LOCUS       sequence-338828-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pylBCD variant in pCDF backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  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 7104)
  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: pJH224
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             307..600
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3689..3733
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3771..3789)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3785..3832
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4003..4794)
                     /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(4795..4886)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4934..5672)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(5882..6964)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6926..6945
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6965..7042)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7088..7104,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatgatgggc
      301 agcagcatgt cggactcaga agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc
      361 aagcctgaga ctcacatcaa tttaaaggtg tccgatgggt cttcagagat cttcttcaag
      421 atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag
      481 gaaatggact ccttaagatt cttgtacgac ggtattagaa ttcaagctga tcagacccct
      541 gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca gattggtggt
      601 catatgatta agaagatggc gactgaagat ctggatcgtt ttggcgaaaa aattattgaa
      661 ggatttcagt tatctgacga cgatttacgc gctttgctta gcctggaatc ggaagaagaa
      721 ttggaaaagc tgtattacgt cgcacgtaaa gtacgcaacc attactttgg aaatcgcgta
      781 ttcctgagct gttttatcta cttcagcaca tactgcaaaa accaatgtgc gttttgctat
      841 tataactgca aaaacgagat taaccgctat cgtttgtccc gcgaagaaat caaagagact
      901 tgcaaggccc ttaaaggtgc gggtttccat atgattgatc tgactatggg ggaggatccc
      961 tattattacg aggaacctga tcgcttcgtg gagctggtcc aaatggttaa gaatgagctt
     1021 ggacttccaa tcatgatttc gccaggagta atggataatg ctaccctttt aaaagcacgc
     1081 gaaaagggag ctaatttctt cgccttatat caagaaacct atgatcaaaa gttgtacaag
     1141 aagttgcgcg taggtcagtc gttcaagggg cgctacaata ctcgtcgttt tgctaaagaa
     1201 caagggtatt gcattgaaga cggtatctta acgggagtcg ggaacgatat cgagtcaacg
     1261 atcaaatctt tacgcggcat gaaatcaaat gatcccgaca tggttcgcgt gatgactttc
     1321 ttaccccagg aaggaactcc ccttgagcgt tttaaggaaa atagtaacct gagcgaactg
     1381 aaaatcattg caatcttgcg tctgatcttt ccgaagtgct taatccccgc gtcgttggac
     1441 ctggaaggga tcgacgggat ggtacaccgt cttaacgcag gtgctaatat cgtgactagc
     1501 atccttccta gtgattcaaa acttgaggga gttgctaact acgatcgtga cctggaagag
     1561 cgcgaccgcg acattaaaag cgtcatcaag cgccttgagg gaatgggcat ggaaccagcg
     1621 ccacaggcgg aattcgagac tgtactgcgc tgctaataag gaggaaaaat gaaaaccatc
     1681 tgcctgattg gtggtaaatt gcagggtttt gaggccgcgt atctgtctaa aaaagccgat
     1741 atgaaagtag tggttatcga caagaacccc caagctctta tccgcaatta cgcagacgag
     1801 tttcattgtt ttgatgttat caaggaacca gagaaacttc ttgaaatcag caaaaatgta
     1861 gacgctatct tgcccgtcaa cgaaaactta gaatgtatta aattcttaag tagtgtaaaa
     1921 gaggaatttt cttgcccagt actgttcgat tttgaggctt accgcatctc gcgcgataag
     1981 aagaaatcga aagagtattt ccgttccatc ggcattccga caccgcaaga caaacctggt
     2041 cgtcctccat acttcgttaa gccaccgtgc gaaagctcgt cggttggtgc tcgcattatc
     2101 tacgatgaag aagaactggg agaattggag cccggcatgc tggtagaaga gtatgtggaa
     2161 ggtgaggtta tttcacttga agtaatcggg gacggtaccc actttgcagt tgttaaagag
     2221 acacttattc acatcgaccg cacctatgac tgccatatgg taactcccct gccatctgat
     2281 ttgtctttcc gtgagatcag ctattcttta gcggccaacc tgcctttgaa ggggatcatg
     2341 gacgttgaag caatttcggg cccgcaaggt ttgaaggtca ttgaaattga ttcacgtttc
     2401 ccctcccaga cccccacagc cgtgtattat tcgtcgggag tgaatttaat tgagctgttg
     2461 tttcgtgcct ttggagaagg ggtggaggaa gttaaaaccc ttccagagga ccgctactgt
     2521 atctacgagc acctgatgct tgcagaaaat ggggccctga tcccagtagg tgagcaggtt
     2581 ctgtcgatgg ggaatgacta tgggaaatac tatgaagaac caggaattga aatcttttta
     2641 tgtcgcggag aggaccccgt gtttacgctt gtcttttggg gaaaggaccg tgaggaagcc
     2701 gaaaacaaaa agcgcactgg tttgctgatt ttgaaggacc gcttcggggc agccgtttaa
     2761 aatggcaaaa atcggataag aataaaaatt tgcccaaagt aaaatttgtt taaaacagaa
     2821 aaaggagata aaaaatggct ttgttgactc ccgaggacct ggagaacatt aaccgtcaac
     2881 ttcacgaagc cgattctacg gtacgtcgtg tgactgggtt agatatcaag ggagtttgta
     2941 aagatttcta tgggactact ccatgctgtg agaaggtcgg tatcgtcccg gtaactagtg
     3001 gcaacggcat cattggcaac ttttctgaaa gtcttcgtgc gatcactgag tactttggat
     3061 ttgactcatt cgtgacgacc atgccagacg ttagtggcta ctatgaggcg gtccgtaatg
     3121 gagcacaaat tattcttatg gcagatgata atacgtttct tgcacacaat ttaaagaacg
     3181 gcaagatcgc taataatcaa ccgtgtaccg gaattatcta cgcggagatc gcctcgcgct
     3241 atatgaaagc cgattccaaa gatgtgcttg ccgtcgggct tggaaaagtg gggtttccag
     3301 gcgctgcaca tttggttaat aaggggtttc gtgtgtatgg gtacgacgcg gataaggcgt
     3361 tgttagaaaa gactgtatca gaccttggga tcacaccttt cgagcctgag aatccccgtc
     3421 gcttctcaat catttttgag gcgaccccgt gtgctgatac agtaccagag tctgtcatca
     3481 gcgacaattg tgtcatctct acgcccggta tcccttgcgc gatcagccgt gaacttcaag
     3541 aaaaatacgg ggtggagttg attatggaac ctctgggtat tggaactgct tctatgctgt
     3601 atagcatttt gtaaatatac atatggcaga tctcaattgg atatcggccg gccacgcgat
     3661 cgctgacgtc ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca
     3721 gcacatggac tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca
     3781 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc
     3841 tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca
     3901 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg
     3961 gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt
     4021 ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa
     4081 gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
     4141 ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt
     4201 tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
     4261 gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc
     4321 aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct
     4381 tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
     4441 aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca
     4501 cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
     4561 tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc
     4621 aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc
     4681 cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
     4741 gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt
     4801 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4861 tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt
     4921 gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca
     4981 ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc
     5041 cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg
     5101 aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc
     5161 gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata
     5221 cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt
     5281 atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc
     5341 agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg
     5401 gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct
     5461 aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt
     5521 ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac
     5581 cttcgatcaa accacctccc caggtggttt tttcgtttac agggcaaaag attacgcgca
     5641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac tgaaccgctc tagatttcag
     5701 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
     5761 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5821 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5881 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5941 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6001 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     6061 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     6121 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     6181 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     6241 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     6301 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     6361 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     6421 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6481 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6541 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6601 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6661 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6721 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6781 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6841 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6901 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6961 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     7021 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     7081 aggaaattaa tacgactcac tata
//
