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pCDFDuet™-1

Bacterial vector with a CloDF13 (CDF) origin for the co-expression of two genes.

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pCDFDuet-1 Sequence and MappCDFDuet-1.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 BamHI (106) NcoI (69) RBS lac operator T7 promoter EcoNI (3754) ACYCDuetUP1 Primer (3722 .. 3740) PfoI (3718) AclI (3626) BstAPI (3610) MluI (3286) ApaI (3083) PspOMI (3079) HpaI (2784) BsmBI (2671) PluTI (2651) SfoI (2649) NarI * (2648) KasI (2647) XbaI (2366) EcoRI (112) Eco53kI (120) SacI (122) 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 RBS NdeI (298) BglII (305) MfeI (311) EcoRV (319) FseI (328) AsiSI (337) ZraI (344) AatII (346) Acc65I (348) KpnI (352) AvaI - BsoBI - PaeR7I - PspXI - XhoI (354) BmeT110I (355) PacI (429) AvrII (433) T7 Terminator Primer (448 .. 466) EcoO109I (478) T7 terminator Bsu36I (517) AgeI (566) DrdI - PflFI - Tth111I (626) BsaAI (715) BglI (843) SspI (1488) BspHI (1519) NheI (1566) BmtI (1570) PciI (1666) NspI (1670) SgrAI (1693) pCDFDuet™-1 3781 bp
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.
NcoI  (69)
1 site
C C A T G G G G T A C C
EcoNI  (3754)
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.
PfoI  (3718)
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  (3626)
1 site
A A C G T T T T G C A A
BstAPI  (3610)
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  (3286)
1 site
A C G C G T T G C G C A
ApaI  (3083)
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  (3079)
1 site
G G G C C C C C C G G G
HpaI  (2784)
1 site
G T T A A C C A A T T G
BsmBI  (2671)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PluTI  (2651)
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  (2649)
1 site
G G C G C C C C G C G G
NarI  (2648)
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  (2647)
1 site
G G C G C C C C G C G G
XbaI  (2366)
1 site
T C T A G A A G A T C T
EcoRI  (112)
1 site
G A A T T C C T T A A G
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
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

The sticky ends produced by AflII are hard to ligate.
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.
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
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
AvaI  (354)
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  (354)
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.
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
BmeT110I  (355)
1 site
C Y C G R G G R G C Y 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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
DrdI  (626)
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.
PflFI  (626)
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  (626)
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.
BsaAI  (715)
1 site
Y A C G T R R T G C A Y
BglI  (843)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
SspI  (1488)
1 site
A A T A T T T T A T A A
BspHI  (1519)
1 site
T C A T G A A G T A C T
NheI  (1566)
1 site
G C T A G C C G A T C G
BmtI  (1570)
1 site
G C T A G C C G A T C G
PciI  (1666)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1670)
1 site
R C A T G Y Y G T A C R
SgrAI  (1693)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
ACYCDuetUP1 Primer
19-mer  /  63% GC
1 binding site
3722 .. 3740  =  19 annealed bases
Tm  =  60°C
DuetUP2 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  56°C
DuetDOWN1 Primer
20-mer  /  50% GC
1 binding site
189 .. 208  =  20 annealed bases
Tm  =  56°C
T7 Terminator Primer
19-mer  /  53% GC
1 binding site
448 .. 466  =  19 annealed bases
Tm  =  56°C
lacI
2559 .. 3641  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
2559 .. 3641  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
SmR
680 .. 1471  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
confers resistance to spectinomycin and
streptomycin
SmR
680 .. 1471  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
confers resistance to spectinomycin and
streptomycin
CloDF13 ori
1611 .. 2349  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
CloDF13 ori
1611 .. 2349  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
AmpR promoter
1472 .. 1563  =  92 bp
AmpR promoter
1472 .. 1563  =  92 bp
lacI promoter
3642 .. 3719  =  78 bp
lacI promoter
3642 .. 3719  =  78 bp
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
462 .. 509  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
3 .. 27  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
3 .. 27  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
233 .. 257  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
233 .. 257  =  25 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
214 .. 232  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3765 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3765 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
RBS
286 .. 291  =  6 bp
ribosome binding site
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