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pET-41 Ek/LIC

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At a Glance

pET-41 Ek/LIC, Image made with PlasMapper

Type

Cloning/Expression

Origin of Replication

f1, pBR322

Copy #

~40

Markers

Kanamycin

Link to Sequence

Novagen

Features

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

Accession Number




Notes

pET-41 is used for rapid, directional cloning of PCR-amplified DNA for high level expression of polypeptides fused with N-terminal GST•Tag, His•Tag, and S•Tag sequences. Using specifically designed primers for amplification, inserts can be efficiently cloned without the need for restriction digestion or ligation.

History

Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions[1][2]. 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. Aslanidis, C & de Jong, PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18 6069-74 PubMed EcoliWiki page
  2. 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 gaggccccaa 
      61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 
     121 tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg gtgctcgagt 
     181 gcggccgcaa gcttgtcgac ggagctcgcc tgcaggcgcg ccaaggcctg tacagaattc 
     241 ggatccccga tatccatggt tgaggagaag cccgggctct tgtcgtcgtc atcaccagaa 
     301 ccaccaccgg tacccagatc tgggctgtcc atgtgctggc gttcgaattt agcagcagcg 
     361 gtttctttca taccaattgc agtactaccg cgtggcacca gacccgcgga gtgatggtga 
     421 tggtgatgac cagaaccact agttgaacca tccgattttg gaggatggtc gccaccacca 
     481 aacgtggctt gccagccctg caaaggccat gctatatact tgctggattt caagtactta 
     541 tcaatttgtg ggatagcttc aatacgtttt ttaaaacaaa ctaattttgg gaacgcatcc 
     601 aggcacattg ggtccatgta taaaacaaca tcaagagcgt catacaacat gaagtcagga 
     661 tgggttacat gatcaccatt taaatatgtt ttatgacata aacgatcttc gaacattttc 
     721 agcatttcag gtagcttgct aagaaaatca actttgagag tttcaaagtc tttactatat 
     781 gcaattctcg aaacaccgta tctaatatcc aaaaccgctc cttcaagcat tgaaatctct 
     841 gcacgctctt ttggacaacc acccaacatg ttgtgcttgt cagctatata acgtatgatg 
     901 gccatagact gtgttaattt aacatcacca tcaatataat aaggaagatt gggaaactcc 
     961 aaacccaatt caaacttttt gtttcgccat ttatcacctt catcgcgctc atacaaatgc 
    1021 tcttcatatt tttcttcaag atattccaaa agaagtcgag tgggttgcac aaggccctta 
    1081 attttccaat aacctagtat aggggacata tgtatatctc cttcttaaag ttaaacaaaa 
    1141 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt 
    1201 tcgcgggatc gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg 
    1261 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg 
    1321 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg 
    1381 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca 
    1441 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc 
    1501 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg 
    1561 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag 
    1621 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct 
    1681 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc 
    1741 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg 
    1801 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt 
    1861 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg 
    1921 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag 
    1981 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct 
    2041 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg 
    2101 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct 
    2161 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag 
    2221 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa 
    2281 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg 
    2341 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg 
    2401 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag 
    2461 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag 
    2521 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg 
    2581 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 
    2641 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac 
    2701 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt 
    2761 ccggtgggcg cggggcatga ctagcatgat cgtgctcctg tcgttgagga cccggctagg 
    2821 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag 
    2881 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg 
    2941 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg 
    3001 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg 
    3061 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc 
    3121 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct 
    3181 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc 
    3241 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt 
    3301 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc 
    3361 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg 
    3421 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 
    3481 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 
    3541 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 
    3601 cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga 
    3661 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 
    3721 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 
    3781 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 
    3841 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 
    3901 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 
    3961 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 
    4021 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 
    4081 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 
    4141 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 
    4201 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 
    4261 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 
    4321 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 
    4381 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 
    4441 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 
    4501 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat 
    4561 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat 
    4621 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca 
    4681 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat 
    4741 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg 
    4801 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta 
    4861 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca 
    4921 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt 
    4981 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg 
    5041 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa 
    5101 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat 
    5161 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat 
    5221 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc 
    5281 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct 
    5341 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat 
    5401 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 
    5461 tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa 
    5521 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 
    5581 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 
    5641 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 
    5701 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 
    5761 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 
    5821 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 
    5881 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca 
    5941 ttcgcca