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pCDF-2 Ek/LIC (linearized)

Linearized bacterial vector with a CloDF13 (CDF) origin for ligation-independent cloning (LIC) to express 6xHis- and S-tagged proteins with an enterokinase site.

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pCDF-2 Ek_LIC (linearized) Sequence and MappCDF-2 Ek_LIC (linearized).dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 T7 promoter EcoNI (3419) PfoI (3383) AclI (3291) BstAPI (3275) MluI (2951) ApaI - BanII (2748) PspOMI (2744) HincII - HpaI (2449) BsmBI (2336) PluTI (2316) SfoI (2314) NarI * (2313) KasI (2312) XbaI (2031) NcoI (3515) ATG PmlI (3540) End (3546) enterokinase site Start (0) AvaI - BsoBI - PaeR7I - XhoI (19) BmeT110I (20) AccI (76) PacI (94) AvrII (98) EcoO109I (143) Bsu36I (182) AgeI (231) DrdI - PflFI - Tth111I (291) NgoMIV (484) NaeI (486) BglI (508) PvuI (758) BfuAI - BspMI (786) SspI (1153) BspHI (1184) NheI (1231) BmtI (1235) PciI (1331) NspI (1335) SgrAI (1358) pCDF-2 Ek/LIC 3546 bp
EcoNI  (3419)
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  (3383)
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  (3291)
1 site
A A C G T T T T G C A A
BstAPI  (3275)
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  (2951)
1 site
A C G C G T T G C G C A
ApaI  (2748)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BanII  (2748)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
PspOMI  (2744)
1 site
G G G C C C C C C G G G
HincII  (2449)
1 site
G T Y R A C C A R Y T G
HpaI  (2449)
1 site
G T T A A C C A A T T G
BsmBI  (2336)
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  (2316)
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  (2314)
1 site
G G C G C C C C G C G G
NarI  (2313)
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  (2312)
1 site
G G C G C C C C G C G G
XbaI  (2031)
1 site
T C T A G A A G A T C T
NcoI  (3515)
1 site
C C A T G G G G T A C C
PmlI  (3540)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
End  (3546)
0 sites
Start  (0)
0 sites
AvaI  (19)
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  (19)
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  (19)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (19)
1 site
C T C G A G G A G C T C
BmeT110I  (20)
1 site
C Y C G R G G R G C Y C
AccI  (76)
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.
PacI  (94)
1 site
T T A A T T A A A A T T A A T T
AvrII  (98)
1 site
C C T A G G G G A T C C
EcoO109I  (143)
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  (182)
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  (231)
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  (291)
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  (291)
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  (291)
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.
NgoMIV  (484)
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  (486)
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.
BglI  (508)
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.
PvuI  (758)
1 site
C G A T C G G C T A G C
BfuAI  (786)
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  (786)
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.
SspI  (1153)
1 site
A A T A T T T T A T A A
BspHI  (1184)
1 site
T C A T G A A G T A C T
NheI  (1231)
1 site
G C T A G C C G A T C G
BmtI  (1235)
1 site
G C T A G C C G A T C G
PciI  (1331)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1335)
1 site
R C A T G Y Y G T A C R
SgrAI  (1358)
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.
lacI
2224 .. 3306  =  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
2224 .. 3306  =  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
345 .. 1136  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
345 .. 1136  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
CloDF13 ori
1276 .. 2014  =  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
1276 .. 2014  =  739 bp
Plasmids containing the CloDF13 (CDF) origin of
replication can be propagated in E. coli cells that
contain additional plasmids with compatible origins.
AmpR promoter
1137 .. 1228  =  92 bp
AmpR promoter
1137 .. 1228  =  92 bp
lacI promoter
3307 .. 3384  =  78 bp
lacI promoter
3307 .. 3384  =  78 bp
T7 terminator
127 .. 174  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
127 .. 174  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
31 .. 75  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
31 .. 75  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
3449 .. 3473  =  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
3449 .. 3473  =  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
3430 .. 3448  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3430 .. 3448  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3523 .. 3540  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3523 .. 3540  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
3544 .. 3558  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
3544 .. 3558  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
3504 .. 3509  =  6 bp
ribosome binding site
RBS
3504 .. 3509  =  6 bp
ribosome binding site
ATG
3517 .. 3519  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3517 .. 3519  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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