pET-30 Ek/LIC
At a Glance
pET-30 Ek-LIC Image made with PlasMapper
<protect>
| Type |
Cloning/Expression[1] |
|---|---|
| Origin of Replication |
pBR322, f1 |
| Copy # |
~40 |
| Markers |
Kanamycin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
</protect>
Notes
pET-30 Ek/LIC is an Ek/LIC version of pET-30b(+). It is designed for cloning and high-level expression of target proteins fused with the His•Tag and STag coding sequences that are cleavable with enterokinase (Ek) protease[1].
History
Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions[2][3]. LIC takes advantage of the 3’→5’ exonuclease activity of T4 DNA polymerase to create very specific 12 to 15 nucleotide single-stranded overhangs in the vector and the insert, so that the vast majority of annealed products consist of the desired molecules. The annealed LIC vector and insert are transformed into competent E. coli cells, and covalent bonds are formed at the vector-insert junctions within the cell to yield circular plasmid. Directional cloning of the insert is achieved with minimal non-recombinant background, and cloning is so efficient that virtually all of the resulting colonies contain the desired recombinant.
Sources
References
See Help:References for how to manage references in EcoliWiki.
- ↑ 1.0 1.1 LaVallie, ER et al. (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (N.Y.) 11 187-93 PubMed
- ↑ Aslanidis, C & de Jong, PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18 6069-74 PubMed
- ↑ Haun, RS et al. (1992) Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. BioTechniques 13 515-8 PubMed
Sequence
1 ATCCGGATAT AGTTCCTCCT TTCAGCAAAA AACCCCTCAA GACCCGTTTA
51 GAGGCCCCAA GGGGTTATGC TAGTTATTGC TCAGCGGTGG CAGCAGCCAA
101 CTCAGCTTCC TTTCGGGCTT TGTTAGCAGC CGGATCTCAG TGGTGGTGGT
151 GGTGGTGCTC GAGTGCGGCC GCAAGCTTGT CGACGGAGCT CGAATTCGGA
201 TCCGATATCG CCATGGTTGA GGAGAAGCCC GGGCTCTTGT CGTCGTCATC
251 GGTACCCAGA TCTGGGCTGT CCATGTGCTG GCGTTCGAAT TTAGCAGCAG
301 CGGTTTCTTT CATACCAGAA CCGCGTGGCA CCAGACCAGA AGAATGATGA
351 TGATGATGGT GCATATGTAT ATCTCCTTCT TAAAGTTAAA CAAAATTATT
401 TCTAGAGGGG AATTGTTATC CGCTCACAAT TCCCCTATAG TGAGTCGTAT
451 TAATTTCGCG GGATCGAGAT CGATCTCGAT CCTCTACGCC GGACGCATCG
501 TGGCCGGCAT CACCGGCGCC ACAGGTGCGG TTGCTGGCGC CTATATCGCC
551 GACATCACCG ATGGGGAAGA TCGGGCTCGC CACTTCGGGC TCATGAGCGC
601 TTGTTTCGGC GTGGGTATGG TGGCAGGCCC CGTGGCCGGG GGACTGTTGG
651 GCGCCATCTC CTTGCATGCA CCATTCCTTG CGGCGGCGGT GCTCAACGGC
701 CTCAACCTAC TACTGGGCTG CTTCCTAATG CAGGAGTCGC ATAAGGGAGA
751 GCGTCGAGAT CCCGGACACC ATCGAATGGC GCAAAACCTT TCGCGGTATG
801 GCATGATAGC GCCCGGAAGA GAGTCAATTC AGGGTGGTGA ATGTGAAACC
851 AGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG
901 TTTCCCGCGT GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA
951 AAAGTGGAAG CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC
1001 ACAACAACTG GCGGGCAAAC AGTCGTTGCT GATTGGCGTT GCCACCTCCA
1051 GTCTGGCCCT GCACGCGCCG TCGCAAATTG TCGCGGCGAT TAAATCTCGC
1101 GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG AACGAAGCGG
1151 CGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA
1201 GTGGGCTGAT CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG
1251 GAAGCTGCCT GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA
1301 GACACCCATC AACAGTATTA TTTTCTCCCA TGAAGACGGT ACGCGACTGG
1351 GCGTGGAGCA TCTGGTCGCA TTGGGTCACC AGCAAATCGC GCTGTTAGCG
1401 GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG GCTGGCATAA
1451 ATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT
1501 GGAGTGCCAT GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC
1551 ATCGTTCCCA CTGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC
1601 AATGCGCGCC ATTACCGAGT CCGGGCTGCG CGTTGGTGCG GACATCTCGG
1651 TAGTGGGATA CGACGATACC GAAGACAGCT CATGTTATAT CCCGCCGTTA
1701 ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA GCGTGGACCG
1751 CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC
1801 CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC
1851 GCCTCTCCCC GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT
1901 TTCCCGACTG GAAAGCGGGC AGTGAGCGCA ACGCAATTAA TGTAAGTTAG
1951 CTCACTCATT AGGCACCGGG ATCTCGACCG ATGCCCTTGA GAGCCTTCAA
2001 CCCAGTCAGC TCCTTCCGGT GGGCGCGGGG CATGACTATC GTCGCCGCAC
2051 TTATGACTGT CTTCTTTATC ATGCAACTCG TAGGACAGGT GCCGGCAGCG
2101 CTCTGGGTCA TTTTCGGCGA GGACCGCTTT CGCTGGAGCG CGACGATGAT
2151 CGGCCTGTCG CTTGCGGTAT TCGGAATCTT GCACGCCCTC GCTCAAGCCT
2201 TCGTCACTGG TCCCGCCACC AAACGTTTCG GCGAGAAGCA GGCCATTATC
2251 GCCGGCATGG CGGCCCCACG GGTGCGCATG ATCGTGCTCC TGTCGTTGAG
2301 GACCCGGCTA GGCTGGCGGG GTTGCCTTAC TGGTTAGCAG AATGAATCAC
2351 CGATACGCGA GCGAACGTGA AGCGACTGCT GCTGCAAAAC GTCTGCGACC
2401 TGAGCAACAA CATGAATGGT CTTCGGTTTC CGTGTTTCGT AAAGTCTGGA
2451 AACGCGGAAG TCAGCGCCCT GCACCATTAT GTTCCGGATC TGCATCGCAG
2501 GATGCTGCTG GCTACCCTGT GGAACACCTA CATCTGTATT AACGAAGCGC
2551 TGGCATTGAC CCTGAGTGAT TTTTCTCTGG TCCCGCCGCA TCCATACCGC
2601 CAGTTGTTTA CCCTCACAAC GTTCCAGTAA CCGGGCATGT TCATCATCAG
2651 TAACCCGTAT CGTGAGCATC CTCTCTCGTT TCATCGGTAT CATTACCCCC
2701 ATGAACAGAA ATCCCCCTTA CACGGAGGCA TCAGTGACCA AACAGGAAAA
2751 AACCGCCCTT AACATGGCCC GCTTTATCAG AAGCCAGACA TTAACGCTTC
2801 TGGAGAAACT CAACGAGCTG GACGCGGATG AACAGGCAGA CATCTGTGAA
2851 TCGCTTCACG ACCACGCTGA TGAGCTTTAC CGCAGCTGCC TCGCGCGTTT
2901 CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
2951 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG
3001 TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA GCCATGACCC AGTCACGTAG
3051 CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG AGCAGATTGT
3101 ACTGAGAGTG CACCATATAT GCGGTGTGAA ATACCGCACA GATGCGTAAG
3151 GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC
3201 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG
3251 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG
3301 AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG
3351 CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC
3401 TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT
3451 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA
3501 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT
3551 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT
3601 GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG
3651 GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG
3701 GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC
3751 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG
3801 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT
3851 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT
3901 TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC
3951 CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT
4001 TAAGGGATTT TGGTCATGAA CAATAAAACT GTCTGCTTAC ATAAACAGTA
4051 ATACAAGGGG TGTTATGAGC CATATTCAAC GGGAAACGTC TTGCTCTAGG
4101 CCGCGATTAA ATTCCAACAT GGATGCTGAT TTATATGGGT ATAAATGGGC
4151 TCGCGATAAT GTCGGGCAAT CAGGTGCGAC AATCTATCGA TTGTATGGGA
4201 AGCCCGATGC GCCAGAGTTG TTTCTGAAAC ATGGCAAAGG TAGCGTTGCC
4251 AATGATGTTA CAGATGAGAT GGTCAGACTA AACTGGCTGA CGGAATTTAT
4301 GCCTCTTCCG ACCATCAAGC ATTTTATCCG TACTCCTGAT GATGCATGGT
4351 TACTCACCAC TGCGATCCCC GGGAAAACAG CATTCCAGGT ATTAGAAGAA
4401 TATCCTGATT CAGGTGAAAA TATTGTTGAT GCGCTGGCAG TGTTCCTGCG
4451 CCGGTTGCAT TCGATTCCTG TTTGTAATTG TCCTTTTAAC AGCGATCGCG
4501 TATTTCGTCT CGCTCAGGCG CAATCACGAA TGAATAACGG TTTGGTTGAT
4551 GCGAGTGATT TTGATGACGA GCGTAATGGC TGGCCTGTTG AACAAGTCTG
4601 GAAAGAAATG CATAAACTTT TGCCATTCTC ACCGGATTCA GTCGTCACTC
4651 ATGGTGATTT CTCACTTGAT AACCTTATTT TTGACGAGGG GAAATTAATA
4701 GGTTGTATTG ATGTTGGACG AGTCGGAATC GCAGACCGAT ACCAGGATCT
4751 TGCCATCCTA TGGAACTGCC TCGGTGAGTT TTCTCCTTCA TTACAGAAAC
4801 GGCTTTTTCA AAAATATGGT ATTGATAATC CTGATATGAA TAAATTGCAG
4851 TTTCATTTGA TGCTCGATGA GTTTTTCTAA GAATTAATTC ATGAGCGGAT
4901 ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA
4951 TTTCCCCGAA AAGTGCCACC TGAAATTGTA AACGTTAATA TTTTGTTAAA
5001 ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG
5051 AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG
5101 AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC
5151 CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG
5201 AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA
5251 AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC
5301 GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA
5351 GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC
5401 GCGCTTAATG CGCCGCTACA GGGCGCGTCC CATTCGCCA
