LOCUS       pET-51b(+)_Ek_LI        5218 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector with an enterokinase site for ligation-independent 
            cloning (LIC) to express proteins tagged with Strep-Tag II.
ACCESSION   .
VERSION     .
KEYWORDS    pET-51b(+) Ek_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5218)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5218)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and KpnI and treat with T4 DNA 
            polymerase plus dTTP.
FEATURES             Location/Qualifiers
     source          1..5218
                     /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(113..139)
                     /label=9xHis
                     /note="9xHis affinity tag"
     CDS             complement(209..223)
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(230..253)
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
     CDS             complement(260..262)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(269..291)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(306..330)
                     /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(331..349)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        662..739
                     /label=lacI promoter
     CDS             740..1819
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1835..1856
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             2394..2582
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    2687..2829
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3015..3603)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3684..3776
                     /label=AmpR promoter
     CDS             3777..4634
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      complement(4752..5207)
                     /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 ggttaattag tggtggtgat
      121 ggtgatgatg gtggtgatga gcggaaattg aggagaagcc cggagctcct gcggccgcaa
      181 gcttgtcgac cgacgtcgga tccggtacct tgtcgtcgtc atctgcaccc ttttcgaact
      241 gcgggtggct ccagcttgcc atggtatatc tccttcttaa agttaaacaa aattatttct
      301 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
      361 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      421 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      481 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      541 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
      601 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
      661 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      721 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
      781 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
      841 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
      901 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
      961 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1021 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1081 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1141 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1201 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1261 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1321 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1381 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1441 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1501 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     1561 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1621 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1681 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1741 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1801 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     1861 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     1921 cgcggggcat gactagcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     1981 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     2041 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     2101 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     2161 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     2221 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     2281 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     2341 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     2401 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     2461 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     2521 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     2581 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     2641 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     2701 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     2761 actgagagtg caccatatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     2821 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2881 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2941 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3001 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3061 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3121 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3181 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3241 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3301 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3361 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3421 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     3481 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3541 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3601 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3661 taagggattt tggtcatgac ctatttgttt atttttctaa atacattcaa atatgtatcc
     3721 gctcatgaga caataaccct gataaatgct tcaatcatga ttgaaaaagg aagagtatga
     3781 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3841 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3901 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3961 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4021 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     4081 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     4141 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     4201 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     4261 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     4321 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     4381 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     4441 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4501 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4561 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4621 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4681 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgccat
     4741 cgtgccgcca gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4801 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4861 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4921 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4981 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     5041 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     5101 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     5161 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
