LOCUS       pET-46_Ek_LIC.        5200 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            6xHis-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-46 Ek_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5200)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5200)
  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..5200
                     /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(125..169)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(203..217)
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(221..238)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(242..244)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(251..273)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(288..312)
                     /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(313..331)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        644..721
                     /label=lacI promoter
     CDS             722..1801
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1817..1838
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2376..2564
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    2669..2811
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(2997..3585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3666..3758
                     /label=AmpR promoter
     CDS             3759..4616
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      complement(4734..5189)
                     /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 cagcagccta ggttaattaa gctgcgctag
      121 tagacgagtc catgtgctgg cgttcaaatt tcgcagcagc ggtttcttta ccagactcga
      181 gatttgagga gaagcccgga ctcttgtcgt cgtcatccac gtgatggtgg tggtgatgtg
      241 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
      301 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc
      361 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
      421 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
      481 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
      541 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
      601 ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg
      661 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
      721 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
      781 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
      841 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
      901 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
      961 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     1021 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     1081 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     1141 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     1201 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     1261 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     1321 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     1381 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     1441 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     1501 cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg aagacagctc
     1561 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     1621 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     1681 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     1741 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     1801 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga
     1861 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactagca
     1921 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     1981 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     2041 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     2101 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     2161 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     2221 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     2281 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     2341 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     2401 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     2461 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     2521 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     2581 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     2641 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     2701 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     2761 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     2821 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2881 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2941 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3001 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3061 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3121 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3181 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3241 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3301 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3361 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3421 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3481 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3541 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3601 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3661 acctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3721 ctgataaatg cttcaatcat gattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4441 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4681 aaaggatcta ggtgaagatc ctttttgata atctcatgcc atcgtgccgc cagaaattgt
     4741 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4801 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4861 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4921 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4981 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     5041 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     5101 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     5161 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
