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pET-30 Ek/LIC

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At a Glance

pET-30 Ek-LIC Image made with PlasMapper

Type

Cloning/Expression[1]

Origin of Replication

pBR322, f1

Copy #

~40

Markers

Kanamycin

Link to Sequence

Novagen

Features

T7 Promoter
lac Operator
His Tag
S Tag
MCS
lacI coding sequence

Accession Number




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

Novagen

References

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See Help:References for how to manage references in EcoliWiki.

  1. 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 EcoliWiki page
  2. Aslanidis, C & de Jong, PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18 6069-74 PubMed EcoliWiki page
  3. Haun, RS et al. (1992) Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. BioTechniques 13 515-8 PubMed EcoliWiki page

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