI (521) AarI (585) SalI (656) AfeI (758) EcoRI (1317) MluI (1536) StuI (1867) PshAI (2466) PaeR7I - PspXI - XhoI (2653) NcoI (2734) AscI (2741) SwaI (2760) AflII (3659) PmlI (3675) BamHI (4122) AvrII (4126) XbaI (4136) PacI (4149) TspMI - XmaI (4155) SmaI (4157) MfeI (4465) BstEII (4737) AhdI (4867) SbfI (4892) PvuI (5026) PmeI (5113) pFGC5941 11,406 bp
BclI  (11,169)
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.
EcoRV  (10,289)
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.
BstZ17I  (9225)
1 site
G T A T A C C A T A T G
PluTI  (9030)
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.
SfoI  (9028)
1 site
G G C G C C C C G C G G
NarI  (9027)
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.
KasI  (9026)
1 site
G G C G C C C C G C G G
EcoNI  (8554)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BsiWI  (8125)
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.
Acc65I  (407)
1 site
G G T A C C C C A T G G
KpnI  (411)
1 site
G G T A C C C C A T G G
FspAI  (521)
1 site
R T G C G C A Y Y A C G C G T R
AarI  (585)
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.
SalI  (656)
1 site
G T C G A C C A G C T G
AfeI  (758)
1 site
A G C G C T T C G C G A
EcoRI  (1317)
1 site
G A A T T C C T T A A G
MluI  (1536)
1 site
A C G C G T T G C G C A
StuI  (1867)
1 site
A G G C C T T C C G G A
PshAI  (2466)
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.
PaeR7I  (2653)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2653)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2653)
1 site
C T C G A G G A G C T C
NcoI  (2734)
1 site
C C A T G G G G T A C C
AscI  (2741)
1 site
G G C G C G C C C C G C G C G G
SwaI  (2760)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AflII  (3659)
1 site
C T T A A G G A A T T C
PmlI  (3675)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BamHI  (4122)
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.
AvrII  (4126)
1 site
C C T A G G G G A T C C
XbaI  (4136)
1 site
T C T A G A A G A T C T
PacI  (4149)
1 site
T T A A T T A A A A T T A A T T
TspMI  (4155)
1 site
C C C G G G G G G C C C
XmaI  (4155)
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.
SmaI  (4157)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
MfeI  (4465)
1 site
C A A T T G G T T A A C
BstEII  (4737)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
AhdI  (4867)
1 site
G A C N N