LOCUS       sequence-339044-        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: pJH576
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      56..83
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        102..193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(194..262,263..1054)
                     /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(412..431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1225..1813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1714..1733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1978..1995
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2010..2150
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2076..2095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2236..2258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2414..3292)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3319..3603
                     /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     3526..3545
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             3652..3945
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(7035..7052)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      7205..7291
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
       61 gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat
      121 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      181 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      241 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      301 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      361 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
      421 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
      481 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
      541 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
      601 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
      661 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
      721 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
      781 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
      841 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      901 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      961 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1021 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1081 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1141 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1201 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1261 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1321 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1381 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1441 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1501 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1561 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1621 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1681 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1741 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1801 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1861 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1921 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     1981 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2041 tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2101 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     2161 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2221 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2281 gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc
     2341 aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct
     2401 gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg
     2461 cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt
     2521 gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa
     2581 gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact
     2641 gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg
     2701 cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc
     2761 gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt
     2821 gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa
     2881 tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc
     2941 ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa
     3001 agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct
     3061 ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt
     3121 tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc
     3181 ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat
     3241 gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca
     3301 tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact
     3361 gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc
     3421 tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg
     3481 gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt
     3541 tatccataag attagcggat cctacctgac gctttttatc gcaactctct actgtttctc
     3601 catacccgtt ttttgggcta acaggaggaa ttaaccatga tgggcagcag catgtcggac
     3661 tcagaagtca atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac
     3721 atcaatttaa aggtgtccga tgggtcttca gagatcttct tcaagatcaa aaagaccact
     3781 cctttaagaa ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta
     3841 agattcttgt acgacggtat tagaattcaa gctgatcaga cccctgaaga tttggacatg
     3901 gaggataacg atattattga ggctcacaga gaacagattg gtggtcatat gattaagaag
     3961 atggcgactg aagatctgga tcgttttggc gaaaaaatta ttgaaggatt tcagttatct
     4021 gacgacgatt tacgcgcttt gcttagcctg gaatcggaag aagaattgga aaagctgtat
     4081 tacgtcgcac gtaaagtacg caaccattac tttggaaatc gcgtattcct gagctgtttt
     4141 atctacttca gcacatactg caaaaaccaa tgtgcgtttt gctattataa ctgcaaaaac
     4201 gagattaacc gctatcgttt gtcccgcgaa gaaatcaaag agacttgcaa ggcccttaaa
     4261 ggtgcgggtt tccatatgat tgatctgact atgggggagg atccctatta ttacgaggaa
     4321 cctgatcgct tcgtggagct ggtccaaatg gttaagaatg agcttggact tccaatcatg
     4381 atttcgccag gagtaatgga taatgctacc cttttaaaag cacgcgaaaa gggagctaat
     4441 ttcttcgcct tatatcaaga aacctatgat caaaagttgt acaagaagtt gcgcgtaggt
     4501 cagtcgttca aggggcgcta caatactcgt cgttttgcta aagaacaagg gtattgcatt
     4561 gaagacggta tcttaacggg agtcgggaac gatatcgagt caacgatcaa atctttacgc
     4621 ggcatgaaat caaatgatcc cgacatggtt cgcgtgatga ctttcttacc ccaggaagga
     4681 actccccttg agcgttttaa ggacaatagt aacctgagcg aactgaaaat cattgcgatc
     4741 ttgcgtctga tctttccgaa gtgcttaatc cccgcgtcgt tggacctgga agggatcgac
     4801 gggatggtac accgtcttaa cgcaggtgct aatatcgtga ctagcatcct tcctagtgat
     4861 tcaaaacttg agggagttgc taactacgat cgtgacctgg aagagcgcga ccgcgacatt
     4921 aaaagcgtca tcaagcgcct tgagggaatg ggcatggaac cagcgccaca ggcggaattc
     4981 gagactgtac tgcgctgcta ataaggagga aaaatgaaaa ccatctgcct gattggtggt
     5041 aaattgcagg gttttgaggc cgcgtatctg tctaaaaaag ccgatatgaa agtagtggtt
     5101 atcgacaaga acccccaagc tcttatccgc aattacgcag acgagtttca ttgttttgat
     5161 gttatcaagg aaccagagaa acttcttgaa atcagcaaaa atgtagacgc tatcttgccc
     5221 gtcaacgaaa acttagaatg tattaaattc ttaagtagtg taaaagagga attttcttgc
     5281 ccagtactgt tcgattttga ggcttaccgc atctcgcgcg ataagaagaa atcgaaagag
     5341 tatttccgtt ccatcggcat tccgacaccg caagacaaac ctggtcgtcc tccatacttc
     5401 gttaagccac cgtgcgaaag ctcgtcggtt ggtgctcgca ttatctacga tgaagaagaa
     5461 ctgggagaat tggagcccgg catgctggta gaagagtatg tggaaggtga ggttatttca
     5521 cttgaagtaa tcggggacgg tacccacttt gcagttgtta aagagacact tattcacatc
     5581 gaccgcacct atgactgcca tatggtaact cccctgccat ctgatttgtc tttccgtgag
     5641 atcagctatt ctttagcggc caacctgcct ttgaagggga tcatggacgt tgaagcaatt
     5701 tcgggcccgc aaggtttgaa ggtcattgaa attgattcac gtttcccctc ccagaccccc
     5761 acagccgtgt attattcgtc gggagtgaat ttaattgagc tgttgtttcg tgcctttgga
     5821 gaaggggtgg aggaagttaa aacccttcca gaggaccgct actgtatcta cgagcacctg
     5881 atgcttgcag aaaatggggc cctgatccca gtaggtgagc aggttctgtc gatggggaat
     5941 gactatggga aatactatga agaaccagga attgaaatct ttttatgtcg cggagaggac
     6001 cccgtgttta cgcttgtctt ttggggaaag gaccgtgagg aagccgaaaa caaaaagcgc
     6061 actggtttgc tgattttgaa ggaccgcttc ggggcagccg tttaaaatgg caaaaatcgg
     6121 ataagaataa aaatttgccc aaagtaaaat ttgtttaaaa cagaaaaagg agataaaaaa
     6181 tggctttgtt gactcccgag gacctggaga acattaaccg tcaacttcac gaagccgatt
     6241 ctacggtacg tcgtgtgact gggttagata tcaagggagt ttgtaaagat ttctatggga
     6301 ctactccatg ctgtgagaag gtcggtatcg tcccggtaac tagtggcaac ggcatcattg
     6361 gcaacttttc tgaaagtctt cgtgcgatca ctgagtactt tggatttgac tcattcgtga
     6421 cgaccatgcc agacgttagt ggctactatg aggcggtccg taatggagca caaattattc
     6481 ttatggcaga tgataatacg tttcttgcac acaatttaaa gaacggcaag atcgctaata
     6541 atcaaccgtg taccggaatt atctacgcgg agatcgcctc gcgctatatg aaagccgatt
     6601 ccaaagatgt gcttgccgtc gggcttggaa aagtggggtt tccaggcgct gcacatttgg
     6661 ttaataaggg gtttcgtgtg tatgggtacg acgcggataa ggcgttgtta gaaaagactg
     6721 tatcagacct tgggatcaca cctttcgagc ctgagaatcc ccgtcgcttc tcaatcattt
     6781 ttgaggcgac cccgtgtgct gatacagtac cagagtctgt catcagcgac aattgtgtca
     6841 tctctacgcc cggtatccct tgcgcgatca gccgtgaact tcaagaaaaa tacggggtgg
     6901 agttgattat ggaacctctg ggtattggaa ctgtttctat gctgtatagc attttgtaaa
     6961 gctcgagatc tgcagctggt accatatggg aattcgaagc ttggctgttt tggcggatga
     7021 gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag
     7081 aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
     7141 aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
     7201 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     7261 gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa
     7321 gcaacgg
//
