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pET-46 Ek/LIC (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC) to express 6xHis-tagged proteins with an enterokinase site.

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pET-46 Ek_LIC (linearized) Sequence and MappET-46 Ek_LIC (linearized).dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 BlpI (5079) DraIII (4757) PsiI (4629) SspI (4549) AhdI (4345) BsaI (4279) BglI (4227) FspI (4122) PvuI (3976) ScaI (3864) AlwNI (3151) PciI (2735) BspQI - SapI (2619) BstZ17I (2506) AvrII (5096) PacI (5106) AvaI - BsoBI - PaeR7I - XhoI (5175) BmeT110I (5176) End (5184) Start (0) enterokinase site PmlI (19) NcoI (40) ATG XbaI (79) T7 promoter BspDI * - ClaI * (148) SgrAI (190) SphI (346) EcoNI (406) BstAPI (554) MluI (871) BclI * (885) BstEII (1052) PspOMI (1078) ApaI (1082) BssHII (1282) HincII - HpaI (1377) PpuMI (1741) Bpu10I (1841) PflFI - Tth111I (2480) pET-46 Ek/LIC 5184 bp
BlpI  (5079)
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  (4757)
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  (4629)
1 site
T T A T A A A A T A T T
SspI  (4549)
1 site
A A T A T T T T A T A A
AhdI  (4345)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
BsaI  (4279)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BglI  (4227)
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.
FspI  (4122)
1 site
T G C G C A A C G C G T
PvuI  (3976)
1 site
C G A T C G G C T A G C
ScaI  (3864)
1 site
A G T A C T T C A T G A
AlwNI  (3151)
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.
PciI  (2735)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2619)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2619)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (2506)
1 site
G T A T A C C A T A T G
AvrII  (5096)
1 site
C C T A G G G G A T C C
PacI  (5106)
1 site
T T A A T T A A A A T T A A T T
AvaI  (5175)
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  (5175)
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  (5175)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (5175)
1 site
C T C G A G G A G C T C
BmeT110I  (5176)
1 site
C Y C G R G G R G C Y C
End  (5184)
0 sites
Start  (0)
0 sites
PmlI  (19)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NcoI  (40)
1 site
C C A T G G G G T A C C
XbaI  (79)
1 site
T C T A G A A G A T C T
BspDI  (148)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (148)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (190)
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  (346)
1 site
G C A T G C C G T A C G
EcoNI  (406)
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  (554)
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  (871)
1 site
A C G C G T T G C G C A
BclI  (885)
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  (1052)
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.
PspOMI  (1078)
1 site
G G G C C C C C C G G G
ApaI  (1082)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (1282)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
HincII  (1377)
1 site
G T Y R A C C A R Y T G
HpaI  (1377)
1 site
G T T A A C C A A T T G
PpuMI  (1741)
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.
Bpu10I  (1841)
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.
PflFI  (2480)
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  (2480)
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.
lacI
521 .. 1603  =  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
521 .. 1603  =  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).
AmpR
3558 .. 4418  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3558 .. 3626  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3558 .. 4418  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3627 .. 4418  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3558 .. 4418  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2796 .. 3384  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2796 .. 3384  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4533 .. 4988  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4533 .. 4988  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
2175 .. 2366  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
2175 .. 2366  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
lacI promoter
443 .. 520  =  78 bp
lacI promoter
443 .. 520  =  78 bp
T7 terminator
5025 .. 5072  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
5025 .. 5072  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
S-Tag
5124 .. 5168  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
5124 .. 5168  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
87 .. 111  =  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
87 .. 111  =  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
112 .. 130  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
112 .. 130  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
20 .. 37  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
20 .. 37  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
2 .. 16  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
2 .. 16  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
51 .. 56  =  6 bp
ribosome binding site
RBS
51 .. 56  =  6 bp
ribosome binding site
ATG
41 .. 43  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
41 .. 43  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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