LOCUS       pET-30_Ek_LIC.        5439 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            6xHis- and S-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-30 Ek_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5439)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5439)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..5439
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    158..216
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(236..250)
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(266..310)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(317..334)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(344..361)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(362..364)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(372..394)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(409..433)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(434..452)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        765..842
                     /label=lacI promoter
     CDS             843..1922
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1938..1959
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2734..2922
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3027..3169
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3355..3943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             4065..4877
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(4973..5428)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        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 gcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
      421 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat cgatctcgat
      481 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
      541 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
      601 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
      661 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
      721 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
      781 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
      841 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
      901 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
      961 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     1021 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     1081 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     1141 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     1201 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     1261 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     1321 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     1381 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     1441 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     1501 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     1561 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     1621 ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc gaagacagct
     1681 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     1741 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     1801 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     1861 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1921 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg
     1981 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     2041 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     2101 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     2161 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     2221 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg
     2281 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     2341 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     2401 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     2461 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     2521 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     2581 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     2641 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     2701 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     2761 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     2821 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     2881 cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     2941 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3001 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     3061 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat
     3121 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     3181 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3241 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3301 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3361 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3421 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3481 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3541 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3601 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3661 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3721 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3781 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3841 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3901 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3961 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     4021 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     4081 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     4141 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     4201 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     4261 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     4321 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     4381 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     4441 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     4501 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     4561 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     4621 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     4681 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     4741 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     4801 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     4861 tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag
     4921 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta
     4981 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5041 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5101 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     5161 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     5221 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     5281 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     5341 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     5401 gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
