LOCUS       62056_19150        8316 bp DNA     circular SYN 26-NOV-2019
DEFINITION  Expression vector pS239D.M, complete sequence.
ACCESSION   MN393464
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8316)
  AUTHORS   Calles B, Goni-Moreno A, de Lorenzo V.
  TITLE     Digitalizing heterologous gene expression in Gram-negative bacteria 
            with a portable on/off module
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8316)
  AUTHORS   Calles B, Goni-Moreno A, de Lorenzo V.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-AUG-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, Darwin, 3, Cantoblanco, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 8316)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (28-AUG-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, Darwin, 3, Cantoblanco, Madrid 28049, Spain"
FEATURES             Location/Qualifiers
     source          1..8316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      5..99
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             231..1043
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     oriT            1213..1321
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             complement(1335..1994)
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     rep_origin      complement(1995..2766)
                     /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"
     terminator      complement(2881..2967)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     gene            complement(2982..5639)
                     /gene="alkS"
                     /label=alkS
     CDS             complement(2982..5639)
                     /codon_start=1
                     /transl_table=11
                     /gene="alkS"
                     /product="AlkS"
                     /label=alkS
                     /note="edited; crc mut"
                     /protein_id="QGN03500.1"
                     /translation="MGMKIKIINNDFPVAKIGLDRITTLVSAKVHNCIYRPRLSIADGT
                     APRVCLYRAPPGYGKTVALAFEWLRHRTTGRPAVWISLRASSYSEFDICAEIIEQLEAF
                     ELVTFSHVREGVSKPTLLRDLASSLWQSTSSNEIETLICLDNINQGLGLPLLHALMEFM
                     LETPKSIRFAVAGNTIKGFSRLKLAGAMQEHTEKDLAFSADEAVALVEAEAVLGVSEVQ
                     IEALVQEMEGWPVLIGFLLKCELPAKSISTVVEIDNYFNDEIFEALPERYRVFLVNSSL
                     LDVVTPDGYNYVFKCVNAASCIKYLSTNYMLLRHVEGEPAQFTLHPVLRDFLQGIAWAE
                     NPAKRSYLLKRAAFWHWRRGEYQYAIRIALRANDCRWAVGMSEGIILDLSFRQGEIDTL
                     RHWLSELPVKDLHKNPIVLICFAWVLYFSQQSARAEKLLKDLITPPDKKNKWQEKGWPQ
                     LVFAIGKATNDEMLLSEELCNKWISLFGDSNAVGKGAALTCLAFIFASEYRFAELEKVL
                     AQAQAVNKFAKQDFAFGWLYVAKLQQALASGKMSWARQLITQARTDSGAQIMETAFTSK
                     MLDALELESNYELCRLDTSEEKFSEILEFIANHGVTDVFFSVCRVVSAWRLGRNDLNGS
                     IEILEWAKAYAAEKNLPRLEVMSQIEIYQRLLFQGVTYINTLQAFEDRKIFSGPHSAPL
                     KARLLLVQSLALSRDQNFHLAAHRALLAIQQARKISAGQLEVRGLLYLAGAQAGGGTLK
                     KAQHNIAYALEMAKQLQCFQTVLDEIRLIKRLIPTSCEVFAAVNLDQAIGAFSLPQMVE
                     IRKSAENKAGDFLTLKQVSVLKLVKEGCSNKQIATKMYVTEDAIKWHMRKIFTILNVKS
                     RTQAIIEAERQGVI"
     regulatory      complement(5666..5771)
                     /label=Km promoter
                     /note="Km promoter"
                     /regulatory_class="promoter"
     regulatory      5975..6035
                     /label=PAlkB
                     /note="PAlkB"
                     /regulatory_class="promoter"
     regulatory      6039..6045
                     /regulatory_class="ribosome_binding_site"
     CDS             6057..7136
                     /label=lacI
                     /note="lac repressor"
     CDS             7137..7154
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             7183..7893
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Pedelacq et al., 2006)"
     misc_feature    7897..7953
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     regulatory      7986..8015
                     /label=synthetic T500 terminator
                     /note="synthetic T500 terminator"
                     /regulatory_class="terminator"
     terminator      complement(8022..8049)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(8089..8135)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     regulatory      complement(8150..8258)
                     /note="anti LacI sRNA; MicC-based"
                     /regulatory_class="other"
     regulatory      complement(8259..8316)
                     /label=PA1/O4s
                     /note="PA1/O4s"
                     /regulatory_class="promoter"
ORIGIN
        1 cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga
       61 acgctcggtt gccgccgggc gttttttatt ggtgagaatc caggggtccc caataattac
      121 gatttaaatt tgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
      181 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
      241 ttcagcgtga aacgagctgt agccgtccgc gtctgaacag caacatggat gcggatctgt
      301 atggctataa atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt tatcgtctgt
      361 atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc gtggcgaacg
      421 atgtgaccga tgaaatggtg cgtctgaact ggctgaccga atttatgccg ctgccgacca
      481 ttaaacattt tattcgcacc ccggatgatg cgtggctgct gaccaccgcg attccgggca
      541 aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt gtggatgcgc
      601 tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg tttaacagcg
      661 atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa caacggcctg gtggatgcga
      721 gcgattttga tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa gaaatgcata
      781 aactgctgcc gtttagcccg gatagcgtgg tgacccacgg cgattttagc ctggataacc
      841 tgattttcga tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg ggcattgcgg
      901 atcgttatca ggatctggcc attctgtgga actgcctggg cgaatttagc ccgagcctgc
      961 aaaaacgtct gtttcagaaa tatggcattg ataatccgga tatgaacaaa ctgcaatttc
     1021 atctgatgct ggatgaattt ttctaataat taattggacc gcggtccgcg cgttgtcctt
     1081 ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt
     1141 tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca
     1201 acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt
     1261 cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc
     1321 gtaggccggc cctaccggcg cggcagcgtt acccgtgtcg gcggctccaa cggctcgcca
     1381 tcgtccagaa aacacggctc atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat
     1441 tcctccgttt cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc gggctggctg
     1501 ggcggctcct cgccggggcc ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg
     1561 cggcgcaggt cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg
     1621 gcggcactga acaccgacag gcgcaactgg tcgcggggct ggccccacgc cacgcggtca
     1681 ttgaccacgt aggccgacac ggtgccgggg ccgttgagct tcacgacgga gatccagcgc
     1741 tcggccacca agtccttgac tgcgtattgg accgtccgca aagaacgtcc gatgagcttg
     1801 gaaagtgtct tctggctgac caccacggcg ttctggtggc ccatctgcgc cacgaggtga
     1861 tgcagcagca ttgccgccgt gggtttcctc gcaataagcc cggcccacgc ctcatgcgct
     1921 ttgcgttccg tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg
     1981 gactgcgtgg ccatgcttat ctccatgcgg taggggtgcc gcacggttgc ggcaccatgc
     2041 gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag
     2101 tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc
     2161 tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata
     2221 tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc
     2281 ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc
     2341 ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg
     2401 gggggcaggc gggcgggctt cgcccttcga ctgcccccac tcgcataggc ttgggtcgtt
     2461 ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac ccgccttcca
     2521 cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg ccggaggctt ttccccagag
     2581 aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc agttggagcc ggtgggtatg
     2641 tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc aagctcgtgg gcaggcgcag
     2701 cctgtccatc agcttgtcca gcagggttgt ccacgggccg agcgaagcga gccagccggt
     2761 ggccgctcgc ggccatcgtc cacatatcca cgggctggca agggagcgca gcgaccgcgc
     2821 agggcgaagc ccggagagca agcccgtagg gggggcgcgc ccagctgtct agggcggcgg
     2881 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     2941 tttcgactga gcctttcgtt ttatttgatg cctttaatta atcagataac accctgacgt
     3001 tcggcttcaa ttattgcttg cgtgcgactc tttacattaa ggatggtaaa tattttcctc
     3061 atatgccact ttatagcatc ttcggtgaca tacatctttg ttgctatttg tttgtttgag
     3121 cacccctctt ttacaagttt caagacactg acctgcttca gtgtcaaaaa atcaccagct
     3181 ttattttctg cggattttct aatctccacc atttgcggca gactaaaagc tccaatcgct
     3241 tgatctaaat taactgctgc gaagacttca catgaagtcg gtattaatct cttaatcaaa
     3301 cgtatttcat ccagcactgt ttgaaagcat tgaagctgtt ttgccatctc caatgcataa
     3361 gcaatgttat gctgagcctt ctttaatgtg ccgccacctg cttgcgctcc ggctaaatac
     3421 aataagccac ggacttccaa ttggcctgcg ctaattttac gggcttgctg aatagccaat
     3481 agcgctctgt gcgcggcaag atgaaagttc tgatctcggg aaagcgcgag tgattgaaca
     3541 agcagcaggc gtgcctttag gggggctgag tgcggtccgg agaaaatctt gcgatcttca
     3601 aaggcttgta acgtatttat gtacgttacg ccttgaaaga gcaagcgttg atagatctca
     3661 atttggctca taacttccaa tcttggtaga tttttttcag ccgcatatgc cttcgcccac
     3721 tccaaaatct caatggaacc attcaggtca ttccttccaa gccgccaagc tgacacaaca
     3781 cggcatacgg aaaaaaacac gtctgtcacc ccgtgattgg caataaactc taaaatttcg
     3841 gagaactttt cttctgaggt gtccaagcgg cacaattcat aatttgactc aagctccaga
     3901 gcgtccagca ttttcgaggt aaacgcggtc tccataatct gcgcgccact gtctgtgcgt
     3961 gcttgagtta taagctgcct ggcccagctc attttcccgc ttgctagcgc ttgttgcagc
     4021 ttggcgacat acagccaacc gaaagcaaaa tcttgttttg caaatttatt cacggcttgg
     4081 gcctgagcca gcaccttctc taactctgca aatctatact cactggcaaa aataaaagcc
     4141 aagcaggtta gcgcggcccc tttcccaact gcgtttgaat ccccaaataa actaatccac
     4201 ttattacaca gctcctcact caaaagcatt tcatcattcg ttgctttacc tattgcaaac
     4261 acaagctgcg gccatccttt ttcttgccat ttgttttttt tatcgggcgg ggtaattagg
     4321 tctttaagta acttctctgc tcgcgcgctt tgctgactga aatacaatac ccacgcgaaa
     4381 caaataagta ctatggggtt cttgtgcaag tccttcactg gcagctccga cagccagtgt
     4441 ctcagcgtat ctatttcgcc ctgacgaaat gacaaatcta aaattattcc ctcactcatg
     4501 ccgacagccc agcgacagtc attcgcccgt agggctattc gtattgcgta ctggtattcg
     4561 cctctacgcc aatgccagaa agctgcgcgc ttaagcaagt aggatctttt agcaggattt
     4621 tcagcccaag caattccttg aagaaaatcg cgcaatactg gatgtagtgt aaactgcgcc
     4681 ggctcacctt ctacatggcg aagtaacatg taattcgtgc tcagatattt aatacatgag
     4741 gccgcattga cgcatttgaa tacataattg tagccatcag gcgtcacgac atcgagcaat
     4801 gaagaattta caagaaaaac acgataccgc tcgggaagcg cctcaaatat ttcatcatta
     4861 aagtaattgt ctatttcaac tactgttgaa atagacttgg ccggcaactc acattttaac
     4921 aaaaacccga taagaacagg ccacccctcc atttcttgca ccaaggcctc tatctgtacc
     4981 tcagaaactc caagaacagc ctctgcctcc actaacgcca ctgcctcgtc tgcgctaaag
     5041 gccaaatctt tctcggtgtg ctcctgcata gcgcctgcaa gtttaagccg cgagaaccct
     5101 tttattgtat tacctgcgac tgcaaacctg atactttttg gtgtttctaa catgaactcc
     5161 ataagcgcgt gcaacaacgg caagcccaag ccctgattaa tattatccaa acaaattagc
     5221 gtttctattt cgttactcga ggtactctgc caaagactgg atgcaaggtc tcgcaagagc
     5281 gtaggcttgc tcacaccctc tctcacatgg ctgaatgtta ccagttcgaa cgcttcaagc
     5341 tgctcaataa tttctgcgca gatatcaaat tcactgtaag aactggctct taaagaaatc
     5401 cacaccgcag ggcgtccggt tgttctgtgg cgtagccact cgaacgcaag agccacggtt
     5461 tttccatatc caggcggggc tctataaagg catactctgg gtgcagttcc atccgcgata
     5521 ctcaatctgg gccgatatat gcagttatgc actttggcac ttactagagt cgtaattcga
     5581 tccagcccga tcttggcaac cgggaaatca ttgttgatga tcttgatctt catgcccatg
     5641 gtttttcctc cttataaagt taatcaacac cccttgtatt actgtttatg taagcagaca
     5701 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     5761 acacaagacg tggcaaaaaa cattatccag aacgggagtg cgccttgagc gacacgaatt
     5821 atgcagtgat ttacgacctg cacagccata ccacagcttc cgatggctgc ctgacgccag
     5881 aagcattggt gcaccgtgca gtcgatgata agctgtcaaa cagcggataa caatttcaca
     5941 caggacgtgt ttttccagca gacgacggag caaaaactac ccgtaggtgt agttggcgca
     6001 agcgtccgat tagctcaggt ttaagatgtc gagagcctag gaggaaaaaa catatggtga
     6061 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     6121 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     6181 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     6241 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     6301 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     6361 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     6421 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     6481 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     6541 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     6601 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     6661 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     6721 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     6781 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     6841 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     6901 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     6961 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     7021 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     7081 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagcatc
     7141 atcaccacca tcattaggat ggtggtgatg ataatggcta gccgtaaagg tgaagaactg
     7201 ttcaccggtg ttgttccgat cctggttgaa ctggatggtg atgttaacgg ccacaaattc
     7261 tctgttcgtg gtgaaggtga aggtgatgca accaacggta aactgaccct gaaattcatc
     7321 tgcactaccg gtaaactgcc ggttccatgg ccgactctgg tgactaccct gacctatggt
     7381 gttcagtgtt tttctcgtta cccggatcac atgaagcagc atgatttctt caaatctgca
     7441 atgccggaag gttatgtaca ggagcgcacc atttctttca aagacgatgg cacctacaaa
     7501 acccgtgcag aggttaaatt tgaaggtgat actctggtga accgtattga actgaaaggc
     7561 attgatttca aagaggacgg caacatcctg ggccacaaac tggaatataa cttcaactcc
     7621 cataacgttt acatcaccgc agacaaacag aagaacggta tcaaagctaa cttcaaaatt
     7681 cgccataacg ttgaagacgg tagcgtacag ctggcggacc actaccagca gaacactccg
     7741 atcggtgatg gtccggttct gctgccggat aaccactacc tgtccaccca gtctaaactg
     7801 tccaaagacc cgaacgaaaa gcgcgaccac atggtgctgc tggagttcgt tactgccgca
     7861 ggtatcacgc acggcatgga tgaactctac aaataagaat tcgagctcgg tacccgggga
     7921 tcctctagag tcgacctgca ggcatgcaag cttgcggccg cgtcgtgact gggaaaaccc
     7981 tggcgcaaag cccgccgaaa ggcgggcttt tctgttataa acgcagaaag gcccacccga
     8041 aggtgagcca gtgtgactct agtagagagc gttcaccgac aaaaacagat aaaacgaaag
     8101 gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggctcgagc tcgagaaaaa
     8161 aagcccggac gactgttcgg gcttgtcttt ttatatgttg gaaaatcagt ggcaatgcaa
     8221 tggcccaaca gaaaatggtg aaaccagtaa cgttatactg aatctaagta tcaatgtgag
     8281 cgctcacaag tcaacactct ttttgataaa actagt
//
