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pET-30 Ek/LIC (linearized)

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

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pET-30 Ek_LIC (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Novagen (EMD Millipore)
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DraIII (4963) PsiI (4835) AsiSI - PvuI (4262) SmaI (4136) TspMI - XmaI (4134) NruI (3919) AcuI (3608) AlwNI (3476) BssS α I (3233) PciI (3060) BspQI - SapI (2944) TatI (2864) BstZ17I (2831) PflFI - Tth111I (2805) BlpI (5285) PaeR7I - PspXI - XhoI (5363) EagI - NotI (5371) HindIII (5378) SalI (5384) Eco53kI (5393) SacI (5395) EcoRI (5397) BamHI (5403) EcoRV (5411) NcoI (5416) End (5423) Start (0) Acc65I (17) KpnI (21) BglII (24) BstBI (51) NdeI (129) XbaI (167) SgrAI (278) SphI (434) BstAPI (642) MluI (959) BclI * (973) BstEII (1140) NmeAIII (1165) PspOMI (1166) ApaI (1170) BssHII (1370) HpaI (1465) PshAI (1804) BglI (2023) FspI - FspAI (2041) PpuMI (2066) pET-30 Ek/LIC 5423 bp
DraIII  (4963)
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  (4835)
1 site
T T A T A A A A T A T T
AsiSI  (4262)
1 site
G C G A T C G C C G C T A G C G
PvuI  (4262)
1 site
C G A T C G G C T A G C
SmaI  (4136)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (4134)
1 site
C C C G G G G G G C C C
XmaI  (4134)
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.
NruI  (3919)
1 site
T C G C G A A G C G C T
AcuI  (3608)
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  (3476)
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  (3233)
1 site
C A C G A G G T G C T C
PciI  (3060)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2944)
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  (2944)
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.
TatI  (2864)
1 site
W G T A C W W C A T G W
BstZ17I  (2831)
1 site
G T A T A C C A T A T G
PflFI  (2805)
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  (2805)
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.
BlpI  (5285)
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.
PaeR7I  (5363)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5363)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5363)
1 site
C T C G A G G A G C T C
EagI  (5371)
1 site
C G G C C G G C C G G C
NotI  (5371)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5378)
1 site
A A G C T T T T C G A A
SalI  (5384)
1 site
G T C G A C C A G C T G
Eco53kI  (5393)
1 site
G A G C T C C T C G A G
SacI  (5395)
1 site
G A G C T C C T C G A G
EcoRI  (5397)
1 site
G A A T T C C T T A A G
BamHI  (5403)
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  (5411)
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  (5416)
1 site
C C A T G G G G T A C C
End  (5423)
0 sites
Start  (0)
0 sites
Acc65I  (17)
1 site
G G T A C C C C A T G G
KpnI  (21)
1 site
G G T A C C C C A T G G
BglII  (24)
1 site
A G A T C T T C T A G A
BstBI  (51)
1 site
T T C G A A A A G C T T
NdeI  (129)
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  (167)
1 site
T C T A G A A G A T C T
SgrAI  (278)
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  (434)
1 site
G C A T G C C G T A C G
BstAPI  (642)
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  (959)
1 site
A C G C G T T G C G C A
BclI  (973)
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  (1140)
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  (1165)
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  (1166)
1 site
G G G C C C C C C G G G
ApaI  (1170)
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  (1370)
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  (1465)
1 site
G T T A A C C A A T T G
PshAI  (1804)
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.
BglI  (2023)
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  (2041)
1 site
T G C G C A A C G C G T
FspAI  (2041)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2066)
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
609 .. 1691  =  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
609 .. 1691  =  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
3831 .. 4646  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
3831 .. 4646  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
ori
3121 .. 3709  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3121 .. 3709  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4739 .. 5194  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4739 .. 5194  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
2500 .. 2691  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
2500 .. 2691  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
lacI promoter
531 .. 608  =  78 bp
lacI promoter
531 .. 608  =  78 bp
MCS
5363 .. 5421  =  59 bp
multiple cloning site
MCS
5363 .. 5421  =  59 bp
multiple cloning site
T7 terminator
5231 .. 5278  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5231 .. 5278  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
S-Tag
32 .. 76  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
32 .. 76  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
lac operator
175 .. 199  =  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
175 .. 199  =  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
200 .. 218  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
200 .. 218  =  19 bp
promoter for bacteriophage T7 RNA polymerase
thrombin site
83 .. 100  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
83 .. 100  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
6xHis
110 .. 127  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
110 .. 127  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5345 .. 5362  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5345 .. 5362  =  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
139 .. 144  =  6 bp
ribosome binding site
RBS
139 .. 144  =  6 bp
ribosome binding site
ATG
128 .. 130  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
128 .. 130  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  1747 .. 2103  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  2467 .. 2691  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  2 .. 859  =  858 bp
ORF:  285 amino acids  =  30.5 kDa  (no start codon)
ORF:  1448 .. 1711  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  732 .. 1691  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3831 .. 4646  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  182 .. 433  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
ORF:  307 .. 570  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1474 .. 1725  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  2100 .. 2468  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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