LOCUS       pET-50b(+).        6733 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing proteins with a cleavable N-terminal
            NusA tag and a cleavable C-terminal S-tag.
ACCESSION   .
VERSION     .
KEYWORDS    pET-50b(+)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6733)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6733)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6733
                     /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(168..212)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(213..230)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     misc_feature    234..308
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(309..332)
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
     CDS             complement(342..359)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(375..1859)
                     /label=NusA
                     /note="transcription elongation protein (N utilization
                     substance protein A)"
     CDS             complement(1866..1883)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1893..1895)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(1903..1925)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(1940..1964)
                     /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(1965..1983)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2296..2373
                     /label=lacI promoter
     CDS             2374..3453
                     /label=lacI
                     /note="lac repressor"
     protein_bind    3469..3490
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             4028..4216
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    4321..4463
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4649..5237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             5359..6171
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      complement(6267..6722)
                     /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 tgtttagcag cctaggttaa ttaagcctcg agagcagcag aagtagagct gtccatgtgc
      181 tggcgttcga atttagcagc agcggtttct ttactaccgc gtggcaccag agcgagctct
      241 gcggccgcaa gcttgtcgac ggacgtcggg cgcgccaagg cctgtacaga attcggatcc
      301 tggtacccgg gtccctgaaa gaggacttca agagccgcgg agtgatggtg atggtgatga
      361 ccagaaccac tagtcgcttc gtcaccgaac cagcaaatat tacgggcagc cataatcagt
      421 gctccggctt tttcgtcggt caacccttcg atatcagcca gatcatcaat gccctgttcg
      481 gcgagatctt ccagcgtaca aacgccacgg gcggccagtt tgaatgccaa atcacgatct
      541 accccttcaa ggttcagcag atcgtcagcc ggtttgttat caccgaggct ttcttcctgg
      601 gcctgtgcaa tggtggccag tgcattttta gcacgctcgc gcagtgcttc aacggtcggc
      661 tcatcaaggc cttcgatttc caacagctct ttcatcggca cataggccaa ttcttccagc
      721 gtcgagaagc cttcttctac cagaacagtc gcgaagtctt cgtcgatgtc gagatatttg
      781 gtgaaggtgt cgatcgctgc gtgcgcttcc gcctgatgct tagcttgcag gtcgtcaacg
      841 gtcatcacgt tgagttccca accgctcagt tgcgaagcca gacgcacgtt ctgaccgtta
      901 cggccaatcg cctgcgccag attaccggct tcaacggcga tgtccatggt gtgtttatct
      961 tcatccacca cgatagaagc aacgtctgcc ggtgccattg cgttaatcac gaactgcgcc
     1021 gggttatcat cccacaggac gatatcgata cgctcgccac ccagttcagt agacaccgcc
     1081 tgaacacgcg cgccacgcat acctacgcaa gcacctaccg gatcgatacg tttatcgttg
     1141 gttttcaccg cgattttcgc acgagaaccc ggatcgcgag ccgctgcttt aatttcaatc
     1201 acttcttcgc cgatttctgg cacttcaata cggaacagtt cgatcagcat ttccggcttg
     1261 gaacgagtga cgaacagttg cgcgccacgc gcttccgggc gaacggaata gagcacgcca
     1321 cgaacgcggt cgccagggcg gaagttttca cgcggcagca tatcttcgcg caggatcacg
     1381 gcttcagcgt tgttgcccag atccagagag atgttgtcgc ggtttacttt tttcaccacg
     1441 ccggtgatga tttcaccttc gtgttcacgg aactgatcaa ccaccatcgc acgttcggct
     1501 tcacgcactt tctgcacgat aacctgtttt gccgtctggg tagtgatacg gtcaaaggta
     1561 acagactcaa tctgatcttc aacgtaatcg cccaggttca ggctttcatc ttcataacgt
     1621 gcggcttcaa gggtgatttc cttggtcggc tgggtgactt catcaacaac taaccagcga
     1681 cggaaagtgt caaaatcacc gcttttgcga tcgatctgta cgcggacgtc gatctcttgt
     1741 tcatattttt tctttgttgc tgtcgccagc gcgctttcca atgcttcgaa aatcttctcg
     1801 cgaggtagcg ccttttcatt ggatacggct tcaactacag ccaaaatttc tttgttcata
     1861 ctagaatggt gatggtggtg atggctgctg cccatatgta tatctccttc ttaaagttaa
     1921 acaaaattat ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta
     1981 ttaatttcgc gggatcgaga tcgatctcga tcctctacgc cggacgcatc gtggccggca
     2041 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     2101 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     2161 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     2221 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     2281 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     2341 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     2401 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     2461 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     2521 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     2581 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     2641 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     2701 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     2761 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     2821 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     2881 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     2941 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     3001 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     3061 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     3121 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggacatctcg
     3181 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     3241 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     3301 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     3361 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3421 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     3481 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     3541 ctccttccgg tgggcgcggg gcatgactag catgatcgtg ctcctgtcgt tgaggacccg
     3601 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     3661 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     3721 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     3781 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     3841 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     3901 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     3961 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     4021 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     4081 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     4141 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     4201 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     4261 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     4321 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     4381 tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg
     4441 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5281 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac
     5341 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     5401 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     5461 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     5521 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     5581 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     5641 tggttactca ccactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct
     5701 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     5761 cctgtttgta attgtccttt taacagtgat cgcgtatttc gtctcgctca ggcgcaatca
     5821 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     5881 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     5941 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     6001 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     6061 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     6121 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     6181 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6241 cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc
     6301 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     6361 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     6421 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     6481 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     6541 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     6601 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     6661 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     6721 gtcccattcg cca
//
