LOCUS       sequence-338825-        7327 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 7327)
  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 7327)
  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: pJH575
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(103..120)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      273..359
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      451..478
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        497..588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(589..657,658..1449)
                     /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(807..826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1620..2208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2109..2128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2373..2390
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2405..2545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2471..2490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2631..2653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2809..3687)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3714..3998
                     /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     3921..3940
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             4047..4340
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MLDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 gtttctatgc tgtatagcat tttgtaaagc tcgagatctg cagctggtac catatgggaa
       61 ttcgaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc
      121 agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
      181 acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc
      241 tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag
      301 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
      361 cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc
      421 cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
      481 cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg
      541 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      601 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      661 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      721 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
      781 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
      841 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      901 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      961 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1021 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1081 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1141 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1201 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1261 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1321 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     1381 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     1441 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1501 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1741 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctggcctttt gctcacatgt
     2281 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2341 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2401 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     2461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     2521 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     2581 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     2641 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg
     2701 cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca
     2761 aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt
     2821 gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc
     2881 ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc
     2941 gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg
     3001 gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg
     3061 cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag
     3121 gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga
     3181 ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag
     3241 ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa
     3301 atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca
     3361 gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc
     3421 gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc
     3481 acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt
     3541 gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc
     3601 gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag
     3661 caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca gagaagaaac
     3721 caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc tcttctcgct
     3781 aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga ccaaagccat
     3841 gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca ttgattattt
     3901 gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag cggatcctac
     3961 ctgacgcttt ttatcgcaac tctctactgt ttctccatac ccgttttttg ggctaacagg
     4021 aggaattaac catgatgggc agcagcatgt tggactcaga agtcaatcaa gaagctaagc
     4081 cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg tccgatgggt
     4141 cttcagagat cttcttcaag atcaaaaaga ccactccttt aagaaggctg atggaagcgt
     4201 tcgctaaaag acagggtaag gaaatggact ccttaagatt cttgtacgac ggtattagaa
     4261 ttcaagctga tcagacccct gaagatttgg acatggagga taacgatatt attgaggctc
     4321 acagagaaca gattggtggt catatgatta agaagatggc gactgaagat ctggatcgtt
     4381 ttggcgaaaa aattattgaa ggatttcagt tatctgacga cgatttacgc gctttgctta
     4441 gcctggaatc ggaagaagaa ttggaaaagc tgtattacgt cgcacgtaaa gtacgcaacc
     4501 attactttgg aaatcgcgta ttcctgagct gttttatcta cttcagcaca tactgcaaaa
     4561 accaatgtgc gttttgctat tataactgca aaaacgagat taaccgctat cgtttgtccc
     4621 gcgaagaaat caaagagact tgcaaggccc ttaaaggtgc gggtttccat atgattgatc
     4681 tgactatggg ggaggatccc tattattacg aggaacctga tcgcttcgtg gagctggtcc
     4741 aaatggttaa gaatgagctt ggacttccaa tcatgatttc gccaggagta atggataatg
     4801 ctaccctttt aaaagcacgc gaaaagggag ctaatttctt cgccttatat caagaaacct
     4861 atgatcaaga gttgtacaag aagttgcgcg taggtcagtc gttcaagagg cgctacaata
     4921 ctcgtcgttt tgctaaagaa caagggtatt gcattgaaga cggtatctta acgggagtcg
     4981 ggaacgatat cgagtcaacg atcaaatctt tacgcggcat gaaatcaaat gatcccgaca
     5041 tggttcgcgt gatgactttc ttaccccagg aaggaactcc ccttgagcgt tttaaggaca
     5101 atagtaacct gagcgaactg aaaatcattg cgatcttgcg tctgatcttt ccgaagtgct
     5161 taatccccgc gtcgttggac ctggaaggga tcgacgggat ggtacaccgt cttaacgcag
     5221 gtgctaatat cgtgactagc atccttccta gtgattcaaa acttgaggga gttgctaact
     5281 acgatcgtga cctggaagag cgcgaccgcg acattaaaag cgtcatcaag cgccttgagg
     5341 gaatgggcat ggaaccagcg ccacaggcgg aattcgagac tgtactgcgc tgctaataag
     5401 gaggaaaaat gaaaaccatc tgcctgattg gtggtaaatt gcagggtttt gaggccgcgt
     5461 atctgtctaa aaaagccgat atgaaagtag tggttatcga caagaacccc caagctctta
     5521 tccgcaatta cgcagacgag tttcattgtt ttgatgttat caaggaacca gagaaacttc
     5581 ttgaaatcag caaaaatgta gacgctatct tgcccgtcaa cgaaaactta gaatgtatta
     5641 aattcttaag tagtgtaaaa gaggaatttt cttgcccagt actgttcgat tttgaggctt
     5701 accgcatctc gcgcgataag aagaaatcga aagagtattt ccgttccatc ggcattccga
     5761 caccgcaaga caaacctggt cgtcctccat acttcgttaa gccaccgtgc gaaagctcgt
     5821 cggttggtgc tcgcattatc tacgatgaag aagaactggg agaattggag cccggcatgc
     5881 tggtagaaga gtatgtggaa ggtgaggtta tttcacttga agtaatcggg gacggtaccc
     5941 actttgcagt tgttaaagag acacttattc acatcgaccg cacctatgac tgccatatgg
     6001 taactcccct gccatctgat ttgtctttcc gtgagatcag ctattcttta gcggccaacc
     6061 tgcctttgaa ggggatcatg gacgttgaag caatttcggg cccgcaaggt ttgaaggtca
     6121 ttgaaattga ttcacgtttc ccctcccaga cccccacagc cgtgtattat tcgtcgggag
     6181 tgaatttaat tgagctgttg tttcgtgcct ttggagaagg ggtggaggaa gttaaaaccc
     6241 ttccagagga ccgctactgt atctacgagc acctgatgct tgcagaaaat ggggccctga
     6301 tcccagtagg tgagcaggtt ctgtcgatgg ggaatgacta tgggaaatac tatgaagaac
     6361 caggaattga aatcttttta tgtcgcggag aggaccccgt gtttacgctt gtcttttggg
     6421 gaaaggaccg tgaggaagcc gaaaacaaaa agcgcactgg tttgctgatt ttgaaggacc
     6481 gcttcggggc agccgtttaa aatggcaaaa atcggataag aataaaaatt tgcccaaagt
     6541 aaaatttgtt taaaacagaa aaaggagata aaaaatggct ttgttgactc ccgaggacct
     6601 ggagaacatt aaccgtcaac ttcacgaagc cgattctacg gtacgtcgtg tgactgggtt
     6661 agatatcaag ggagtttgta aagatttcta tgggactact ccatgctgtg agaaggtcgg
     6721 tatcgtcccg gtaactagtg gcaacggcat cattggcaac ttttctgaaa gtcttcgtgc
     6781 gatcactgag tactttggat ttgactcatt cgtgacgacc atgccagacg ttagtggcta
     6841 ctatgaggcg gtccgtaatg gagcacaaat tattcttatg gcagatgata atacgtttct
     6901 tgcacacaat ttaaagaacg gcaagatcgc taataatcaa ccgtgtaccg gaattatcta
     6961 cgcggagatc gcctcgcgct atatgaaagc cgattccaaa gatgtgcttg ccgtcgggct
     7021 tggaaaagtg gggtttccag gcgctgcaca tttggttaat aaggggtttc gtgtgtatgg
     7081 gtacgacgcg gataaggcgt tgttagaaaa gactgtatca gaccttggga tcacaccttt
     7141 cgagcctgag aatccccgtc gcttctcaat catttttgag gcgaccccgt gtgctgatac
     7201 agtaccagag tctgtcatca gcgacaattg tgtcatctct acgcccggta tcccttgcgc
     7261 gatcagccgt gaacttcaag aaaaatacgg ggtggagttg attatggaac ctctgggtat
     7321 tggaact
//
