pET-32 Ek_LIC

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

Sequence Author: MilliporeSigma (Novagen)

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6xHis BlpI (80) T7 terminator 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) 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) thrombin site 6xHis 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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
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
2 segments
   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
2 segments
   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
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
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
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
thrombin site
317 .. 334  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
317 .. 334  =  18 bp
6 amino acids  =  627.7 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
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
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
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
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
RBS
718 .. 723  =  6 bp
ribosome binding site
RBS
718 .. 723  =  6 bp
ribosome binding site
ORF:  2326 .. 2682  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  3046 .. 3270  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  344 .. 1438  =  1095 bp
ORF:  364 amino acids  =  39.8 kDa
ORF:  2027 .. 2290  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4589 .. 4855  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1311 .. 2270  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  137 .. 709  =  573 bp
ORF:  190 amino acids  =  20.5 kDa
ORF:  761 .. 1012  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  886 .. 1149  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  2053 .. 2304  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4459 .. 5319  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2679 .. 3047  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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