pRSFDuet-1

Bacterial vector with an RSF 1030 origin for the co-expression of two genes.

Sequence Author: MilliporeSigma (Novagen)

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EcoRI (112) BamHI (106) BtgI - NcoI (69) RBS lac operator T7 promoter ACYCDuetUP1 Primer (3770 .. 3788) PfoI (3766) AclI (3674) BstAPI (3658) MluI (3334) BclI * (3320) BstEII (3152) ApaI (3131) PspOMI (3127) HpaI (2832) PluTI (2699) SfoI (2697) NarI * (2696) KasI (2695) AcuI (2474) XbaI (2414) BssSI - BssSαI (1944) Eco53kI (120) SacI (122) BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) SalI (137) HindIII (143) NotI (150) AflII (163) DuetUP2 Primer (189 .. 208) BsrGI - TatI (190) DuetDOWN1 Primer (189 .. 208) T7 promoter lac operator RBS NdeI (298) BglII (305) MfeI (311) EcoRV (319) NgoMIV (324) NaeI (326) FseI (328) AsiSI - PvuI (337) ZraI (344) AatII (346) Acc65I (348) KpnI (352) PaeR7I - PspXI - XhoI (354) PacI (429) AvrII (433) T7 Terminator Primer (448 .. 466) EcoO109I (478) T7 terminator Bsu36I (517) AgeI (566) DrdI (637) BsaI (645) XmnI (733) Bpu10I (1102) TspMI - XmaI (1246) SmaI (1248) BspDI - ClaI (1429) NruI (1465) SphI (1654) AfeI (1658) BspQI - SapI (1666) PciI (1782) pRSFDuet™-1 3829 bp
EcoRI  (112)
1 site
G A A T T C C T T A A G
BamHI  (106)
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.
BtgI  (69)
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  (69)
1 site
C C A T G G G G T A C C
PfoI  (3766)
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.
AclI  (3674)
1 site
A A C G T T T T G C A A
BstAPI  (3658)
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.
MluI  (3334)
1 site
A C G C G T T G C G C A
BclI  (3320)
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.
BstEII  (3152)
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.
ApaI  (3131)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (3127)
1 site
G G G C C C C C C G G G
HpaI  (2832)
1 site
G T T A A C C A A T T G
PluTI  (2699)
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  (2697)
1 site
G G C G C C C C G C G G
NarI  (2696)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (2695)
1 site
G G C G C C C C G C G G
AcuI  (2474)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
XbaI  (2414)
1 site
T C T A G A A G A T C T
BssSI  (1944)
1 site
C A C G A G G T G C T C
BssSαI  (1944)
1 site
C A C G A G G T G C T C
Eco53kI  (120)
1 site
G A G C T C C T C G A G
SacI  (122)
1 site
G A G C T C C T C G A G
BfuAI  (124)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (124)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
AscI  (125)
1 site
G G C G C G C C C C G C G C G G
PstI  (135)
1 site
C T G C A G G A C G T C
SbfI  (135)
1 site
C C T G C A G G G G A C G T C C
SalI  (137)
1 site
G T C G A C C A G C T G
HindIII  (143)
1 site
A A G C T T T T C G A A
NotI  (150)
1 site
G C G G C C G C C G C C G G C G
AflII  (163)
1 site
C T T A A G G A A T T C
BsrGI  (190)
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.
TatI  (190)
1 site
W G T A C W W C A T G W
NdeI  (298)
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.
BglII  (305)
1 site
A G A T C T T C T A G A
MfeI  (311)
1 site
C A A T T G G T T A A C
EcoRV  (319)
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.
NgoMIV  (324)
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.
NaeI  (326)
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.
FseI  (328)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
AsiSI  (337)
1 site
G C G A T C G C C G C T A G C G
PvuI  (337)
1 site
C G A T C G G C T A G C
ZraI  (344)
1 site
G A C G T C C T G C A G
AatII  (346)
1 site
G A C G T C C T G C A G
Acc65I  (348)
1 site
G G T A C C C C A T G G
KpnI  (352)
1 site
G G T A C C C C A T G G
PaeR7I  (354)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (354)
1 site
V C T C G A G B B G A G C T C V
XhoI  (354)
1 site
C T C G A G G A G C T C
PacI  (429)
1 site
T T A A T T A A A A T T A A T T
AvrII  (433)
1 site
C C T A G G G G A T C C
EcoO109I  (478)
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.
Bsu36I  (517)
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.
AgeI  (566)
1 site
A C C G G T T G G C C A
DrdI  (637)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BsaI  (645)
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.
XmnI  (733)
1 site
G A A N N N N T T C C T T N N N N A A G
Bpu10I  (1102)
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.
TspMI  (1246)
1 site
C C C G G G G G G