LOCUS       sequence-338574-        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: pJH19
FEATURES             Location/Qualifiers
     source          1..8181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             980..2059
                     /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"
     CDS             2123..3103
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     CDS             complement(5381..6172)
                     /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(6173..6244)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6305..6893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6794..6813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7047..7064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7079..7219
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7145..7164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7305..7327
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7321..7512)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gttctgaggg tggcggtact aaacctcctg agtacggtga tacacctatt ccgggctata
       61 cttatatcaa ccctctcgac ggcacttatc cgcctggtac tgagcaaaac cccgctaatc
      121 ctaatccttc tcttgaggag tctcagcctc ttaatacttt catgtttcag aataataggt
      181 tccgaaatag gcagggggca ttaactgttt atacgggcac tgttactcaa ggcactgacc
      241 ccgttaaaac ttattaccag tacactcctg tatcatcaaa agccatgtat gacgcttact
      301 ggaacggtaa attcagagac tgcgctttcc attctggctt taatgaggat ccattcgttt
      361 gtgaatatca aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct
      421 ctggtggtgg ttctggtggc ggctctgagg gtggtggctc tgagggtggc ggttctgagg
      481 gtggcggctc tgagggaggc ggttccggtg gtggctctgg ttccggtgat tttgattatg
      541 aaaagatggc aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac
      601 agtctgacgc taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg
      661 gtttcattgg tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg
      721 gctctaattc ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt
      781 tccgtcaata tttaccttcc ctccctcaat cggttgaatg tcgccctttt gtctttggcg
      841 ctggtaaacc ttacgagttc agtatcgact gcgataagat caacctgttc cgcggtgtct
      901 ttgcgtttct tttatatgtt gccaccttta tgtatgtatt ttctacgttt gctaacatac
      961 tgcgtaataa ggagtcttaa tgaaatttgg aaactttttg cttacatacc aacctcccca
     1021 attttcccaa acagaggtaa tgaaacgttt ggttaaatta ggtcgcatct ctgaggagtg
     1081 tggttttgat accgtatggt tactggagca tcatttcacg gagtttggtt tgcttggtaa
     1141 cccttatgtc gctgctgcat atttacttgg cgcgactaaa aaattgaatg taggaactgc
     1201 cgctattgtt cttcccacag cccatccagt acgccaactt gaagatgtga atttattgga
     1261 tcaaatgtca aaaggacgat ttcggtttgg tatttgccga gggctttaca acaaggactt
     1321 tcgcgtattc ggcacagata tgaataacag tcgcgcctta gcggaatgct ggtacgggct
     1381 gataaagaat ggcatgacag agggatatat ggaagctgat aatgaacata tcaagttcca
     1441 taaggtaaaa gtaaaccccg cggcgtatag cagaggtggc gcaccggttt atgtggtggc
     1501 tgaatcagct tcgacgactg agtgggctgc tcaatttggc ctaccgatga tattaagttg
     1561 gattataaat actaacgaaa agaaagcaca acttgagctt tataatgaag tggctcaaga
     1621 atatgggcac gatattcata atatcgacca ttgcttatca tatataacat ctgtagatca
     1681 tgactcaatt aaagcgaaag agatttgccg gaaatttctg gggcattggt atgattctta
     1741 tgtgaatgct acgactattt ttgatgattc agaccaaaca agaggttatg atttcaataa
     1801 agggcagtgg cgtgactttg tattaaaagg acataaagat actaatcgcc gtattgatta
     1861 cagttacgaa atcaatcccg tgggaacgcc gcaggaatgt attgacataa ttcaaaaaga
     1921 cattgatgct acaggaatat caaatatttg ttgtggattt gaagctaatg gaacagtaga
     1981 cgaaattatt gcttccatga agctcttcca gtctgatgtc atgccatttc ttaaagaaaa
     2041 acaacgttcg ctattatatt atggcggtgg cggtagcggc ggtggcggta gcggcggtgg
     2101 cggtagcggc ggtggcggta gcaaatttgg attgttcttc cttaacttca tcaattcaac
     2161 aactgttcaa gaacagagta tagttcgcat gcaggaaata acggagtatg ttgataagtt
     2221 gaattttgaa cagattttag tgtatgaaaa tcatttttca gataatggtg ttgtcggcgc
     2281 tcctctgact gtttctggtt ttctgctcgg tttaacagag aaaattaaaa ttggttcatt
     2341 aaatcacatc attacaactc atcatcctgt ccgcatagcg gaggaagctt gcttattgga
     2401 tcagttaagt gaagggagat ttattttagg gtttagtgat tgcgaaaaaa aagatgaaat
     2461 gcattttttt aatcgcccgg ttgaatatca acagcaacta tttgaagagt gttatgaaat
     2521 cattaacgat gctttaacaa caggctattg taatccagat aacgattttt atagcttccc
     2581 taaaatatct gtaaatcccc atgcttatac gccaggcgga cctcggaaat atgtaacagc
     2641 aaccagtcat catattgttg agtgggcggc caaaaaaggt attcctctca tctttaagtg
     2701 ggatgattct aatgatgtta gatatgaata tgctgaaaga tataaagccg ttgcggataa
     2761 atatgacgtt gacctatcag agatagacca tcagttaatg atattagtta actataacga
     2821 agatagtaat aaagctaaac aagagacgcg tgcatttatt agtgattatg ttcttgaaat
     2881 gcaccctaat gaaaatttcg aaaataaact tgaagaaata attgcagaaa acgctgtcgg
     2941 aaattatacg gagtgtataa ctgcggctaa gttggcaatt gaaaagtgtg gtgcgaaaag
     3001 tgtattgctg tcctttgaac caatgaatga tttgatgagc caaaaaaatg taatcaatat
     3061 tgttgatgat aatattaaga agtaccacac ggaatatacc taaacggagc caatgtacgc
     3121 aaaaaacccc gcttcagcgg ggttttttcg ccaaaaaaaa ccccgccctg tcaggggcgg
     3181 ggtttttttt tagggcccta cgttcgatgc ttctttgagc gaacgatcaa aaataagtgc
     3241 cttcccatca aaaaaatatt ctcaacataa aaaactttgt gtaatacttg taacgctaca
     3301 tggagattaa ctcaatctag agggtaacta gtatggataa gaagccgctg gatgttctga
     3361 tctctgcgac cggtctgtgg atgtcccgta ccggcacgct gcacaagatc aagcactatg
     3421 aggtttctcg ttctaaaatc tacatcgaaa tggcgtgtgg tgaccatctg gttgtgaaca
     3481 actctcgttc ttgtcgtacc gcacgtgcat tccgtcatca taaataccgt aaaacctgca
     3541 aacgttgtcg tgtttctgac gaagatatca acaacttcct gacccgttct accgaaggca
     3601 aaacctctgt taaagttaaa gttgtttctg cgccgaaagt gaaaaaagcg atgccgaaat
     3661 ctgtttctcg tgcgccgaaa ccgctggaaa atccggtttc tgcgaaagcg tctaccgaca
     3721 cctctcgttc tgttccgtct ccggcgaaat ctaccccgaa ctctccggtt ccgacctctg
     3781 caagtgcccc cgcacttacg aagagccaga ctgacaggct tgaagtcctg ttaaacccaa
     3841 aagatgagat ttccctgaat tccggcaagc ctttcaggga gcttgagtcc gaattgctct
     3901 ctcgcagaaa aaaagacctg cagcagatct acgcggaaga aagggagaat tatctgggga
     3961 aactcgagcg tgaaattacc aggttctttg tggacagggg ttttctggaa ataaaatccc
     4021 cgatcctgat ccctcttgag tatatcgaaa ggatgggcat tgataatgat accgaacttt
     4081 caaaacagat cttcagggtt gacaagaact tctgcctgag acccatgctt gctccaaacc
     4141 tttacaacta cctgcgcaag cttgacaggg ccctgcctga tccaataaaa atttttgaaa
     4201 taggcccatg ctacagaaaa gagtccgacg gcaaagaaca cctcgaagag tttaccatgc
     4261 tgaacttctg ccagatggga tcgggatgca cacgggaaaa tcttgaaagc ataattacgg
     4321 acttcctgaa ccacctggga attgatttca agatcgtagg cgattcctgc atggtctatg
     4381 gggataccct tgatgtaatg cacggagacc tggaactttc ctctgcagta gtcggaccca
     4441 taccgcttga ccgggaatgg ggtattgata aaccctggat aggggcaggt ttcgggctcg
     4501 aacgccttct aaaggttaaa cacgacttta aaaatatcaa gagagctgca aggtccgagt
     4561 cttactataa cgggatttct accaacctgt aacctaggct gctgccaccg ctgagatcta
     4621 tggcccgcgc ccacaggaag gagctaccgg cagcggtgcg gactgttgta actcataata
     4681 agaaatgagg ccgctcatgg cgttctgttg cccgtctcgc tggtgaaaag aaaaaccacc
     4741 ctggctaact aagcggcctg ctgactttct cgccgatcaa atgactgtcc gtgtgcttct
     4801 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga
     4861 tcagtgcacg gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct
     4921 attaagggat tgacgagggc gtatctgcgc agtaagatgc gccccgcatt cggaaacctg
     4981 atcatgtaga tcgaatggac tctaaatccg ttcagccggg ttagattccc ggggtttccg
     5041 ccaggatccg tcgacctgca gatccttagc gaaagctaag gatttttttt aagcttctgc
     5101 cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat
     5161 aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     5221 taagcattct gccgacgttc atcggatccc gggcccgtcg actgcagagg cctgcatgca
     5281 agcttggcgt aatcatgatg gacttaatta acggcactcc tcagcaaata taatgaccct
     5341 cttgataacc caagagggca ttttttaatg cccatggcgt ttatttgccg actaccttgg
     5401 tgatctcgcc tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc
     5461 gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg
     5521 ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta
     5581 ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt
     5641 cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag
     5701 gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg
     5761 cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa
     5821 gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg
     5881 gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc
     5941 caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa
     6001 gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca
     6061 ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc
     6121 caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc
     6181 tttttcaata ttattgaagc atttatcagg gttattgttt catgagcgta tacatatttg
     6241 aatgtattta gaaaaatatt aattgcgttg cgctcactgc ccgctttcca ggccaaatag
     6301 gccgttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6361 taccagcggt ggtttgtttg ccggatcaag agctaccacc tctttttccg aaggtaactg
     6421 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6481 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6541 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6601 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6661 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6721 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6781 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6841 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6901 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6961 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7021 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7081 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     7141 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     7201 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     7261 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     7321 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     7381 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     7441 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     7501 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     7561 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacaaa gaggacatcc
     7621 ggtcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttgc tgaggagact
     7681 tagggaccct gatgaaattc gccacttgtt agtgtaattc gctaactcat cctggcatgt
     7741 tgctgttgat tcttcaatca gatctttata aatcaaaaag ataaaaaatt ggcacgcaaa
     7801 ttgtattaac agttcagcag gacaatcctg aacgcagaaa tcaagaggac aacattacct
     7861 gcaggtgcag taaggaggaa aaaaaaatga aaaaattatt attcgcaatt cctttagttg
     7921 ttcctttcta ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa tagcatacag
     7981 aaaattcatt tactaacgtc tggaaagacg acaaaacttt agatcgttac gctaactatg
     8041 agggctgtct gtggaatgct acaggcgttg tagtttgtac tggtgacgaa actcagtgtt
     8101 acggtacatg ggttcctatt gggcttgcta tccctgaaaa tgagggtggt ggctctgagg
     8161 gtggcggttc tgagggtggc g
//
