LOCUS       sequence-338790-        8179 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 8179)
  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 8179)
  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: pJH190
FEATURES             Location/Qualifiers
     source          1..8179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3251..4042)
                     /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(4043..4114)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4175..4763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4664..4683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4917..4934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4949..5089
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5015..5034)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5175..5197
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5191..5382)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             7031..8110
                     /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             join(8174..8179,1..975)
                     /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"
ORIGIN
        1 ggattgttct tccttaactt catcaattca acaactgttc aagaacagag tatagttcgc
       61 atgcaggaaa taacggagta tgttgataag ttgaattttg aacagatttt agtgtatgaa
      121 aatcattttt cagataatgg tgttgtcggc gctcctctga ctgtttctgg ttttctgctc
      181 ggtttaacag agaaaattaa aattggttca ttaaatcaca tcattacaac tcatcatcct
      241 gtccgcatag cggaggaagc ttgcttattg gatcagttaa gtgaagggag atttatttta
      301 gggtttagtg attgcgaaaa aaaagatgaa atgcattttt ttaatcgccc ggttgaatat
      361 caacagcaac tatttgaaga gtgttatgaa atcattaacg atgctttaac aacaggctat
      421 tgtaatccag ataacgattt ttatagcttc cctaaaatat ctgtaaatcc ccatgcttat
      481 acgccaggcg gacctcggaa atatgtaaca gcaaccagtc atcatattgt tgagtgggcg
      541 gccaaaaaag gtattcctct catctttaag tgggatgatt ctaatgatgt tagatatgaa
      601 tatgctgaaa gatataaagc cgttgcggat aaatatgacg ttgacctatc agagatagac
      661 catcagttaa tgatattagt taactataac gaagatagta ataaagctaa acaagagacg
      721 cgtgcattta ttagtgatta tgttcttgaa atgcacccta atgaaaattt cgaaaataaa
      781 cttgaagaaa taattgcaga aaacgctgtc ggaaattata cggagtgtat aactgcggct
      841 aagttggcaa ttgaaaagtg tggtgcgaaa agtgtattgc tgtcctttga accaatgaat
      901 gatttgatga gccaaaaaaa tgtaatcaat attgttgatg ataatattaa gaagtaccac
      961 acggaatata cctaaacgga gccaatgtac gcaaaaaacc ccgcttcagc ggggtttttt
     1021 cgccaaaaaa aaccccgccc tgtcaggggc ggggtttttt tttagggccc tacgttcgat
     1081 gcttctttga gcgaacgatc aaaaataagt gccttcccat caaaaaaata tagacaacat
     1141 aaaaaacttt gtgttatagt tgtaacgcta catggagatt aactcaaaag gaggaaaaaa
     1201 aaatgaataa taagccgctg gatgttctga tctctgcgac cggtctgtgg atgtcccgta
     1261 ccggcacgct gcacaagatc aagcactatg aggtttctcg ttctaaaatc tacatcgaaa
     1321 tggcgtgtgg tgaccatctg gttgtgaaca actctcgctc ttgccgtccc gcacgtgcat
     1381 tccgttatca taaataccgt aaaacctgca aacgttgtcg tgtttctgac gaagatatca
     1441 acaacttcct gacccgttct accgaaggca aaacctctgt taaagttaaa gttgtttctg
     1501 cgccgaaagt gaaaaaagcg atgccgaaat ctgtttctcg tgcgccgaaa ccgctggaaa
     1561 atccggtttc tgcgaaagcg tctaccgaca cctctcgttc tgttccgtct ccggcgaaat
     1621 ctaccccgaa ctctccggtt ccgacctctg caagtgcccc cgcacttacg aagagccaga
     1681 ctgacaggct tgaagtcctg ttaaacccaa aagatgagat ttccctgaat tccggcaagc
     1741 ctttcaggga gcttgagtcc gaattgctct ctcgcagaaa aaaagacctg cagcagatct
     1801 acgcggaaga aagggagaat tatctgggga aactcgagcg tgaaattacc aggttctttg
     1861 tggacagggg ttttctggaa ataaaatccc cgatcctgat ccctcttgag tatatcgaaa
     1921 ggatgggcat tgataatgat accgaacttt caaaacagat cttcagggtt gacaagaact
     1981 tctgcctgag acccatgctt gctccaaacc tttacaacta cctgcgcaag cttgacaggg
     2041 ccctgcctga tccaataaaa atttttgaaa taggcccatg ctacagaaaa gagtccgacg
     2101 gcaaagaaca cctcgaagag tttaccatgc tgaacttctg ccagatggga tcgggatgca
     2161 cacgggaaaa tcttgaaagc ataattacgg acttcctgaa ccacctggga attgatttca
     2221 agatcgtagg cgattcctgc atggtctatg gggataccct tgatgtaatg cacggagacc
     2281 tggaactttc ctctgcagta gtcggaccca taccgcttga ccgggaatgg ggtattgata
     2341 aaccctggat aggggcaggt ttcgggctcg aacgccttct aaaggttaaa cacgacttta
     2401 aaaatatcaa gagagctgca aggtccgagt cttactataa cgggatttct accaacctgt
     2461 aacctaggct gctgccaccg ctgagatcta tggcccgcgc ccacaggaag gagctaccgg
     2521 cagcggtgcg gactgttgta actcataata agaaatgagg ccgctcatgg cgttctgttg
     2581 cccgtctcgc tggtgaaaag aaaaaccacc ctggctaact aagcggcctg ctgactttct
     2641 cgccgatcaa atgactgtcc gtgtgcttct caaatgcctg aggccagttt gctcaggctc
     2701 tccccgtgga ggtaataatt gacgatatga tcagtgcacg gctaactaag cggcctgctg
     2761 actttctcgc cgatcaaaag gcattttgct attaagggat tgacgagggc gtatctgcgc
     2821 agtaagatgc gccccgcatt cggaaacctg atcatgtaga tcgaatggac tctaaatccg
     2881 ttcagccggg ttagattccc ggggtttccg ccaggatccg tcgacctgca gatccttagc
     2941 gaaagctaag gatttttttt aagcttctgc cattcatccg cttattatca cttattcagg
     3001 cgtagcaacc aggcgtttaa gggcaccaat aactgcctta aaaaaattac gccccgccct
     3061 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacgttc atcggatccc
     3121 gggcccgtcg actgcagagg cctgcatgca agcttggcgt aatcatgatg gacttaatta
     3181 acggcactcc tcagcaaata taatgaccct cttgataacc caagagggca ttttttaatg
     3241 cccatggcgt ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt
     3301 cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc
     3361 tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag
     3421 cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa
     3481 gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt
     3541 catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg
     3601 gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt
     3661 cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt
     3721 gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga
     3781 cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt
     3841 tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa
     3901 tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca
     3961 acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt
     4021 cggcgatcac cgcttccctc atactcttcc tttttcaata ttattgaagc atttatcagg
     4081 gttattgttt catgagcgta tacatatttg aatgtattta gaaaaatatt aattgcgttg
     4141 cgctcactgc ccgctttcca ggccaaatag gccgttgaga tccttttttt ctgcgcgtaa
     4201 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4261 agctaccacc tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4321 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4381 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4441 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4501 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4561 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4621 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4681 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4741 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4801 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4861 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4921 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4981 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5041 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     5101 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5161 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5221 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     5281 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     5341 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     5401 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     5461 ggttactgat gatgaacaaa gaggacatcc ggtcaaataa aacgaaaggc tcagtcgaaa
     5521 gactgggcct ttcgttttgc tgaggagact tagggaccct gatgaaattc gccacttgtt
     5581 agtgtaattc gctaactcat cctggcatgt tgctgttgat tcttcaatca gatctttata
     5641 aatcaaaaag ataaaaaatt ggcacgcaaa ttgtattaac agttcagcag gacaatcctg
     5701 aacgcagaaa tcaagaggac aacattacct gcaggtgcag taaggaggaa aaaaaaatga
     5761 aaaaattatt attcgcaatt cctttagttg ttcctttcta ttctcactcc gctgaaactg
     5821 ttgaaagttg tttagcaaaa tagcatacag aaaattcatt tactaacgtc tggaaagacg
     5881 acaaaacttt agatcgttac gctaactatg agggctgtct gtggaatgct acaggcgttg
     5941 tagtttgtac tggtgacgaa actcagtgtt acggtacatg ggttcctatt gggcttgcta
     6001 tccctgaaaa tgagggtggt ggctctgagg gtggcggttc tgagggtggc ggttctgagg
     6061 gtggcggtac taaacctcct gagtacggtg atacacctat tccgggctat acttatatca
     6121 accctctcga cggcacttat ccgcctggta ctgagcaaaa ccccgctaat cctaatcctt
     6181 ctcttgagga gtctcagcct cttaatactt tcatgtttca gaataatagg ttccgaaata
     6241 ggcagggggc attaactgtt tatacgggca ctgttactca aggcactgac cccgttaaaa
     6301 cttattacca gtacactcct gtatcatcaa aagccatgta tgacgcttac tggaacggta
     6361 aattcagaga ctgcgctttc cattctggct ttaatgagga tccattcgtt tgtgaatatc
     6421 aaggccaatc gtctgacctg cctcaacctc ctgtcaatgc tggcggcggc tctggtggtg
     6481 gttctggtgg cggctctgag ggtggtggct ctgagggtgg cggttctgag ggtggcggct
     6541 ctgagggagg cggttccggt ggtggctctg gttccggtga ttttgattat gaaaagatgg
     6601 caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg
     6661 ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg
     6721 gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt
     6781 cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat
     6841 atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac
     6901 cttacgagtt cagtatcgac tgcgataaga tcaacctgtt ccgcggtgtc tttgcgtttc
     6961 ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata
     7021 aggagtctta atgaaatttg gaaacttttt gcttacatac caacctcccc aattttccca
     7081 aacagaggta atgaaacgtt tggttaaatt aggtcgcatc tctgaggagt gtggttttga
     7141 taccgtatgg ttactggagc atcatttcac ggagtttggt ttgcttggta acccttatgt
     7201 cgctgctgca tatttacttg gcgcgactaa aaaattgaat gtaggaactg ccgctattgt
     7261 tcttcccaca gcccatccag tacgccaact tgaagatgtg aatttattgg atcaaatgtc
     7321 aaaaggacga tttcggtttg gtatttgccg agggctttac aacaaggact ttcgcgtatt
     7381 cggcacagat atgaataaca gtcgcgcctt agcggaatgc tggtacgggc tgataaagaa
     7441 tggcatgaca gagggatata tggaagctga taatgaacat atcaagttcc ataaggtaaa
     7501 agtaaacccc gcggcgtata gcagaggtgg cgcaccggtt tatgtggtgg ctgaatcagc
     7561 ttcgacgact gagtgggctg ctcaatttgg cctaccgatg atattaagtt ggattataaa
     7621 tactaacgaa aagaaagcac aacttgagct ttataatgaa gtggctcaag aatatgggca
     7681 cgatattcat aatatcgacc attgcttatc atatataaca tctgtagatc atgactcaat
     7741 taaagcgaaa gagatttgcc ggaaatttct ggggcattgg tatgattctt atgtgaatgc
     7801 tacgactatt tttgatgatt cagaccaaac aagaggttat gatttcaata aagggcagtg
     7861 gcgtgacttt gtattaaaag gacataaaga tactaatcgc cgtattgatt acagttacga
     7921 aatcaatccc gtgggaacgc cgcaggaatg tattgacata attcaaaaag acattgatgc
     7981 tacaggaata tcaaatattt gttgtggatt tgaagctaat ggaacagtag acgaaattat
     8041 tgcttccatg aagctcttcc agtctgatgt catgccattt cttaaagaaa aacaacgttc
     8101 gctattatat tatggcggtg gcggtagcgg cggtggcggt agcggcggtg gcggtagcgg
     8161 cggtggcggt agcaaattt
//
