LOCUS       sequence-339080-        7169 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 7169)
  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 7169)
  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: pJH223
FEATURES             Location/Qualifiers
     source          1..7169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..23
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    24..48
                     /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             104..121
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        235..253
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    254..278
                     /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             328..621
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3710..3754
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3792..3810)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3806..3853
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4024..4815)
                     /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(4816..4907)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(5968..7050)
                     /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     7012..7031
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7051..7128)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata
       61 attttgttta actttaataa ggagatatac catgggcagc agccatcacc atcatcacca
      121 cagccaggat ccgaattcga gctcggcgcg cctgcaggtc gacaagcttg cggccgcata
      181 atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg aaattaatac
      241 gactcactat aggggaattg tgagcggata acaattcccc atcttagtat attagttaag
      301 tataagaagg agatgatggg cagcagcatg tcggactcag aagtcaatca agaagctaag
      361 ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt gtccgatggg
      421 tcttcagaga tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg
      481 ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga
      541 attcaagctg atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct
      601 cacagagaac agattggtgg tcatatgatt aagaagatgg cgactgaaga tctggatcgt
      661 tttggcgaaa aaattattga aggatttcag ttatctgacg acgatttacg cgctttgctt
      721 agcctggaat cggaagaaga attggaaaag ctgtattacg tcgcacgtaa agtacgcaac
      781 cattactttg gaaatcgcgt attcctgagc tgttttatct acttcagcac atactgcaaa
      841 aaccaatgtg cgttttgcta ttataactgc aaaaacgaga ttaaccgcta tcgtttgtcc
      901 cgcgaagaaa tcaaagagac ttgcaaggcc cttaaaggtg cgggtttcca tatgattgat
      961 ctgactatgg gggaggatcc ctattattac gaggaacctg atcgcttcgt ggagctggtc
     1021 caaatggtta agaatgagct tggacttcca atcatgattt cgccaggagt aatggataat
     1081 gctacccttt taaaagcacg cgaaaaggga gctaatttct tcgccttata tcaagaaacc
     1141 tatgatcaag agttgtacaa gaagttgcgc gtaggtcagt cgttcaaggg gcgctacaat
     1201 actcgtcgtt ttgctaaaga acaagggtat tgcattgaag acggtatctt aacgggagtc
     1261 gggaacgata tcgagtcaac gatcaaatct ttacgcggca tgaaatcaaa tgatcccgac
     1321 atggttcgcg tgatgacttt cttaccccag gaaggaactc cccttgagcg ttttaaggac
     1381 aatagtaacc tgagcgaact gaaaatcatt gcgatcttgc gtctgatctt tccgaagtgc
     1441 ttaatccccg cgtcgttgga cctggaaggg atcgacggga tggtacaccg tcttaacgca
     1501 ggtgctaata tcgtgactag catccttcct agtgattcaa aacttgaggg agttgctaac
     1561 tacgatcgtg acctggaaga gcgcgaccgc gacattaaaa gcgtcatcaa gcgccttgag
     1621 ggaatgggca tggaaccagc gccacaggcg gaattcgaga ctgtactgcg ctgctaataa
     1681 ggaggaaaaa tgaaaaccat ctgcctgatt ggtggtaaat tgcagggttt tgaggccgcg
     1741 tatctgtcta aaaaagccga tatgaaagta gtggttatcg acaagaaccc ccaagctctt
     1801 atccgcaatt acgcagacga gtttcattgt tttgatgtta tcaaggaacc agagaaactt
     1861 cttgaaatca gcaaaaatgt agacgctatc ttgcccgtca acgaaaactt agaatgtatt
     1921 aaattcttaa gtagtgtaaa agaggaattt tcttgcccag tactgttcga ttttgaggct
     1981 taccgcatct cgcgcgataa gaagaaatcg aaagagtatt tccgttccat cggcattccg
     2041 acaccgcaag acaaacctgg tcgtcctcca tacttcgtta agccaccgtg cgaaagctcg
     2101 tcggttggtg ctcgcattat ctacgatgaa gaagaactgg gagaattgga gcccggcatg
     2161 ctggtagaag agtatgtgga aggtgaggtt atttcacttg aagtaatcgg ggacggtacc
     2221 cactttgcag ttgttaaaga gacacttatt cacatcgacc gcacctatga ctgccatatg
     2281 gtaactcccc tgccatctga tttgtctttc cgtgagatca gctattcttt agcggccaac
     2341 ctgcctttga aggggatcat ggacgttgaa gcaatttcgg gcccgcaagg tttgaaggtc
     2401 attgaaattg attcacgttt cccctcccag acccccacag ccgtgtatta ttcgtcggga
     2461 gtgaatttaa ttgagctgtt gtttcgtgcc tttggagaag gggtggagga agttaaaacc
     2521 cttccagagg accgctactg tatctacgag cacctgatgc ttgcagaaaa tggggccctg
     2581 atcccagtag gtgagcaggt tctgtcgatg gggaatgact atgggaaata ctatgaagaa
     2641 ccaggaattg aaatcttttt atgtcgcgga gaggaccccg tgtttacgct tgtcttttgg
     2701 ggaaaggacc gtgaggaagc cgaaaacaaa aagcgcactg gtttgctgat tttgaaggac
     2761 cgcttcgggg cagccgttta aaatggcaaa aatcggataa gaataaaaat ttgcccaaag
     2821 taaaatttgt ttaaaacaga aaaaggagat aaaaaatggc tttgttgact cccgaggacc
     2881 tggagaacat taaccgtcaa cttcacgaag ccgattctac ggtacgtcgt gtgactgggt
     2941 tagatatcaa gggagtttgt aaagatttct atgggactac tccatgctgt gagaaggtcg
     3001 gtatcgtccc ggtaactagt ggcaacggca tcattggcaa cttttctgaa agtcttcgtg
     3061 cgatcactga gtactttgga tttgactcat tcgtgacgac catgccagac gttagtggct
     3121 actatgaggc ggtccgtaat ggagcacaaa ttattcttat ggcagatgat aatacgtttc
     3181 ttgcacacaa tttaaagaac ggcaagatcg ctaataatca accgtgtacc ggaattatct
     3241 acgcggagat cgcctcgcgc tatatgaaag ccgattccaa agatgtgctt gccgtcgggc
     3301 ttggaaaagt ggggtttcca ggcgctgcac atttggttaa taaggggttt cgtgtgtatg
     3361 ggtacgacgc ggataaggcg ttgttagaaa agactgtatc agaccttggg atcacacctt
     3421 tcgagcctga gaatccccgt cgcttctcaa tcatttttga ggcgaccccg tgtgctgata
     3481 cagtaccaga gtctgtcatc agcgacaatt gtgtcatctc tacgcccggt atcccttgcg
     3541 cgatcagccg tgaacttcaa gaaaaatacg gggtggagtt gattatggaa cctctgggta
     3601 ttggaactgt ttctatgctg tatagcattt tgtaaatata catatggcag atctcaattg
     3661 gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta aagaaaccgc
     3721 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     3781 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     3841 gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt
     3901 agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     3961 accgacgacc gggtcatcgt ggccggatct tgcggcccct cggcttgaac gaattgttag
     4021 acattatttg ccgactacct tggtgatctc gcctttcacg tagtggacaa attcttccaa
     4081 ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca agataagcct gtctagcttc
     4141 aagtatgacg ggctgatact gggccggcag gcgctccatt gcccagtcgg cagcgacatc
     4201 cttcggcgcg attttgccgg ttactgcgct gtaccaaatg cgggacaacg taagcactac
     4261 atttcgctca tcgccagccc agtcgggcgg cgagttccat agcgttaagg tttcatttag
     4321 cgcctcaaat agatcctgtt caggaaccgg atcaaagagt tcctccgccg ctggacctac
     4381 caaggcaacg ctatgttctc ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt
     4441 ggctggctcg aagatacctg caagaatgtc attgcgctgc cattctccaa attgcagttc
     4501 gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg tgacttctac
     4561 agcgcggaga atctcgctct ctccagggga agccgaagtt tccaaaaggt cgttgatcaa
     4621 agctcgccgc gttgtttcat caagccttac ggtcaccgta accagcaaat caatatcact
     4681 gtgtggcttc aggccgccat ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg
     4741 ttcgagatgg cgctcgatga cgccaactac ctctgatagt tgagtcgata cttcggcgat
     4801 caccgcttcc ctcatactct tcctttttca atattattga agcatttatc agggttattg
     4861 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag ctagctcact
     4921 cggtcgctac gctccgggcg tgagactgcg gcgggcgctg cggacacata caaagttacc
     4981 cacagattcc gtggataagc aggggactaa catgtgaggc aaaacagcag ggccgcgccg
     5041 gtggcgtttt tccataggct ccgccctcct gccagagttc acataaacag acgcttttcc
     5101 ggtgcatctg tgggagccgt gaggctcaac catgaatctg acagtacggg cgaaacccga
     5161 caggacttaa agatccccac cgtttccggc gggtcgctcc ctcttgcgct ctcctgttcc
     5221 gaccctgccg tttaccggat acctgttccg cctttctccc ttacgggaag tgtggcgctt
     5281 tctcatagct cacacactgg tatctcggct cggtgtaggt cgttcgctcc aagctgggct
     5341 gtaagcaaga actccccgtt cagcccgact gctgcgcctt atccggtaac tgttcacttg
     5401 agtccaaccc ggaaaagcac ggtaaaacgc cactggcagc agccattggt aactgggagt
     5461 tcgcagagga tttgtttagc taaacacgcg gttgctcttg aagtgtgcgc caaagtccgg
     5521 ctacactgga aggacagatt tggttgctgt gctctgcgaa agccagttac cacggttaag
     5581 cagttcccca actgacttaa ccttcgatca aaccacctcc ccaggtggtt ttttcgaagg
     5641 ttaagtcagt tggggaactg cttaaccttc gatcaaacca cctccccagg tggttttttc
     5701 gtttacaggg caaaagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5761 ttctactgaa ccgctctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     5821 cagccccata cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag
     5881 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5941 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     6001 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     6061 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     6121 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     6181 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     6241 taccgagatg tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     6301 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     6361 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     6421 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     6481 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     6541 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     6601 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     6661 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     6721 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     6781 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6841 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6901 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6961 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     7021 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     7081 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     7141 gctctccctt atgcgactcc tgcattagg
//
