LOCUS       sequence-338575-        8181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory Plasmid for PACE of pylBCD variants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8181)
  AUTHORS   Ho JML, Miller CA, Smith KA, Mattia JR, Bennett MR
  TITLE     Improved pyrrolysine biosynthesis through phage assisted 
            non-continuous directed evolution of the complete pathway.
  JOURNAL   Nat Commun. 2021 Jun 24;12(1):3914. doi: 10.1038/s41467-021-24183-9.
  PUBMED    34168131
REFERENCE   2  (bases 1 to 8181)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-06-24"; volume: "12(1):3914. doi"; pages: " 
            10.1038/s41467-021-24183-9"
COMMENT     Sequence Label: pJH77
FEATURES             Location/Qualifiers
     source          1..8181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             15..995
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEHILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     CDS             complement(3273..4064)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(4065..4136)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4197..4785
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4686..4705
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4939..4956
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4971..5111
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5037..5056)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5197..5219
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5213..5404)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             7053..8132
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKEKQRSLLY"
ORIGIN
        1 gcggtggcgg tagcaaattt ggattgttct tccttaactt catcaattca acaactgttc
       61 aagaacagag tatagttcgc atgcaggaaa taacggagta tgttgataag ttgaattttg
      121 aacatatttt agtgtatgaa aatcattttt cagataatgg tgttgtcggc gctcctctga
      181 ctgtttctgg ttttctgctc ggtttaacag agaaaattaa aattggttca ttaaatcaca
      241 tcattacaac tcatcatcct gtccgcatag cggaggaagc ttgcttattg gatcagttaa
      301 gtgaagggag atttatttta gggtttagtg attgcgaaaa aaaagatgaa atgcattttt
      361 ttaatcgccc ggttgaatat caacagcaac tatttgaaga gtgttatgaa atcattaacg
      421 atgctttaac aacaggctat tgtaatccag ataacgattt ttatagcttc cctaaaatat
      481 ctgtaaatcc ccatgcttat acgccaggcg gacctcggaa atatgtaaca gcaaccagtc
      541 atcatattgt tgagtgggcg gccaaaaaag gtattcctct catctttaag tgggatgatt
      601 ctaatgatgt tagatatgaa tatgctgaaa gatataaagc cgttgcggat aaatatgacg
      661 ttgacctatc agagatagac catcagttaa tgatattagt taactataac gaagatagta
      721 ataaagctaa acaagagacg cgtgcattta ttagtgatta tgttcttgaa atgcacccta
      781 atgaaaattt cgaaaataaa cttgaagaaa taattgcaga aaacgctgtc ggaaattata
      841 cggagtgtat aactgcggct aagttggcaa ttgaaaagtg tggtgcgaaa agtgtattgc
      901 tgtcctttga accaatgaat gatttgatga gccaaaaaaa tgtaatcaat attgttgatg
      961 ataatattaa gaagtaccac acggaatata cctaaacgga gccaatgtac gcaaaaaacc
     1021 ccgcttcagc ggggtttttt cgccaaaaaa aaccccgccc tgtcaggggc ggggtttttt
     1081 tttagggccc tacgttcgat gcttctttga gcgaacgatc aaaaataagt gccttcccat
     1141 caaaaaaata ttctcaacat aaaaaacttt gtgtaatact tgtaacgcta catggagatt
     1201 aactcaatct agagggtaac tagtatggat aagaagccgc tggatgttct gatctctgcg
     1261 accggtctgt ggatgtcccg taccggcacg ctgcacaaga tcaagcacta tgaggtttct
     1321 cgttctaaaa tctacatcga aatggcgtgt ggtgaccatc tggttgtgaa caactctcgt
     1381 tcttgtcgta ccgcacgtgc attccgtcat cataaatacc gtaaaacctg caaacgttgt
     1441 cgtgtttctg acgaagatat caacaacttc ctgacccgtt ctaccgaagg caaaacctct
     1501 gttaaagtta aagttgtttc tgcgccgaaa gtgaaaaaag cgatgccgaa atctgtttct
     1561 cgtgcgccga aaccgctgga aaatccggtt tctgcgaaag cgtctaccga cacctctcgt
     1621 tctgttccgt ctccggcgaa atctaccccg aactctccgg ttccgacctc tgcaagtgcc
     1681 cccgcactta cgaagagcca gactgacagg cttgaagtcc tgttaaaccc aaaagatgag
     1741 atttccctga attccggcaa gcctttcagg gagcttgagt ccgaattgct ctctcgcaga
     1801 aaaaaagacc tgcagcagat ctacgcggaa gaaagggaga attatctggg gaaactcgag
     1861 cgtgaaatta ccaggttctt tgtggacagg ggttttctgg aaataaaatc cccgatcctg
     1921 atccctcttg agtatatcga aaggatgggc attgataatg ataccgaact ttcaaaacag
     1981 atcttcaggg ttgacaagaa cttctgcctg agacccatgc ttgctccaaa cctttacaac
     2041 tacctgcgca agcttgacag ggccctgcct gatccaataa aaatttttga aataggccca
     2101 tgctacagaa aagagtccga cggcaaagaa cacctcgaag agtttaccat gctgaacttc
     2161 tgccagatgg gatcgggatg cacacgggaa aatcttgaaa gcataattac ggacttcctg
     2221 aaccacctgg gaattgattt caagatcgta ggcgattcct gcatggtcta tggggatacc
     2281 cttgatgtaa tgcacggaga cctggaactt tcctctgcag tagtcggacc cataccgctt
     2341 gaccgggaat ggggtattga taaaccctgg ataggggcag gtttcgggct cgaacgcctt
     2401 ctaaaggtta aacacgactt taaaaatatc aagagagctg caaggtccga gtcttactat
     2461 aacgggattt ctaccaacct gtaacctagg ctgctgccac cgctgagatc tatggcccgc
     2521 gcccacagga aggagctacc ggcagcggtg cggactgttg taactcataa taagaaatga
     2581 ggccgctcat ggcgttctgt tgcccgtctc gctggtgaaa agaaaaacca ccctggctaa
     2641 ctaagcggcc tgctgacttt ctcgccgatc aaatgactgt ccgtgtgctt ctcaaatgcc
     2701 tgaggccagt ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcagtgca
     2761 cggctaacta agcggcctgc tgactttctc gccgatcaaa aggcattttg ctattaaggg
     2821 attgacgagg gcgtatctgc gcagtaagat gcgccccgca ttcggaaacc tgatcatgta
     2881 gatcgaatgg actctaaatc cgttcagccg ggttagattc ccggggtttc cgccaggatc
     2941 cgtcgacctg cagatcctta gcgaaagcta aggatttttt ttaagcttct gccattcatc
     3001 cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct
     3061 taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt
     3121 ctgccgacgt tcatcggatc ccgggcccgt cgactgcaga ggcctgcatg caagcttggc
     3181 gtaatcatga tggacttaat taacggcact cctcagcaaa tataatgacc ctcttgataa
     3241 cccaagaggg cattttttaa tgcccatggc gtttatttgc cgactacctt ggtgatctcg
     3301 cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
     3361 tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg
     3421 cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
     3481 taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc
     3541 gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga
     3601 tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
     3661 agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca
     3721 ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
     3781 tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa
     3841 gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg
     3901 gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
     3961 ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc
     4021 tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa
     4081 tattattgaa gcatttatca gggttattgt ttcatgagcg tatacatatt tgaatgtatt
     4141 tagaaaaata ttaattgcgt tgcgctcact gcccgctttc caggccaaat aggccgttga
     4201 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4261 gtggtttgtt tgccggatca agagctacca cctctttttc cgaaggtaac tggcttcagc
     4321 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4381 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4441 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4501 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4561 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4621 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4681 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4741 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4801 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4861 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4921 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4981 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     5041 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     5101 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5161 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5221 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     5281 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     5341 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5401 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5461 gagagaggat gctcacgata cgggttactg atgatgaaca aagaggacat ccggtcaaat
     5521 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt gctgaggaga cttagggacc
     5581 ctgatgaaat tcgccacttg ttagtgtaat tcgctaactc atcctggcat gttgctgttg
     5641 attcttcaat cagatcttta taaatcaaaa agataaaaaa ttggcacgca aattgtatta
     5701 acagttcagc aggacaatcc tgaacgcaga aatcaagagg acaacattac ctgcaggtgc
     5761 agtaaggagg aaaaaaaaat gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc
     5821 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aatagcatac agaaaattca
     5881 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt
     5941 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca
     6001 tgggttccta ttgggcttgc tatctaggaa aatgagggtg gtggctctga gggtggcggt
     6061 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct
     6121 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa
     6181 aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt
     6241 cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact
     6301 caaggcactg accccgttaa aacttattag cagtacactc ctgtatcatc aaaagccatg
     6361 tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag
     6421 gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat
     6481 gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt
     6541 ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt
     6601 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat
     6661 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt
     6721 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact
     6781 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct
     6841 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct
     6901 tttgtctttg gcgctggtaa accttacgag ttcagtatcg actgcgataa gatcaacctg
     6961 ttccgcggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg
     7021 tttgctaaca tactgcgtaa taaggagtct taatgaaatt tggaaacttt ttgcttacat
     7081 accaacctcc ccaattttcc caaacagagg taatgaaacg tttggttaaa ttaggtcgca
     7141 tctctgagga gtgtggtttt gataccgtat ggttactgga gcatcatttc acggagtttg
     7201 gtttgcttgg taacccttat gtcgctgctg catatttact tggcgcgact aaaaaattga
     7261 atgtaggaac tgccgctatt gttcttccca cagcccatcc agtacgccaa cttgaagatg
     7321 tgaatttatt ggatcaaatg tcaaaaggac gatttcggtt tggtatttgc cgagggcttt
     7381 acaacaagga ctttcgcgta ttcggcacag atatgaataa cagtcgcgcc ttagcggaat
     7441 gctggtacgg gctgataaag aatggcatga cagagggata tatggaagct gataatgaac
     7501 atatcaagtt ccataaggta aaagtaaacc ccgcggcgta tagcagaggt ggcgcaccgg
     7561 tttatgtggt ggctgaatca gcttcgacga ctgagtgggc tgctcaattt ggcctaccga
     7621 tgatattaag ttggattata aatactaacg aaaagaaagc acaacttgag ctttataatg
     7681 aagtggctca agaatatggg cacgatattc ataatatcga ccattgctta tcatatataa
     7741 catctgtaga tcatgactca attaaagcga aagagatttg ccggaaattt ctggggcatt
     7801 ggtatgattc ttatgtgaat gctacgacta tttttgatga ttcagaccaa acaagaggtt
     7861 atgatttcaa taaagggcag tggcgtgact ttgtattaaa aggacataaa gatactaatc
     7921 gccgtattga ttacagttac gaaatcaatc ccgtgggaac gccgcaggaa tgtattgaca
     7981 taattcaaaa agacattgat gctacaggaa tatcaaatat ttgttgtgga tttgaagcta
     8041 atggaacagt agacgaaatt attgcttcca tgaagctctt ccagtctgat gtcatgccat
     8101 ttcttaaaga aaaacaacgt tcgctattat attatggcgg tggcggtagc ggcggtggcg
     8161 gtagcggcgg tggcggtagc g
//
