LOCUS       40924_36998        6389 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRH52, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6389)
  AUTHORS   Ghosh IN, Landick R.
  TITLE     OptSSeq: High-throughput sequencing readout of growth enrichment 
            defines optimal gene expression elements for homoethanologenesis
  JOURNAL   ACS Synth Biol (2016) In press
  PUBMED    27404024
REFERENCE   2  (bases 1 to 6389)
  AUTHORS   Ghosh IN, Landick RC.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUL-2016) DOE Great Lakes Bioenergy Research Center, 
            University of Wisconsin-Madison, 1552 University Avenue, Madison, WI
            53726, United States
REFERENCE   3  (bases 1 to 6389)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6389)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol (2016) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-JUL-2016) DOE Great Lakes Bioenergy Research Center, University 
            of Wisconsin-Madison, 1552 University Avenue, Madison, WI 53726, 
            United States"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     Cloning vector recieved with thanks from Haft, R.J.
FEATURES             Location/Qualifiers
     source          1..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        163..240
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             241..1320
                     /label=lacI
                     /note="lac repressor"
     regulatory      1326..1373
                     /label=B. subtilis glnA terminator
                     /note="B. subtilis glnA terminator"
                     /regulatory_class="terminator"
     protein_bind    complement(1385..1404)
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    1404..1425
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     regulatory      1440..1468
                     /note="Trc promoter (Ptrc); Trc promoter (Ptrc)"
                     /regulatory_class="promoter"
     protein_bind    1477..1493
                     /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        1543..1561
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     RBS             1597..1619
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1627..2340
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
     regulatory      2400..2451
                     /label=Phage 22 terminator
                     /note="Phage 22 terminator"
                     /regulatory_class="terminator"
     CDS             complement(2657..3316)
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     rep_origin      complement(3317..4086)
                     /direction=LEFT
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     promoter        5464..5555
                     /label=AmpR promoter
     CDS             5556..6344
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
ORIGIN
        1 cacgatgacc cggtcgtcgg ttcagggcag ggtcgttaaa tagccgctta tgtctattgc
       61 tggtttaccg gtttattgac taccggaagc agtgtgaccg tgtgcttctt actacgtcag
      121 tgtcgtgcca tagatctcat aagggagagc gtcgagatcc cggacaccat cgaatggtgc
      181 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
      241 gtgaaaccag ttactctgta tgatgttgct gaatacgccg gtgtatctta ccagactgtc
      301 tcccgcgttg tgaatcaggc ttctcatgtg agcgcgaaaa ctcgcgaaaa agtcgaagca
      361 gcgatggccg agctgaatta catcccgaac cgtgttgctc aacaactggc tggcaaacaa
      421 tccctgctga tcggcgtggc tacttcctct ctggccctgc acgcaccgtc ccagatcgtt
      481 gccgcgatca aaagccgtgc ggaccaactg ggcgcttctg ttgtagttag catggtggaa
      541 cgctctggtg ttgaagcctg caaagctgct gttcacaacc tgctggcgca gcgtgtctcc
      601 ggcctgatca tcaactatcc gctggatgac caggatgcaa tcgccgttga agcagcttgc
      661 actaatgttc cggccctgtt cctggatgtt tctgaccaga ccccgattaa ctctatcatc
      721 ttctctcacg aagatggtac tcgtctgggc gtcgaacatc tggtggctct gggccaccaa
      781 cagatcgcgc tgctggcggg ccctctgtcc tctgtatctg ctcgcctgcg tctggcgggc
      841 tggcacaaat acctgacccg taaccagatc cagccgattg cagagcgcga aggtgattgg
      901 tccgcgatgt ctggcttcca acagaccatg caaatgctga acgaaggtat tgttccgact
      961 gcgatgctgg tagctaacga ccagatggct ctgggtgcta tgcgtgcaat caccgaatcc
     1021 ggcctgcgtg tgggcgcgga catttctgtg gtgggttacg atgacaccga agatagctcc
     1081 tgctacattc cgccactgac tactatcaaa caggatttcc gtctgctggg tcagacttcc
     1141 gttgatcgtc tgctgcaact gtctcagggt caggctgtaa aaggcaacca actgctgcct
     1201 gtttccctgg ttaaacgtaa aacgaccctg gcgcccaata cgcaaaccgc ctctccccgc
     1261 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1321 taactactca atcccttggc actaaaagtg tcaggggatt ttttatgtta atacaccatc
     1381 acagaattgt gagcgctcac aattaatgtg agttagctca ctcattaggc accccaggct
     1441 tgacacttta tgcttccgct cgtataatgt gtggaattgt gagcggataa caatttcagg
     1501 tcaccaggaa acataccatg gccatgatta cgccaagcgc gcaattaacc ctcactaaag
     1561 ggaactaaag ctgggtaccg ggcccctaga aataattttg tttaacttta agaaggagat
     1621 atacatatga gcaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
     1681 gatggtgatg ttaatgggca caaattttct gtccgtggag agggtgaagg tgatgctaca
     1741 aacggaaaac tcacccttaa atttatttgc actactggaa aactacctgt tccgtggcca
     1801 acacttgtca ctactctgac ctatggtgtt caatgctttt cccgttatcc ggatcacatg
     1861 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga acgcactata
     1921 tctttcaaag atgacgggac ctacaagacg cgtgctgaag tcaagtttga aggtgatacc
     1981 cttgttaatc gtatcgagtt aaagggtatt gattttaaag aagatggaaa cattcttgga
     2041 cacaaactcg agtacaactt taactcacac aatgtataca tcacggcaga caaacaaaag
     2101 aatggaatca aagctaactt caaaattcgc cacaacgttg aagatggttc cgttcaacta
     2161 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     2221 cattacctgt cgacacaatc tgtcctttcg aaagatccca acgaaaagcg tgaccacatg
     2281 gtccttcttg agtttgtaac tgctgctggg attacacatg gcatggatga gctctacaaa
     2341 ctcgagtaag cttgatatcg aattcctgca gcccggggga tccctagttc tagaaataag
     2401 ttaaataaag ccctgagttt aaccgctcgg ggctttttgc gttttaagca cactagagcg
     2461 gccatgcatc ccagagcctg ataaaaacgg tgaatccgtt agcgaggtgc cgccggcttc
     2521 cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag
     2581 gtatagggcg gcgaggcggc tacagccgat agtctggaac agcgcactta cgggttgctg
     2641 cgcaacccaa gtgctaccgg cgcggcagcg tgacccgtgt cggcggctcc aacggctcgc
     2701 catcgtccag aaaacacggc tcatcgggca tcggcaggcg ctgctgcccg cgccgttccc
     2761 attcctccgt ttcggtcaag gctggcaggt ctggttccat gcccggaatg ccgggctggc
     2821 tgggcggctc ctcgccgggg ccggtcggta gttgctgctc gcccggatac agggtcggga
     2881 tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg gtcgtcgtga tcaaccacca
     2941 cggcggcact gaacaccgac aggcgcaact ggtcgcgggg ctggccccac gccacgcggt
     3001 cattgaccac gtaggccgac acggtgccgg ggccgttgag cttcacgacg gagatccagc
     3061 gctcggccac caagtccttg actgcgtatt ggaccgtccg caaagaacgt ccgatgagct
     3121 tggaaagtgt cttctggctg accaccacgg cgttctggtg gcccatctgc gccacgaggt
     3181 gatgcagcag cattgccgcc gtgggtttcc tcgcaataag cccggcccac gcctcatgcg
     3241 ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt ggcttgaatg ccgatttctc
     3301 tggactgcgt ggccatgctt atctccatgc ggtagggtgc cgcacggttg cggcaccatg
     3361 cgcaatcagc tgcaactttt cggcagcgcg acaacaatta tgcgttgcgt aaaagtggca
     3421 gtcaattaca gattttcttt aacctacgca atgagctatt gcggggggtg ccgcaatgag
     3481 ctgttgcgta cccccctttt ttaagttgtt gatttttaag tctttcgcat ttcgccctat
     3541 atctagttct ttggtgccca aagaagggca cccctgcggg gttcccccac gccttcggcg
     3601 cggctccccc tccggcaaaa agtggcccct ccggggcttg ttgatcgact gcgcggcctt
     3661 cggccttgcc caaggtggcg ctgccccctt ggaacccccg cactcgccgc cgtgaggctc
     3721 ggggggcagg cgggcgggct tcgccttcga ctgcccccac tcgcataggc ttgggtcgtt
     3781 ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac ccgccttcca
     3841 cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg ccggaggctt ttccccagag
     3901 aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc agttggagcc ggtgggtatg
     3961 tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc aagctcgtgg gcaggcgcag
     4021 cctgtccatc agcttgtcca gcagggttgt ccacgggccg agcgaagcga gccagccggt
     4081 ggccgctcgc ggccatcgtc cacatatcca cgggctggca agggagcgca gcgaccgcgc
     4141 agggcgaagc ccggagagca agcccgtagg gcgccgcagc cgccgtaggc ggtcacgact
     4201 ttgcgaagca aagtctagtg agtatactca agcattgagt ggcccgccgg aggcaccgcc
     4261 ttgcgctgcc cccgtcgagc cggttggaca ccaaaaggga ggggcaggca tggcggcata
     4321 cgcgatcatg cgatgcaaga agctggcgaa aatgggcaac gtggcggcca gtctcaagca
     4381 cgcctaccgc gagcgcgaga cgcccaacgc tgacgccagc aggacgccag agaacgagca
     4441 ctgggcggcc agcagcaccg atgaagcgat gggccgactg cgcgagttgc tgccagagaa
     4501 gcggcgcaag gacgctgtgt tggcggtcga gtacgtcatg acggccagcc cggaatggtg
     4561 gaagtcggcc agccaagaac agcaggcggc gttcttcgag aaggcgcaca agtggctggc
     4621 ggacaagtac ggggcggatc gcatcgtgac ggccagcatc caccgtgacg aaaccagccc
     4681 gcacatgacc gcgttcgtgg tgccgctgac gcaggacggc aggctgtcgg ccaaggagtt
     4741 catcggcaac aaagcgcaga tgacccgcga ccagaccacg tttgcggccg ctgtggccga
     4801 tctagggctg caacggggca tcgagggcag caaggcacgt cacacgcgca ttcaggcgtt
     4861 ctacgaggcc ctggagcggc caccagtggg ccacgtcacc atcagcccgc aagcggtcga
     4921 gccacgcgcc tatgcaccgc agggattggc cgaaaagctg ggaatctcaa agcgcgttga
     4981 gacgccggaa gccgtggccg accggctgac aaaagcggtt cggcaggggt atgagcctgc
     5041 cctacaggcc gccgcaggag cgcgtgagat gcgcaagaag gccgatcaag cccaagagac
     5101 ggcccgagac cttcgggagc gcctgaagcc cgttctggac gccctggggc cgttgaatcg
     5161 ggatatgcag gccaaggccg ccgcgatcat caaggccgtg ggcgaaaagc tgctgacgga
     5221 acagcgggaa gtccagcgcc agaaacaggc ccagcgccag caggaacgcg ggcgcgcaca
     5281 tttccccgaa aagtgccacc tggcggcgtt gtgacaattt accgaacaac tccgcggccg
     5341 ggaagccgat ctcggcttga acgaattcct gcttatccac ggaatctgtg ggtaactttg
     5401 tatgtgtccg cagcgcccgc cgcagtctca cgcccggagc gtagcgaccg agtgagctag
     5461 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5521 gataaatgct tcaataatat tgaaaaagga agagtatgag ggaagcggtg atcgccgaag
     5581 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc
     5641 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg
     5701 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg
     5761 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
     5821 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat
     5881 ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca
     5941 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
     6001 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa
     6061 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta
     6121 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg
     6181 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac
     6241 aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat
     6301 ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt ctaacaattc
     6361 gttcaagccg aggggccgca agatccggc
//
