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pET-32 Ek/LIC

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At a Glance

pET-32 Ek/LIC, Image made with PlasMapper

Type

Cloning/Expression[1]

Origin of Replication

pBR322, f1

Copy #

~40

Markers

Ampicillin

Link to Sequence

Novagen

Features

T7 Promoter
Trx Tag
His Tag
S Tag
MCS
lacI coding sequence

Accession Number




Notes

The pET-32 Ek/LIC vector is an Ek/LIC version of pET-32b(+). It is designed for cloning and high-level expression of target proteins fused with the 109aa Trx•Tag thioredoxin protein[1], and His•Tag and STag coding sequences that are cleavable with enterokinase (Ek) protease.

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, thus the 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

You can help EcoliWiki by providing database accessions or, if none are available, the sequence of this plasmid.

      1    atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 
      181 cgacggagct cgaattcgga tccgatatcg ccatggttga ggagaagccc gggctcttgt 
      241 cgtcgtcatc ggtacccaga tctgggctgt ccatgtgctg gcgttcgaat ttagcagcag 
      301 cggtttcttt cataccagaa ccgcgtggca ccagaccaga agaatgatga tgatgatggt 
      361 gcatatggcc agaaccagaa ccggccaggt tagcgtcgag gaactctttc aactgacctt 
      421 tagacagtgc acccactttg gttgccgcca cttcaccgtt tttgaacagc agcagagtcg 
      481 ggataccacg gatgccatat ttcggcgcag tgccagggtt ttgatcgatg ttcagttttg 
      541 caacggtcag tttgccctga tattcgtcag cgatttcatc cagaatcggg gcgatcattt 
      601 tgcacggacc gcaccactct gcccagaaat cgacgaggat cgccccgtcc gctttgagta 
      661 catccgtgtc aaaactgtcg tcagtcaggt gaataatttt atcgctcata tgtatatctc 
      721 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc 
      781 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg 
      841 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat 
      901 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg 
      961 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt 
     1021 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga 
     1081 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg 
     1141 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa 
     1201 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga 
     1261 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc 
     1321 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg 
     1381 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat 
     1441 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg 
     1501 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta 
     1561 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg 
     1621 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag 
     1681 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt 
     1741 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc 
     1801 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg 
     1861 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg 
     1921 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg 
     1981 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc 
     2041 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 
     2101 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga 
     2161 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 
     2221 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 
     2281 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc 
     2341 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg 
     2401 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc 
     2461 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga 
     2521 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag 
     2581 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg 
     2641 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata 
     2701 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga 
     2761 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc 
     2821 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct 
     2881 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat 
     2941 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc 
     3001 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc 
     3061 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt 
     3121 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag 
     3181 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg 
     3241 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 
     3301 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc 
     3361 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta 
     3421 actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt gtgaaatacc 
     3481 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 
     3541 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 
     3601 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 
     3661 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 
     3721 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 
     3781 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 
     3841 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 
     3901 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 
     3961 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 
     4021 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 
     4081 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 
     4141 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 
     4201 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 
     4261 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 
     4321 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 
     4381 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 
     4441 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 
     4501 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 
     4561 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 
     4621 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 
     4681 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 
     4741 agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc 
     4801 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 
     4861 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 
     4921 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 
     4981 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 
     5041 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 
     5101 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 
     5161 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 
     5221 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 
     5281 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 
     5341 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 
     5401 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa 
     5461 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca 
     5521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag 
     5581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg 
     5641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt 
     5701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag 
     5761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc 
     5821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc 
     5881 gcttaatgcg ccgctacagg gcgcgtccca ttcgcca