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Plasmid Files

pET-41 Ek/LIC

Bacterial vector for ligation-independent cloning (LIC) to express GST-tagged proteins with an enterokinase site.

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pET-41 Ek_LIC Sequence and MappET-41 Ek_LIC.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 EagI - NotI (182) PaeR7I - PspXI - XhoI (174) 8xHis AvrII (131) BlpI (80) DraIII (5705) PsiI (5577) AsiSI - PvuI (5004) NruI (4661) AcuI (4350) AlwNI (4218) BssS α I (3975) BstZ17I (3573) PflFI - Tth111I (3547) HindIII (189) SalI (195) Eco53kI (204) SacI (206) PstI - SbfI (214) AscI (216) StuI (226) BsrGI (229) EcoRI (235) BamHI (241) EcoRV (251) NcoI (254) SrfI (273) BseRI (277) enterokinase site AgeI (306) Acc65I (309) KpnI (313) BglII (316) S-Tag MfeI (374) thrombin site SacII (407) SpeI (438) SwaI (681) MscI (901) NdeI (1108) RBS XbaI (1146) T7 promoter SgrAI (1257) SphI (1413) BstAPI (1621) MluI (1938) BstEII (2119) NmeAIII (2144) PspOMI (2145) ApaI (2149) HpaI (2444) PpuMI (2808) pET-41 Ek/LIC 5947 bp
EagI  (182)
1 site
C G G C C G G C C G G C
NotI  (182)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (174)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (174)
1 site
V C T C G A G B B G A G C T C V
XhoI  (174)
1 site
C T C G A G G A G C T C
AvrII  (131)
1 site
C C T A G G G G A T C C
BlpI  (80)
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.
DraIII  (5705)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PsiI  (5577)
1 site
T T A T A A A A T A T T
AsiSI  (5004)
1 site
G C G A T C G C C G C T A G C G
PvuI  (5004)
1 site
C G A T C G G C T A G C
NruI  (4661)
1 site
T C G C G A A G C G C T
AcuI  (4350)
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).
AlwNI  (4218)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BssSαI  (3975)
1 site
C A C G A G G T G C T C
BstZ17I  (3573)
1 site
G T A T A C C A T A T G
PflFI  (3547)
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  (3547)
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.
HindIII  (189)
1 site
A A G C T T T T C G A A
SalI  (195)
1 site
G T C G A C C A G C T G
Eco53kI  (204)
1 site
G A G C T C C T C G A G
SacI  (206)
1 site
G A G C T C C T C G A G
PstI  (214)
1 site
C T G C A G G A C G T C
SbfI  (214)
1 site
C C T G C A G G G G A C G T C C
AscI  (216)
1 site
G G C G C G C C C C G C G C G G
StuI  (226)
1 site
A G G C C T T C C G G A
BsrGI  (229)
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.
EcoRI  (235)
1 site
G A A T T C C T T A A G
BamHI  (241)
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.
EcoRV  (251)
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.
NcoI  (254)
1 site
C C A T G G G G T A C C
SrfI  (273)
1 site
G C C C G G G C C G G G C C C G
BseRI  (277)
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.
AgeI  (306)
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.
Acc65I  (309)
1 site
G G T A C C C C A T G G
KpnI  (313)
1 site
G G T A C C C C A T G G
BglII  (316)
1 site
A G A T C T T C T A G A
MfeI  (374)
1 site
C A A T T G G T T A A C
SacII  (407)
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.
SpeI  (438)
1 site
A C T A G T T G A T C A
SwaI  (681)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
MscI  (901)
1 site
T G G C C A A C C G G T
NdeI  (1108)
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.
XbaI  (1146)
1 site
T C T A G A A G A T C T
SgrAI  (1257)
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.
SphI  (1413)
1 site
G C A T G C C G T A C G
BstAPI  (1621)
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  (1938)
1 site
A C G C G T T G C G C A
BstEII  (2119)
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.
NmeAIII  (2144)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PspOMI  (2145)
1 site
G G G C C C C C C G G G
ApaI  (2149)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
HpaI  (2444)
1 site
G T T A A C C A A T T G
PpuMI  (2808)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
1588 .. 2670  =  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
1588 .. 2670  =  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).
KanR
4573 .. 5388  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
4573 .. 5388  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
GST
456 .. 1109  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
456 .. 1109  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
3863 .. 4451  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3863 .. 4451  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5481 .. 5936  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5481 .. 5936  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
3242 .. 3433  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
3242 .. 3433  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
MCS
174 .. 259  =  86 bp
multiple cloning site
MCS
174 .. 259  =  86 bp
multiple cloning site
lacI promoter
1510 .. 1587  =  78 bp
lacI promoter
1510 .. 1587  =  78 bp
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
324 .. 368  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
324 .. 368  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
1154 .. 1178  =  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
1154 .. 1178  =  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).
8xHis
150 .. 173  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
150 .. 173  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
T7 promoter
1179 .. 1197  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1179 .. 1197  =  19 bp
promoter for bacteriophage T7 RNA polymerase
thrombin site
384 .. 401  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
384 .. 401  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
6xHis
411 .. 428  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
411 .. 428  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
279 .. 293  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
279 .. 293  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
1118 .. 1123  =  6 bp
ribosome binding site
RBS
1118 .. 1123  =  6 bp
ribosome binding site
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