LOCUS       sequence-338567-        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: pJH61
FEATURES             Location/Qualifiers
     source          1..8179
                     /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="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     CDS             complement(3271..4062)
                     /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(4063..4134)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4195..4783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4684..4703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4937..4954
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4969..5109
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5035..5054)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5195..5217
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5211..5402)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             7051..8130
                     /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 aacagatttt 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 tagacaacat aaaaaacttt gtgttatagt tgtaacgcta catggagatt
     1201 aactcaaaag gaggaaaaaa aaatggataa gaagccgctg gatgttctga tctctgcgac
     1261 cggtctgtgg atgtcccgta ccggcacgct gcacaagatc aagcactatg aggtttctcg
     1321 ttctaaaatc tacatcgaaa tggcgtgtgg tgaccatctg gttgtgaaca actctcgttc
     1381 ttgtcgtacc gcacgtgcat tccgtcatca taaataccgt aaaacctgca aacgttgtcg
     1441 tgtttctgac gaagatatca acaacttcct gacccgttct accgaaggca aaacctctgt
     1501 taaagttaaa gttgtttctg cgccgaaagt gaaaaaagcg atgccgaaat ctgtttctcg
     1561 tgcgccgaaa ccgctggaaa atccggtttc tgcgaaagcg tctaccgaca cctctcgttc
     1621 tgttccgtct ccggcgaaat ctaccccgaa ctctccggtt ccgacctctg caagtgcccc
     1681 cgcacttacg aagagccaga ctgacaggct tgaagtcctg ttaaacccaa aagatgagat
     1741 ttccctgaat tccggcaagc ctttcaggga gcttgagtcc gaattgctct ctcgcagaaa
     1801 aaaagacctg cagcagatct acgcggaaga aagggagaat tatctgggga aactcgagcg
     1861 tgaaattacc aggttctttg tggacagggg ttttctggaa ataaaatccc cgatcctgat
     1921 ccctcttgag tatatcgaaa ggatgggcat tgataatgat accgaacttt caaaacagat
     1981 cttcagggtt gacaagaact tctgcctgag acccatgctt gctccaaacc tttacaacta
     2041 cctgcgcaag cttgacaggg ccctgcctga tccaataaaa atttttgaaa taggcccatg
     2101 ctacagaaaa gagtccgacg gcaaagaaca cctcgaagag tttaccatgc tgaacttctg
     2161 ccagatggga tcgggatgca cacgggaaaa tcttgaaagc ataattacgg acttcctgaa
     2221 ccacctggga attgatttca agatcgtagg cgattcctgc atggtctatg gggataccct
     2281 tgatgtaatg cacggagacc tggaactttc ctctgcagta gtcggaccca taccgcttga
     2341 ccgggaatgg ggtattgata aaccctggat aggggcaggt ttcgggctcg aacgccttct
     2401 aaaggttaaa cacgacttta aaaatatcaa gagagctgca aggtccgagt cttactataa
     2461 cgggatttct accaacctgt aacctaggct gctgccaccg ctgagatcta tggcccgcgc
     2521 ccacaggaag gagctaccgg cagcggtgcg gactgttgta actcataata agaaatgagg
     2581 ccgctcatgg cgttctgttg cccgtctcgc tggtgaaaag aaaaaccacc ctggctaact
     2641 aagcggcctg ctgactttct cgccgatcaa atgactgtcc gtgtgcttct caaatgcctg
     2701 aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga tcagtgcacg
     2761 gctaactaag cggcctgctg actttctcgc cgatcaaaag gcattttgct attaagggat
     2821 tgacgagggc gtatctgcgc agtaagatgc gccccgcatt cggaaacctg atcatgtaga
     2881 tcgaatggac tctaaatccg ttcagccggg ttagattccc ggggtttccg ccaggatccg
     2941 tcgacctgca gatccttagc gaaagctaag gatttttttt aagcttctgc cattcatccg
     3001 cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat aactgcctta
     3061 aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     3121 gccgacgttc atcggatccc gggcccgtcg actgcagagg cctgcatgca agcttggcgt
     3181 aatcatgatg gacttaatta acggcactcc tcagcaaata taatgaccct cttgataacc
     3241 caagagggca ttttttaatg cccatggcgt ttatttgccg actaccttgg tgatctcgcc
     3301 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
     3361 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     3421 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     3481 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     3541 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     3601 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     3661 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     3721 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     3781 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     3841 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     3901 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     3961 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     4021 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     4081 ttattgaagc atttatcagg gttattgttt catgagcgta tacatatttg aatgtattta
     4141 gaaaaatatt aattgcgttg cgctcactgc ccgctttcca ggccaaatag gccgttgaga
     4201 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4261 ggtttgtttg ccggatcaag agctaccacc tctttttccg aaggtaactg gcttcagcag
     4321 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4381 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4441 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4501 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4561 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4621 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4681 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4741 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4801 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4861 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4921 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4981 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     5041 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     5101 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5161 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5221 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5281 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5341 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5401 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5461 gagaggatgc tcacgatacg ggttactgat gatgaacaaa gaggacatcc ggtcaaataa
     5521 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttgc tgaggagact tagggaccct
     5581 gatgaaattc gccacttgtt agtgtaattc gctaactcat cctggcatgt tgctgttgat
     5641 tcttcaatca gatctttata aatcaaaaag ataaaaaatt ggcacgcaaa ttgtattaac
     5701 agttcagcag gacaatcctg aacgcagaaa tcaagaggac aacattacct gcaggtgcag
     5761 taaggaggaa aaaaaaatga aaaaattatt attcgcaatt cctttagttg ttcctttcta
     5821 ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa tagcatacag aaaattcatt
     5881 tactaacgtc tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct
     5941 gtggaatgct acaggcgttg tagtttgtac tggtgacgaa actcagtgtt acggtacatg
     6001 ggttcctatt gggcttgcta tccctgaaaa tgagggtggt ggctctgagg gtggcggttc
     6061 tgagggtggc ggttctgagg gtggcggtac taaacctcct gagtacggtg atacacctat
     6121 tccgggctat acttatatca accctctcga cggcacttat ccgcctggta ctgagcaaaa
     6181 ccccgctaat cctaatcctt ctcttgagga gtctcagcct cttaatactt tcatgtttca
     6241 gaataatagg ttccgaaata ggcagggggc attaactgtt tatacgggca ctgttactca
     6301 aggcactgac cccgttaaaa cttattacca gtacactcct gtatcatcaa aagccatgta
     6361 tgacgcttac tggaacggta aattcagaga ctgcgctttc cattctggct ttaatgagga
     6421 tccattcgtt tgtgaatatc aaggccaatc gtctgacctg cctcaacctc ctgtcaatgc
     6481 tggcggcggc tctggtggtg gttctggtgg cggctctgag ggtggtggct ctgagggtgg
     6541 cggttctgag ggtggcggct ctgagggagg cggttccggt ggtggctctg gttccggtga
     6601 ttttgattat gaaaagatgg caaacgctaa taagggggct atgaccgaaa atgccgatga
     6661 aaacgcgcta cagtctgacg ctaaaggcaa acttgattct gtcgctactg attacggtgc
     6721 tgctatcgat ggtttcattg gtgacgtttc cggccttgct aatggtaatg gtgctactgg
     6781 tgattttgct ggctctaatt cccaaatggc tcaagtcggt gacggtgata attcaccttt
     6841 aatgaataat ttccgtcaat atttaccttc cctccctcaa tcggttgaat gtcgcccttt
     6901 tgtctttggc gctggtaaac cttacgagtt cagtatcgac tgcgataaga tcaacctgtt
     6961 ccgcggtgtc tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt
     7021 tgctaacata ctgcgtaata aggagtctta atgaaatttg gaaacttttt gcttacatac
     7081 caacctcccc aattttccca aacagaggta atgaaacgtt tggttaaatt aggtcgcatc
     7141 tctgaggagt gtggttttga taccgtatgg ttactggagc atcatttcac ggagtttggt
     7201 ttgcttggta acccttatgt cgctgctgca tatttacttg gcgcgactaa aaaattgaat
     7261 gtaggaactg ccgctattgt tcttcccaca gcccatccag tacgccaact tgaagatgtg
     7321 aatttattgg atcaaatgtc aaaaggacga tttcggtttg gtatttgccg agggctttac
     7381 aacaaggact ttcgcgtatt cggcacagat atgaataaca gtcgcgcctt agcggaatgc
     7441 tggtacgggc tgataaagaa tggcatgaca gagggatata tggaagctga taatgaacat
     7501 atcaagttcc ataaggtaaa agtaaacccc gcggcgtata gcagaggtgg cgcaccggtt
     7561 tatgtggtgg ctgaatcagc ttcgacgact gagtgggctg ctcaatttgg cctaccgatg
     7621 atattaagtt ggattataaa tactaacgaa aagaaagcac aacttgagct ttataatgaa
     7681 gtggctcaag aatatgggca cgatattcat aatatcgacc attgcttatc atatataaca
     7741 tctgtagatc atgactcaat taaagcgaaa gagatttgcc ggaaatttct ggggcattgg
     7801 tatgattctt atgtgaatgc tacgactatt tttgatgatt cagaccaaac aagaggttat
     7861 gatttcaata aagggcagtg gcgtgacttt gtattaaaag gacataaaga tactaatcgc
     7921 cgtattgatt acagttacga aatcaatccc gtgggaacgc cgcaggaatg tattgacata
     7981 attcaaaaag acattgatgc tacaggaata tcaaatattt gttgtggatt tgaagctaat
     8041 ggaacagtag acgaaattat tgcttccatg aagctcttcc agtctgatgt catgccattt
     8101 cttaaagaaa aacaacgttc gctattatat tatggcggtg gcggtagcgg cggtggcggt
     8161 agcggcggtg gcggtagcg
//
