LOCUS       sequence-338628-        7316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pylBCD variant in pBAD backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7316)
  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 7316)
  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: pJH462
FEATURES             Location/Qualifiers
     source          1..7316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      37..64
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        83..174
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(175..243,244..1035)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(393..412)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1206..1794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1695..1714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1948..1965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1980..2120
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2046..2065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2206..2228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2384..3262)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3289..3573
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     3496..3515
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3622..3915
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(7005..7022)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      7175..7261
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
       61 tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg
      121 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      181 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      241 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      301 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      361 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
      421 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      481 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
      541 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
      601 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
      661 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
      721 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
      781 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
      841 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
      901 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
      961 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1021 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1081 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1141 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1201 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1261 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1321 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1381 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1441 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1501 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1561 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1621 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1681 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1741 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     1801 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     1861 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     1921 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     1981 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2041 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2101 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2161 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2221 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga
     2281 aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc
     2341 tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg
     2401 ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc
     2461 attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg
     2521 tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct
     2581 cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg
     2641 gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct gccaggtgat
     2701 cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg agcgactcgt
     2761 taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc agcagctccg
     2821 aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg
     2881 gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac ggccagttaa
     2941 gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac cattcgcgag
     3001 cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa atatcacccg
     3061 gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga atggtgagat
     3121 tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg
     3181 cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc ggcagcaggg
     3241 gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa gaaaccaatt
     3301 gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc tcgctaacca
     3361 aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa
     3421 aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg
     3481 gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac
     3541 gctttttatc gcaactctct actgtttctc catacccgtt ttttgggcta acaggaggaa
     3601 ttaaccatga tgggcagcag catgtcggac tcagaagtca atcaagaagc taagccagag
     3661 gtcaagccag aagtcaagcc tgagactcac atcaatttaa aggtgtccga tgggtcttca
     3721 gagatcttct tcaagatcaa aaagaccact cctttaagaa ggctgatgga agcgttcgct
     3781 aaaagacagg gtaaggaaat ggactcctta agattcttgt acgacggtat tagaattcaa
     3841 gctgatcaga cccctgaaga tttggacatg gaggataacg atattattga ggctcacaga
     3901 gaacagattg gtggtcatat gattaagaag atggcgactg aagatctgga tcgttttggc
     3961 gaaaaaatta ttgaaggatt tcagttatct gacgacgatt tacgcgcttt gcttagcctg
     4021 gaatcggaag aagaattgga aaagctgtat tacgtcgcac gtaaagtacg caaccattac
     4081 tttggaaatc gcgtattcct gaactgtttt atctacttca gcacatactg caaaaaccaa
     4141 tgtgcgtttt gctattataa ctgcaaaaac gagattaacc gctatcgttt gtcccgcgaa
     4201 gaaatcaaag agacttgcaa ggcccttaaa ggtgcgggtt tccatatgat tgatctgact
     4261 atgggggagg atccctatta ttacgaggaa cctgatcgct tcgtggagct ggtccaaatg
     4321 gttaagaatg agcttggact tccaatcatg atttcgccag gagtaatgga taatgctacc
     4381 cttttaaaag cacgcgaaaa gggagctaat ttcttcgcct tatatcaaga aacctatgat
     4441 caagagttgt acgagaagtt gcgcgtaggt cagtcgttca aggggcgcta caatactcgt
     4501 cgttttgcta aagaacaagg gtattgcatt gaagacggta tcttaacggg agtcgggaac
     4561 gatatcgagt caacgatcaa atctttacgc ggcatgaaat caaatgatcc cgacatggtt
     4621 cgcgtgatga ctttcttacc ccaggaagga actccccttg agcgttttaa ggaaaatagt
     4681 aacctgagcg aactgaaaat cattgcgatc ttgcgtctga tctttccgaa gtgcttaatc
     4741 cccgcgtcgt tggacctgga agggatcgac gggatggtac accgtcttaa cgcaggtgct
     4801 aatatcgtga ctagcatcct tcctagtgat tcaaaacttg agggagttgc taactacgat
     4861 cgtgacctgg aagagcgcga ccgcgacatt aaaagcgtca tcaagcgcct tgagggaatg
     4921 ggcatggaac cagcgccaca ggcggaattc gagactgtac tgcgctgcta ataaggagga
     4981 aaaatgaaaa ccatctgcct gattggtggt aaattgcagg gttttgaggc cgcgtatctg
     5041 tctaaaaaag ccgatatgaa agtagtggtt atcgacaaga acccccaagc tcttatccgc
     5101 aattacgcag acgagtttca ttgttttgat gttatcaagg aaccagagaa acttcttgaa
     5161 atcagcaaaa atgtagacgc tatcttgccc gtcaacgaaa acttagaatg tattaaattc
     5221 ttaagtagtg taaaagagga attttcttgc ccagtactgt tcgattttga ggcttaccgc
     5281 atctcgcgcg ataagaagaa atcgaaagag tatttccgtt ccatcggcat tccgacaccg
     5341 caagacaaac ctggtcgtcc tccatacttc gttaagccac cgtgcgaaag ctcgtcggtt
     5401 ggtgctcgca ttatctacga tgaagaagaa ctgggagaat tggagcccgg catgctggta
     5461 gaagagtatg tggaaggtga ggttatttca cttgaagtaa tcggggacgg tacccacttt
     5521 gcagttgtta aagagacact tattcacatc gaccgcacct atgactgcca tatggtaact
     5581 cccctgccat ctgatttgtc tttccgtgag atcagctatt ctttagcggc caacctgcct
     5641 ttgaagggga tcatggacgt tgaagcaatt tcgggcccgc aaggtttgaa ggtcattgaa
     5701 attgattcac gtttcccctc ccagaccccc acagccgtgt attattcgtc gggagtgaat
     5761 ttaattgagc tgttgtttcg tgcctttgga gaaggggtgg aggaagttaa aacccttcca
     5821 gaggaccgct actgtatcta cgagcacctg atgcttgcag aaaatggggc cctgatccca
     5881 gtaggtgagc aggttctgtc gatggggaat gactatggga aatactatga agaaccagga
     5941 attgaaatct ttttatgtcg cggagaggac cccgtgttta cgcttgtctt ttggggaaag
     6001 gaccgtgagg aagccgaaaa caaaaagcgc actggtttgc tgattttgaa ggaccgcttc
     6061 ggggcagccg tttaaaatgg caaaaatcgg ataagaataa aaatttgccc aaagtaaaat
     6121 ttgtttaaaa cagaaaaagg agataaaaaa tggctttgtt gactcccgag gacctggaga
     6181 acattaaccg tcaacttcac gaagccgatt ctacggtacg tcgtgtgact gggttagata
     6241 tcaagggagt ttgtaaagat ttctatggga ctactccatg ctgtgagaag gtcggtatcg
     6301 tcccggtaac tagtggcaac ggcatcattg gcaacttttc tgaaagtctt cgtgcgatca
     6361 ctgagtactt tggatttgac tcattcgtga cgaccatgcc agacgttagt ggctactatg
     6421 aggcggtccg taatggagca caaattattc ttatggcaga tgataatacg tttcttgcac
     6481 acaatttaaa gaacggcaag atcgctaata atcaaccgtg taccggaatt atctacgcgg
     6541 agatcgcctc gcgctatatg aaagccgatt ccaaagatgt gcttgccgtc gggcttggaa
     6601 aagtggggtt tccaggcgct gcacatttgg ttaataaggg gtttcgtgtg tatgggtacg
     6661 acgcggataa ggcgttgtta gaaaagactg tatcagacct tgggatcaca cctttcgagc
     6721 ctgagaatcc ccgtcgcttc tcaatcattt ttgaggcgac cccgtgtgct gatacagtac
     6781 cagagtctgt catcagcgac aattgtgtca tctctacgcc cggtatccct tgcgcgatca
     6841 gccgtgaact tcaagaaaaa tacggggtgg agttgattat ggaacctctg ggtattggaa
     6901 ctgcttctat gctgtatagc attttgtaaa gctcgagatc tgcagctggt accatatggg
     6961 aattcgaagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     7021 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     7081 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     7141 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     7201 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     7261 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcag
//
