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Plasmid Files

pACYCDuet™-1

Bacterial vector with a p15A origin for the co-expression of two genes.

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pACYCDuet-1 Sequence and MappACYCDuet-1.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 Eco53kI (120) EcoRI (112) BamHI (106) NcoI (69) RBS lac operator T7 promoter EcoNI (3981) ACYCDuetUP1 Primer (3949 .. 3967) lacI promoter BstAPI (3837) ApaLI (3533) AflIII - MluI (3513) BclI * (3499) BstEII (3331) ApaI (3310) PspOMI (3306) BciVI (3061) HpaI (3011) PluTI (2878) SfoI (2876) NarI * (2875) KasI (2874) AcuI (2653) XbaI (2593) NspI (2330) BssS α I (2128) SacI (122) BfuAI - BspMI (124) AscI (125) PstI - SbfI (135) SalI (137) HindIII (143) NotI (150) AflII (163) DuetUP2 Primer (189 .. 208) BsrGI (190) DuetDOWN1 Primer (189 .. 208) T7 promoter 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) AvaI - BsoBI - PaeR7I - PspXI - XhoI (354) BmeT110I (355) PacI (429) AvrII (433) BlpI (451) T7 Terminator Primer (448 .. 466) EcoO109I (478) T7 terminator Bsu36I (517) DrdI - PflFI - Tth111I (626) ScaI (757) MscI (907) BspEI (1174) Bpu10I (1398) BsaAI (1481) AfeI (1751) NheI (1752) BmtI (1756) BstZ17I (1765) XmnI (1808) SgrAI (1838) SacII (2004) pACYCDuet™-1 4008 bp
Eco53kI  (120)
1 site
G A G C T C C T C G A G
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.
NcoI  (69)
1 site
C C A T G G G G T A C C
EcoNI  (3981)
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.
BstAPI  (3837)
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.
ApaLI  (3533)
1 site
G T G C A C C A C G T G
AflIII  (3513)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (3513)
1 site
A C G C G T T G C G C A
BclI  (3499)
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  (3331)
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  (3310)
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  (3306)
1 site
G G G C C C C C C G G G
BciVI  (3061)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
HpaI  (3011)
1 site
G T T A A C C A A T T G
PluTI  (2878)
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  (2876)
1 site
G G C G C C C C G C G G
NarI  (2875)
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  (2874)
1 site
G G C G C C C C G C G G
AcuI  (2653)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
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  (2593)
1 site
T C T A G A A G A T C T
NspI  (2330)
1 site
R C A T G Y Y G T A C R
BssSαI  (2128)
1 site
C A C G A G G T G C T C
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

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.
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
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
BlpI  (451)
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  (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.
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.
ScaI  (757)
1 site
A G T A C T T C A T G A
MscI  (907)
1 site
T G G C C A A C C G G T
BspEI  (1174)
1 site
T C C G G A A G G C C T
Bpu10I  (1398)
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.
BsaAI  (1481)
1 site
Y A C G T R R T G C A Y
AfeI  (1751)
1 site
A G C G C T T C G C G A
NheI  (1752)
1 site
G C T A G C C G A T C G
BmtI  (1756)
1 site
G C T A G C C G A T C G
BstZ17I  (1765)
1 site
G T A T A C C A T A T G
XmnI  (1808)
1 site
G A A N N N N T T C C T T N N N N A A G
SgrAI  (1838)
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.
SacII  (2004)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
ACYCDuetUP1 Primer
19-mer  /  63% GC
1 binding site
3949 .. 3967  =  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
2786 .. 3868  =  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
2786 .. 3868  =  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).
CmR
729 .. 1388  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
729 .. 1388  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
2017 .. 2561  =  545 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
p15A ori
2017 .. 2561  =  545 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
MCS-2
297 .. 438  =  142 bp
multiple cloning site 2
cat promoter
1389 .. 1491  =  103 bp
promoter of the E. coli cat gene
cat promoter
1389 .. 1491  =  103 bp
promoter of the E. coli cat gene
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
MCS-1
69 .. 168  =  100 bp
multiple cloning site 1
lacI promoter
3869 .. 3946  =  78 bp
lacI promoter
3869 .. 3946  =  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
3992 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3992 .. 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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