LOCUS       sequence-341959-        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: pJH191
FEATURES             Location/Qualifiers
     source          1..8179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3256..4047)
                     /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(4048..4119)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4180..4768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4669..4688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4922..4939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4954..5094
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5020..5039)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5180..5202
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5196..5387)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             7036..8115
                     /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(8179,1..980)
                     /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 aatttggatt gttcttcctt aacttcatca attcaacaac tgttcaagaa cagagtatag
       61 ttcgcatgca ggaaataacg gagtatgttg ataagttgaa ttttgaacag attttagtgt
      121 atgaaaatca tttttcagat aatggtgttg tcggcgctcc tctgactgtt tctggttttc
      181 tgctcggttt aacagagaaa attaaaattg gttcattaaa tcacatcatt acaactcatc
      241 atcctgtccg catagcggag gaagcttgct tattggatca gttaagtgaa gggagattta
      301 ttttagggtt tagtgattgc gaaaaaaaag atgaaatgca tttttttaat cgcccggttg
      361 aatatcaaca gcaactattt gaagagtgtt atgaaatcat taacgatgct ttaacaacag
      421 gctattgtaa tccagataac gatttttata gcttccctaa aatatctgta aatccccatg
      481 cttatacgcc aggcggacct cggaaatatg taacagcaac cagtcatcat attgttgagt
      541 gggcggccaa aaaaggtatt cctctcatct ttaagtggga tgattctaat gatgttagat
      601 atgaatatgc tgaaagatat aaagccgttg cggataaata tgacgttgac ctatcagaga
      661 tagaccatca gttaatgata ttagttaact ataacgaaga tagtaataaa gctaaacaag
      721 agacgcgtgc atttattagt gattatgttc ttgaaatgca ccctaatgaa aatttcgaaa
      781 ataaacttga agaaataatt gcagaaaacg ctgtcggaaa ttatacggag tgtataactg
      841 cggctaagtt ggcaattgaa aagtgtggtg cgaaaagtgt attgctgtcc tttgaaccaa
      901 tgaatgattt gatgagccaa aaaaatgtaa tcaatattgt tgatgataat attaagaagt
      961 accacacgga atatacctaa acggagccaa tgtacgcaaa aaaccccgct tcagcggggt
     1021 tttttcgcca aaaaaaaccc cgccctgtca ggggcggggt ttttttttag ggccctacgt
     1081 tcgatgcttc tttgagcgaa cgatcaaaaa taagtgcctt cccatcaaaa aaatatagac
     1141 aacataaaaa actttgtgtt atagttgtaa cgctacatgg agattaactc aaaaggagga
     1201 aaaaaaaatg aataataagc cgctggatgt tctgatctct gcgaccggtc tgtggatgtc
     1261 ccgtaccggc acgctgcaca agatcaagca ctatgaggtt tctcgttcta aaatctacat
     1321 cgaaatggcg tgtggtgacc atctggttgt gaacaactct cgctcttgcc gtcccgcacg
     1381 tgcattccgt tatcataaat accgtaaaac ctgcaaacgt tgtcgtgttt ctgacgaaga
     1441 tatcaacaac ttcctgaccc gttctaccga aggcaaaacc tctgttaaag ttaaagttgt
     1501 ttctgcgccg aaagtgaaaa aagcgatgcc gaaatctgtt tctcgtgcgc cgaaaccgct
     1561 ggaaaatccg gtttctgcga aagcgtctac cgacacctct cgttctgttc cgtctccggc
     1621 gaaatctacc ccgaactctc cggttccgac ctctgcaagt gcccccgcac ttacgaagag
     1681 ccagactgac aggcttgaag tcctgttaaa cccaaaagat gagatttccc tgaattccgg
     1741 caagcctttc agggagcttg agtccgaatt gctctctcgc agaaaaaaag acctgcagca
     1801 gatctacgcg gaagaaaggg agaattatct ggggaaactc gagcgtgaaa ttaccaggtt
     1861 ctttgtggac aggggttttc tggaaataaa atccccgatc ctgatccctc ttgagtatat
     1921 cgaaaggatg ggcattgata atgataccga actttcaaaa cagatcttca gggttgacaa
     1981 gaacttctgc ctgagaccca tgcttgctcc aaacctttac aactacctgc gcaagcttga
     2041 cagggccctg cctgatccaa taaaaatttt tgaaataggc ccatgctaca gaaaagagtc
     2101 cgacggcaaa gaacacctcg aagagtttac catgctgaac ttctgccaga tgggatcggg
     2161 atgcacacgg gaaaatcttg aaagcataat tacggacttc ctgaaccacc tgggaattga
     2221 tttcaagatc gtaggcgatt cctgcatggt ctattgggat acccttgatg taatgcacgg
     2281 agacctggaa ctttcctctg cagtagtcgg acccataccg cttgaccggg aatggggtat
     2341 tgataaaccc tggatagggg caggtttcgg gctcgaacgc cttctaaagg ttaaacacga
     2401 ctttaaaaat atcaagagag ctgcaaggtc cgagtcttac tataacggga tttctaccaa
     2461 cctgtaacct aggctgctgc caccgctgag atctatggcc cgcgcccaca ggaaggagct
     2521 accggcagcg gtgcggactg ttgtaactca taataagaaa tgaggccgct catggcgttc
     2581 tgttgcccgt ctcgctggtg aaaagaaaaa ccaccctggc taactaagcg gcctgctgac
     2641 tttctcgccg atcaaatgac tgtccgtgtg cttctcaaat gcctgaggcc agtttgctca
     2701 ggctctcccc gtggaggtaa taattgacga tatgatcagt gcacggctaa ctaagcggcc
     2761 tgctgacttt ctcgccgatc aaaaggcatt ttgctattaa gggattgacg agggcgtatc
     2821 tgcgcagtaa gatgcgcccc gcattcggaa acctgatcat gtagatcgaa tggactctaa
     2881 atccgttcag ccgggttaga ttcccggggt ttccgccagg atccgtcgac ctgcagatcc
     2941 ttagcgaaag ctaaggattt tttttaagct tctgccattc atccgcttat tatcacttat
     3001 tcaggcgtag caaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc
     3061 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga cgttcatcgg
     3121 atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca tgatggactt
     3181 aattaacggc actcctcagc aaatataatg accctcttga taacccaaga gggcattttt
     3241 taatgcccat ggcgtttatt tgccgactac cttggtgatc tcgcctttca cgtagtggac
     3301 aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc
     3361 ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc
     3421 ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa
     3481 cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa
     3541 ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc
     3601 cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc
     3661 aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc
     3721 aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat
     3781 ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag
     3841 gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa
     3901 atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc
     3961 cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga
     4021 tacttcggcg atcaccgctt ccctcatact cttccttttt caatattatt gaagcattta
     4081 tcagggttat tgtttcatga gcgtatacat atttgaatgt atttagaaaa atattaattg
     4141 cgttgcgctc actgcccgct ttccaggcca aataggccgt tgagatcctt tttttctgcg
     4201 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4261 tcaagagcta ccacctcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4321 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4381 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4441 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4501 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4561 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4621 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4681 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4741 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4801 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4861 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4921 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     4981 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc
     5041 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5101 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5161 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5221 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5281 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5341 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5401 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5461 atacgggtta ctgatgatga acaaagagga catccggtca aataaaacga aaggctcagt
     5521 cgaaagactg ggcctttcgt tttgctgagg agacttaggg accctgatga aattcgccac
     5581 ttgttagtgt aattcgctaa ctcatcctgg catgttgctg ttgattcttc aatcagatct
     5641 ttataaatca aaaagataaa aaattggcac gcaaattgta ttaacagttc agcaggacaa
     5701 tcctgaacgc agaaatcaag aggacaacat tacctgcagg tgcagtaagg aggaaaaaaa
     5761 aatgaaaaaa ttattattcg caattccttt agttgttcct ttctattctc actccgctga
     5821 aactgttgaa agttgtttag caaaatagca tacagaaaat tcatttacta acgtctggaa
     5881 agacgacaaa actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg
     5941 cgttgtagtt tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct
     6001 tgctatccct gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggttc
     6061 tgagggtggc ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta
     6121 tatcaaccct ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa
     6181 tccttctctt gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg
     6241 aaataggcag ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt
     6301 taaaacttat tagcagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa
     6361 cggtaaattc agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga
     6421 atatcaaggc caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg
     6481 tggtggttct ggtggcggct ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg
     6541 cggctctgag ggaggcggtt ccggtggtgg ctctggttcc ggtgattttg attatgaaaa
     6601 gatggcaaac gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc
     6661 tgacgctaaa ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt
     6721 cattggtgac gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc
     6781 taattcccaa atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg
     6841 tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg
     6901 taaaccttac gagttcagta tcgactgcga taagatcaac ctgttccgcg gtgtctttgc
     6961 gtttctttta tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg
     7021 taataaggag tcttaatgaa atttggaaac tttttgctta cataccaacc tccccaattt
     7081 tcccaaacag aggtaatgaa acgtttggtt aaattaggtc gcatctctga ggagtgtggt
     7141 tttgataccg tatggttact ggagcatcat ttcacggagt ttggtttgct tggtaaccct
     7201 tatgtcgctg ctgcatattt acttggcgcg actaaaaaat tgaatgtagg aactgccgct
     7261 attgttcttc ccacagccca tccagtacgc caacttgaag atgtgaattt attggatcaa
     7321 atgtcaaaag gacgatttcg gtttggtatt tgccgagggc tttacaacaa ggactttcgc
     7381 gtattcggca cagatatgaa taacagtcgc gccttagcgg aatgctggta cgggctgata
     7441 aagaatggca tgacagaggg atatatggaa gctgataatg aacatatcaa gttccataag
     7501 gtaaaagtaa accccgcggc gtatagcaga ggtggcgcac cggtttatgt ggtggctgaa
     7561 tcagcttcga cgactgagtg ggctgctcaa tttggcctac cgatgatatt aagttggatt
     7621 ataaatacta acgaaaagaa agcacaactt gagctttata atgaagtggc tcaagaatat
     7681 gggcacgata ttcataatat cgaccattgc ttatcatata taacatctgt agatcatgac
     7741 tcaattaaag cgaaagagat ttgccggaaa tttctggggc attggtatga ttcttatgtg
     7801 aatgctacga ctatttttga tgattcagac caaacaagag gttatgattt caataaaggg
     7861 cagtggcgtg actttgtatt aaaaggacat aaagatacta atcgccgtat tgattacagt
     7921 tacgaaatca atcccgtggg aacgccgcag gaatgtattg acataattca aaaagacatt
     7981 gatgctacag gaatatcaaa tatttgttgt ggatttgaag ctaatggaac agtagacgaa
     8041 attattgctt ccatgaagct cttccagtct gatgtcatgc catttcttaa agaaaaacaa
     8101 cgttcgctat tatattatgg cggtggcggt agcggcggtg gcggtagcgg cggtggcggt
     8161 agcggcggtg gcggtagca
//
