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pET-32 Ek/LIC

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

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pET-32 Ek_LIC Sequence and MappET-32 Ek_LIC.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 BlpI (80) DraIII (5675) PsiI (5547) ScaI (5012) PvuI (4902) PstI (4777) BsaI (4593) AhdI (4532) AlwNI (4055) PciI (3639) BspQI - SapI (3523) BstZ17I (3410) PflFI - Tth111I (3384) 6xHis PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) EcoRV (206) NcoI (211) TspMI - XmaI (228) SmaI - SrfI (230) BseRI (234) enterokinase site Acc65I (251) KpnI (255) BglII (258) BstBI (285) S-Tag thrombin site MscI (368) RsrII (606) RBS XbaI (746) T7 promoter SgrAI (857) SphI (1013) EcoNI (1073) BstAPI (1221) MluI (1538) BclI * (1552) BstEII (1719) PspOMI (1745) ApaI (1749) BssHII (1949) HpaI (2044) PshAI (2383) FspAI (2620) PpuMI (2645) Bpu10I (2745) pET-32 Ek/LIC 5917 bp
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  (5675)
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  (5547)
1 site
T T A T A A A A T A T T
ScaI  (5012)
1 site
A G T A C T T C A T G A
PvuI  (4902)
1 site
C G A T C G G C T A G C
PstI  (4777)
1 site
C T G C A G G A C G T C
BsaI  (4593)
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.
AhdI  (4532)
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.
AlwNI  (4055)
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  (3639)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3523)
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  (3523)
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  (3410)
1 site
G T A T A C C A T A T G
PflFI  (3384)
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  (3384)
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.
PaeR7I  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
HindIII  (173)
1 site
A A G C T T T T C G A A
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
EcoRI  (192)
1 site
G A A T T C C T T A A G
BamHI  (198)
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  (206)
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  (211)
1 site
C C A T G G G G T A C C
TspMI  (228)
1 site
C C C G G G G G G C C C
XmaI  (228)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (230)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (230)
1 site
G C C C G G G C C G G G C C C G
BseRI  (234)
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.
Acc65I  (251)
1 site
G G T A C C C C A T G G
KpnI  (255)
1 site
G G T A C C C C A T G G
BglII  (258)
1 site
A G A T C T T C T A G A
BstBI  (285)
1 site
T T C G A A A A G C T T
MscI  (368)
1 site
T G G C C A A C C G G T
RsrII  (606)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
XbaI  (746)
1 site
T C T A G A A G A T C T
SgrAI  (857)
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  (1013)
1 site
G C A T G C C G T A C G
EcoNI  (1073)
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  (1221)
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  (1538)
1 site
A C G C G T T G C G C A
BclI  (1552)
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  (1719)
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  (1745)
1 site
G G G C C C C C C G G G
ApaI  (1749)
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  (1949)
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.
HpaI  (2044)
1 site
G T T A A C C A A T T G
PshAI  (2383)
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.
FspAI  (2620)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2645)
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  (2745)
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.
lacI
1188 .. 2270  =  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
1188 .. 2270  =  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
4459 .. 5319  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4459 .. 5250  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4459 .. 5319  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5251 .. 5319  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4459 .. 5319  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3700 .. 4288  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3700 .. 4288  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
5451 .. 5906  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
5451 .. 5906  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
TrxA
383 .. 709  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
TrxA
383 .. 709  =  327 bp
109 amino acids  =  11.8 kDa
Product: E. coli thioredoxin
rop
3079 .. 3270  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
3079 .. 3270  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
AmpR promoter
5320 .. 5424  =  105 bp
AmpR promoter
5320 .. 5424  =  105 bp
lacI promoter
1110 .. 1187  =  78 bp
lacI promoter
1110 .. 1187  =  78 bp
MCS
158 .. 216  =  59 bp
multiple cloning site
MCS
158 .. 216  =  59 bp
multiple cloning site
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
266 .. 310  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
S-Tag
266 .. 310  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from
pancreatic ribonuclease A
lac operator
754 .. 778  =  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
754 .. 778  =  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
779 .. 797  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
779 .. 797  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
thrombin site
317 .. 334  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
317 .. 334  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
6xHis
344 .. 361  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
344 .. 361  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
236 .. 250  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
236 .. 250  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
RBS
718 .. 723  =  6 bp
ribosome binding site
RBS
718 .. 723  =  6 bp
ribosome binding site
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