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Plasmid Files

pCDF-2 Ek/LIC

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 Sequence and MappCDF-2 Ek_LIC.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 T7 promoter EcoNI (3534) PfoI (3498) AclI (3406) BstAPI (3390) MluI (3066) ApaI - BanII (2863) PspOMI (2859) HincII - HpaI (2564) BsmBI (2451) PluTI (2431) SfoI (2429) NarI * (2428) KasI (2427) XbaI (2146) NcoI (69) ATG 6xHis PmlI (94) enterokinase site PshAI (111) BseRI (115) AvaI - BsoBI - PaeR7I - XhoI (134) BmeT110I (135) AccI (191) PacI (209) AvrII (213) EcoO109I (258) Bsu36I (297) AgeI (346) DrdI - PflFI - Tth111I (406) NgoMIV (599) NaeI (601) BglI (623) PvuI (873) BfuAI - BspMI (901) SspI (1268) BspHI (1299) NheI (1346) BmtI (1350) PciI (1446) NspI (1450) SgrAI (1473) pCDF-2 Ek/LIC 3561 bp
EcoNI  (3534)
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  (3498)
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  (3406)
1 site
A A C G T T T T G C A A
BstAPI  (3390)
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  (3066)
1 site
A C G C G T T G C G C A
ApaI  (2863)
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  (2863)
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  (2859)
1 site
G G G C C C C C C G G G
HincII  (2564)
1 site
G T Y R A C C A R Y T G
HpaI  (2564)
1 site
G T T A A C C A A T T G
BsmBI  (2451)
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  (2431)
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  (2429)
1 site
G G C G C C C C G C G G
NarI  (2428)
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  (2427)
1 site
G G C G C C C C G C G G
XbaI  (2146)
1 site
T C T A G A A G A T C T
NcoI  (69)
1 site
C C A T G G G G T A C C
PmlI  (94)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PshAI  (111)
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.
BseRI  (115)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
AvaI  (134)
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  (134)
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  (134)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (134)
1 site
C T C G A G G A G C T C
BmeT110I  (135)
1 site
C Y C G R G G R G C Y C
AccI  (191)
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  (209)
1 site
T T A A T T A A A A T T A A T T
AvrII  (213)
1 site
C C T A G G G G A T C C
EcoO109I  (258)
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  (297)
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  (346)
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  (406)
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  (406)
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  (406)
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  (599)
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  (601)
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  (623)
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  (873)
1 site
C G A T C G G C T A G C
BfuAI  (901)
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  (901)
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  (1268)
1 site
A A T A T T T T A T A A
BspHI  (1299)
1 site
T C A T G A A G T A C T
NheI  (1346)
1 site
G C T A G C C G A T C G
BmtI  (1350)
1 site
G C T A G C C G A T C G
PciI  (1446)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1450)
1 site
R C A T G Y Y G T A C R
SgrAI  (1473)
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
2339 .. 3421  =  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
2339 .. 3421  =  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
460 .. 1251  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
460 .. 1251  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
CloDF13 ori
1391 .. 2129  =  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
1391 .. 2129  =  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
1252 .. 1343  =  92 bp
AmpR promoter
1252 .. 1343  =  92 bp
lacI promoter
3422 .. 3499  =  78 bp
lacI promoter
3422 .. 3499  =  78 bp
T7 terminator
242 .. 289  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
242 .. 289  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
146 .. 190  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
146 .. 190  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
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).
T7 promoter
3545 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3545 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
77 .. 94  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
77 .. 94  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
98 .. 112  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
98 .. 112  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
58 .. 63  =  6 bp
ribosome binding site
RBS
58 .. 63  =  6 bp
ribosome binding site
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
71 .. 73  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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