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pET-43.1 Ek/LIC

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At a Glance

pET-43.1 Ek/LIC, Image made with PlasMapper

Type

Cloning/Expression

Origin of Replication

pBR322, fl

Copy #

~40

Markers

Ampicillin

Link to Sequence

Novagen

Features

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

Accession Number




Notes

pET-43.1 Ek/LIC is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides fused with N-terminal Nus•Tag, His•Tag and S•Tag sequences.

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 tcagcggata catatttgaa tgtatttaga aaaataaaca 
      121 aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttaatttgat 
      181 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt 
      241 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga 
      301 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc 
      361 cctactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag 
      421 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt ctgttttatc 
      481 agcctaggaa cgcccaactt aattaacatt agtggtggtg gtggtggtgc tcgagatcct 
      541 ctgggtcttc aggagcgagt tctggctggc ttgcacgtgt atacagctgt gcggccgcaa 
      601 gcttcgaacg cgtatcgatg gtaccgtcga cgtcctgcag gcgcgcctgt acagaattcg 
      661 gatccacgag ctcagatatc gttgaggaga agcccggact cttgtcgtcg tcatcaatcg 
      721 taccagaacc cgcgctgccc cgggggacca gaccagttgg cggtggcgag tccatgtgct 
      781 ggcgttcaaa tttcgcagca gcggtttctt tacccgcgga gtgatggtga tggtgatgac 
      841 cagaaccact agtcgcttcg tcaccgaacc agcaaatatt acgggcagcc ataatcagtg 
      901 ctccggcttt ttcgtcggtc aacccttcga tatcagccag atcatcaatg ccctgttcgg 
      961 cgagatcttc cagcgtacaa acgccacggg cggccagttt gaatgccaaa tcacgatcta 
     1021 ccccttcaag gttcagcaga tcgtcagccg gtttgttatc accgaggctt tcttcctggg 
     1081 cctgtgcaat ggtggccagt gcatttttag cacgctcgcg cagtgcttca acggtcggct 
     1141 catcaaggcc ttcgatttcc aacagctctt tcatcggcac ataggccaat tcttccagcg 
     1201 tcgagaagcc ttcttctacc agaacagtcg cgaagtcttc gtcgatgtcg agatatttgg 
     1261 tgaaggtgtc gatcgctgcg tgcgcttccg cctgatgctt agcttgcagg tcgtcaacgg 
     1321 tcatcacgtt gagttcccaa ccgctcagtt gcgaagccag acgcacgttc tgaccgttac 
     1381 ggccaatcgc ctgcgccaga ttaccggctt caacggcgat gtccatggtg tgtttatctt 
     1441 catccaccac gatagaagca acgtctgccg gtgccattgc gttaatcacg aactgcgccg 
     1501 ggttatcatc ccacaggacg atatcgatac gctcgccacc cagttcagta gacaccgcct 
     1561 gaacacgcgc gccacgcata cctacgcaag cacctaccgg atcgatacgt ttatcgttgg 
     1621 ttttcaccgc gattttcgca cgagaacccg gatcgcgagc cgctgcttta atttcaatca 
     1681 cttcttcgcc gatttctggc acttcaatac ggaacagttc gatcagcatt tccggcttgg 
     1741 aacgagtgac gaacagttgc gcgccacgcg cttccgggcg aacggaatag agcacgccac 
     1801 gaacgcggtc gccagggcgg aagttttcac gcggcagcat atcttcgcgc aggatcacgg 
     1861 cttcagcgtt gttgcccaga tccagagaga tgttgtcgcg gtttactttt ttcaccacgc 
     1921 cggtgatgat ttcaccttcg tgttcacgga actgatcaac caccatcgca cgttcggctt 
     1981 cacgcacttt ctgcacgata acctgttttg ccgtctgggt agtgatacgg tcaaaggtaa 
     2041 cagactcaat ctgatcttca acgtaatcgc ccaggttcag gctttcatct tcataacgtg 
     2101 cggcttcaag ggtgatttcc ttggtcggct gggtgacttc atcaacaact aaccagcgac 
     2161 ggaaagtgtc aaaatcaccg cttttgcgat cgatctgtac gcggacgtcg atctcttgtt 
     2221 catatttttt ctttgttgct gtcgccagcg cgctttccaa tgcttcgaaa atcttctcgc 
     2281 gaggtagcgc cttttcattg gatacggctt caactacagc caaaatttct ttgttcatat 
     2341 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca 
     2401 caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta 
     2461 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat 
     2521 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt 
     2581 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca 
     2641 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct 
     2701 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa 
     2761 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga 
     2821 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc 
     2881 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga 
     2941 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt 
     3001 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg 
     3061 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa 
     3121 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc 
     3181 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta 
     3241 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct 
     3301 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa 
     3361 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc 
     3421 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg 
     3481 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga 
     3541 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc 
     3601 tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt 
     3661 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg 
     3721 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct 
     3781 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt 
     3841 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 
     3901 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc 
     3961 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tagcatgatc 
     4021 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat 
     4081 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga 
     4141 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca 
     4201 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga 
     4261 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc 
     4321 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca 
     4381 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg 
     4441 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac 
     4501 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac 
     4561 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc 
     4621 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 
     4681 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 
     4741 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg 
     4801 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatatgcg 
     4861 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 
     4921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 
     4981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 
     5041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 
     5101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 
     5161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 
     5221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 
     5281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 
     5341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 
     5401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 
     5461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 
     5521 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 
     5581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 
     5641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 
     5701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaccta 
     5761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 
     5821 aaatgcttca atcatgattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 
     5881 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 
     5941 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 
     6001 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 
     6061 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 
     6121 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 
     6181 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 
     6241 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 
     6301 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 
     6361 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 
     6421 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 
     6481 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 
     6541 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 
     6601 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 
     6661 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 
     6721 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 
     6781 atctaggtga agatcctttt tgataatctc atgccatcgt gccgccagaa attgtaaacg 
     6841 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 
     6901 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 
     6961 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 
     7021 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 
     7081 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag 
     7141 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 
     7201 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 
     7261 ttaatgcgcc gctacagggc gcgtcccatt cgcca