LOCUS       sequence-338850-        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: pJH463
FEATURES             Location/Qualifiers
     source          1..7316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             49..342
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3432..3449)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3602..3688
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3780..3807
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3826..3917
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3918..3986,3987..4778)
                     /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(4136..4155)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4949..5537
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5438..5457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5691..5708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5723..5863
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5789..5808)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5949..5971
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6127..7005)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        7032..7316
                     /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     7239..7258
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 acccgttttt tgggctaaca ggaggaatta accatgatgg gcagcagcat gtcggactca
       61 gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga gactcacatc
      121 aatttaaagg tgtccgatgg gtcttcagag atcttcttca agatcaaaaa gaccactcct
      181 ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga ctccttaaga
      241 ttcttgtacg acggtattag aattcaagct gatcagaccc ctgaagattt ggacatggag
      301 gataacgata ttattgaggc tcacagagaa cagattggtg gtcatatgat taagaagatg
      361 gcgactgaag atctggatcg ttttggcgaa aaaattattg aaggatttca gttatctgac
      421 gacgatttac gcgctttgct tagcctggaa tcggaagaag aattggaaaa gctgtattac
      481 gtcgcacgta aagtacgcaa ccattacttt ggaaatcgcg tattcctgag ctgttttatc
      541 tacttcagca catactgcaa aaaccaatgt gcgttttgct attataactg caaaaacgag
      601 attaaccgct atcgtttgtc ccgcgaagaa atcaaagaga cttgcaaggc ccttaaaggt
      661 gcgggtttcc atatgattga tctgactatg ggggaggatc cctattatta caaggaacct
      721 gatcgcttcg tggagctggt ccaaatggtt aagaatgagc ttggacttcc aatcatgatt
      781 tcgccaggag taatggataa tgctaccctt ttaaaagcac gcgaaaaggg agctaatttc
      841 ttcgccttat atcaagaaac ctatgatcaa gagttgtaca agaagttgcg cgtaggtcag
      901 tcgttcaagc ggcgctacaa tactcgtcgt tttgctaaag aacaagggta ttgcattgaa
      961 gacggtatct taacgggagt cgggaacgat atcgagtcaa cgatcaaatc tttacgcggc
     1021 atgaaatcaa atgatcccga catggttcgc gtgatgactt tcttacccca ggaaggaact
     1081 ccccttgagc gttttaagga aaatagtaac ctgagcgaac tgaaaatcat tgcgatcttg
     1141 cgtctgatct ttccgaagtg cttaatcccc gcgtcgttgg acctggaagg gatcgacggg
     1201 atggtacacc gtcttaacgc aggtgctaat atcgtgacta gcatccttcc tagtgattca
     1261 aaacttgagg gagttgctaa ctacgatcgt gacctggaag agcgcgaccg cgacattaaa
     1321 agcgtcatca agcgccttga gggaatgggc atggaaccag cgccacaggc ggaattcgag
     1381 actgtactgc gctgctaata aggaggaaaa atgaaaacca tctgcctgat tggtggtaaa
     1441 ttgcagggtt ttgaggccgc gtatctgtct aaaaaagccg atatgaaagt agtggttatc
     1501 gacaagaacc cccaagctct tatccgcaat tacgcagacg agtttcattg ttttgatgtt
     1561 atcaaggaac cagagaaact tcttgaaatc agcaaaaatg tagacgctat cttgcccgtc
     1621 aacgaaaact tagaatgtat taaattctta agtagtgtaa aagaggaatt ttcttgccca
     1681 gtactgttcg attttgaggc ttaccgcatc tcgcgcgata agaagaaatc gaaagagtat
     1741 ttccgttcca tcggcattcc gacaccgcaa gacaaacctg gtcgtcctcc atacttcgtt
     1801 aagccaccgt gcgaaagctc gtcggttggt gctcgcatta tctacgatga agaagaactg
     1861 ggagaattgg agcccggcat gctggtagaa gagtatgtgg aaggtgaggt tatttcactt
     1921 gaagtaatcg gggacggtac ccactttgca gttgttaaag agacacttat tcacatcgac
     1981 cgcacctatg actgccatat ggtaactccc ctgccatctg atttgtcttt ccgtgagatc
     2041 agctattctt tagcggccaa cctgcctttg aaggggatca tggacgttga agcaatttcg
     2101 ggcccgcaag gtttgaaggt cattgaaatt gattcacgtt tcccctccca gacccccaca
     2161 gccgtgtatt attcgtcggg agtgaattta attgagctgt tgtttcgtgc ctttggagaa
     2221 ggggtggagg aagttaaaac ccttccagag gaccgctact gtatctacga gcacctgatg
     2281 cttgcagaaa atggggccct gatcccagta ggtgagcagg ttctgtcgat ggggaatgac
     2341 tatgggaaat actatgaaga accaggaatt gaaatctttt tatgtcgcgg agaggacccc
     2401 gtgtttacgc ttgtcttttg gggaaaggac cgtgaggaag ccgaaaacaa aaagcgcact
     2461 ggtttgctga ttttgaagga ccgcttcggg gcagccgttt aaaatggcaa aaatcggata
     2521 agaataaaaa tttgcccaaa gtaaaatttg tttaaaacag aaaaaggaga taaaaaatgg
     2581 ctttgttgac tcccgaggac ctggagaaca ttaaccgtca acttcacgaa gccgattcta
     2641 cggtacgtcg tgtgactggg ttagatatca agggagtttg taaagatttc tatgggacta
     2701 ctccatgctg tgagaaggtc ggtatcgtcc cggtaactag tggcaacggc atcattggca
     2761 acttttctga aagtcttcgt gcgatcactg agtactttgg atttgactca ttcgtgacga
     2821 ccatgccaga cgttagtggc tactatgagg cggtccgtaa tggagcacaa attattctta
     2881 tggcagatga taatacgttt cttgcacaca atttaaagaa cggcaagatc gctaataatc
     2941 aaccgtgtac cggaattatc tacgcggaga tcgcctcgcg ctatatgaaa gccgattcca
     3001 aagatgtgct tgccgtcggg cttggaaaag tggggtttcc aggcgctgca catttggtta
     3061 ataaggggtt tcgtgtgtat gggtacgacg cggataaggc gttgttagaa aagactgtat
     3121 cagaccttgg gatcacacct ttcgagcctg agaatccccg tcgcttctca atcatttttg
     3181 aggcgacccc gtgtgctgat acagtaccag agtctgtcat cagcgacaat tgtgtcatct
     3241 ctacgcccgg tatcccttgc gcgatcagcc gtgaacttca agaaaaatac ggggtggagt
     3301 tgattatgga acctctgggt attggaactg cttctatgct gtatagcatt ttgtaaagct
     3361 cgagatctgc agctggtacc atatgggaat tcgaagcttg gctgttttgg cggatgagag
     3421 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     3481 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     3541 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     3601 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3661 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     3721 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     3781 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct
     3841 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3901 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3961 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4021 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4081 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4141 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     4201 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4261 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4321 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4381 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4441 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4501 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4561 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4621 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4681 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4741 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4801 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4861 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4921 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4981 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5041 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     5101 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5161 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5221 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5281 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5341 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5401 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5461 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5521 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5581 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5641 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5701 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5761 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     5821 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     5881 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5941 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     6001 gcagcagatc aattcgcgcg cgaaggcgaa gcggcatgca taatgtgcct gtcaaatgga
     6061 cgaagcaggg attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt
     6121 taccaattat gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa
     6181 ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc
     6241 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt
     6301 cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg
     6361 gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc
     6421 aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc
     6481 catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag
     6541 cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg
     6601 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt
     6661 ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac
     6721 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg
     6781 cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac
     6841 cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat
     6901 aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt
     6961 aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc
     7021 gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt
     7081 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca ttctgtaaca
     7141 aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc acggcagaaa
     7201 agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat
     7261 aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt ctccat
//
