LOCUS       sequence-338573-        5792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Microscopy inclusion body assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  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 5792)
  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: pJH60
FEATURES             Location/Qualifiers
     source          1..5792
                     /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             2333..2377
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2415..2433)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2429..2476
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2647..3438)
                     /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(3439..3530)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(4591..5673)
                     /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     5635..5654
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5674..5751)
                     /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 agatgaaaac catctgcctg attggtggta aattgcaggg ttttgaggcc
      361 gcgtatctgt ctaaaaaagc cgatatgaaa gtagtggtta tcgacaagaa cccccaagct
      421 cttatccgca attacgcaga cgagtttcat tgttttgatg ttatcaagga accagagaaa
      481 cttcttgaaa tcagcaaaaa tgtagacgct atcttgcccg tcaacgaaaa cttagaatgt
      541 attaaattct taagtagtgt aaaagaggaa ttttcttgcc cagtactgtt cgattttgag
      601 gcttaccgca tctcgcgcga taagaagaaa tcgaaagagt atttccgttc catcggcatt
      661 ccgacaccgc aagacaaacc tggtcgtcct ccatacttcg ttaagccacc gtgcgaaagc
      721 tcgtcggttg gtgctcgcat tatctacgat gaagaagaac tgggagaatt ggagcccggc
      781 atgctggtag aagagtatgt ggaaggtgag gttatttcac ttgaagtaat cggggacggt
      841 acccactttg cagttgttaa agagacactt attcacatcg accgcaccta tgactgccat
      901 atggtaactc ccctgccatc tgatttgtct ttccgtgaga tcagctattc tttagcggcc
      961 aacctgcctt tgaaggggat catggacgtt gaagcaattt cgggcccgca aggtttgaag
     1021 gtcattgaaa ttgattcacg tttcccctcc cagaccccca cagccgtgta ttattcgtcg
     1081 ggagtgaatt taattgagct gttgtttcgt gcctttggag aaggggtgga ggaagttaaa
     1141 acccttccag aggaccgcta ctgtatctac gagcacctga tgcttgcaga aaatggggcc
     1201 ctgatcccag taggtgagca ggttctgtcg atggggaatg actatgggaa atactatgaa
     1261 gaaccaggaa ttgaaatctt tttatgtcgc ggagaggacc ccgtgtttac gcttgtcttt
     1321 tggggaaagg accgtgagga agccgaaaac aaaaagcgca ctggtttgct gattttgaag
     1381 gaccgcttcg gggcagccgt ttaaaatggc aaaaatcgga taagaataaa aatttgccca
     1441 aagtaaaatt tgtttaaaac agaaaaagga gataaaaaat ggctttgttg actcccgagg
     1501 acctggagaa cattaaccgt caacttcacg aagccgattc tacggtacgt cgtgtgactg
     1561 ggttagatat caagggagtt tgtaaagatt tctatgggac tactccatgc tgtgagaagg
     1621 tcggtatcgt cccggtaact agtggcaacg gcatcattgg caacttttct gaaagtcttc
     1681 gtgcgatcac tgagtacttt ggatttgact cattcgtgac gaccatgcca gacgttagtg
     1741 gctactatga ggcggtccgt aatggagcac aaattattct tatggcagat gataatacgt
     1801 ttcttgcaca caatttaaag aacggcaaga tcgctaataa tcaaccgtgt accggaatta
     1861 tctacgcgga gatcgcctcg cgctatatga aagccgattc caaagatgtg cttgccgtcg
     1921 ggcttggaaa agtggggttt ccaggcgctg cacatttggt taataagggg tttcgtgtgt
     1981 atgggtacga cgcggataag gcgttgttag aaaagactgt atcagacctt gggatcacac
     2041 ctttcgagcc tgagaatccc cgtcgcttct caatcatttt tgaggcgacc ccgtgtgctg
     2101 atacagtacc agagtctgtc atcagcgaca attgtgtcat ctctacgccc ggtatccctt
     2161 gcgcgatcag ccgtgaactt caagaaaaat acggggtgga gttgattatg gaacctctgg
     2221 gtattggaac tgcttctatg ctgtatagca ttttgtaaat atacatatgg cagatctcaa
     2281 ttggatatcg gccggccacg cgatcgctga cgtcggtacc ctcgagtctg gtaaagaaac
     2341 cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac
     2401 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     2461 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     2521 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     2581 tgaaccgacg accgggtcat cgtggccgga tcttgcggcc cctcggcttg aacgaattgt
     2641 tagacattat ttgccgacta ccttggtgat ctcgcctttc acgtagtgga caaattcttc
     2701 caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt ccaagataag cctgtctagc
     2761 ttcaagtatg acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac
     2821 atccttcggc gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac
     2881 tacatttcgc tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt
     2941 tagcgcctca aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc
     3001 taccaaggca acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat
     3061 cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag
     3121 ttcgcgctta gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc
     3181 tacagcgcgg agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat
     3241 caaagctcgc cgcgttgttt catcaagcct tacggtcacc gtaaccagca aatcaatatc
     3301 actgtgtggc ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt
     3361 cggttcgaga tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc
     3421 gatcaccgct tccctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3481 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa tagctagctc
     3541 actcggtcgc tacgctccgg gcgtgagact gcggcgggcg ctgcggacac atacaaagtt
     3601 acccacagat tccgtggata agcaggggac taacatgtga ggcaaaacag cagggccgcg
     3661 ccggtggcgt ttttccatag gctccgccct cctgccagag ttcacataaa cagacgcttt
     3721 tccggtgcat ctgtgggagc cgtgaggctc aaccatgaat ctgacagtac gggcgaaacc
     3781 cgacaggact taaagatccc caccgtttcc ggcgggtcgc tccctcttgc gctctcctgt
     3841 tccgaccctg ccgtttaccg gatacctgtt ccgcctttct cccttacggg aagtgtggcg
     3901 ctttctcata gctcacacac tggtatctcg gctcggtgta ggtcgttcgc tccaagctgg
     3961 gctgtaagca agaactcccc gttcagcccg actgctgcgc cttatccggt aactgttcac
     4021 ttgagtccaa cccggaaaag cacggtaaaa cgccactggc agcagccatt ggtaactggg
     4081 agttcgcaga ggatttgttt agctaaacac gcggttgctc ttgaagtgtg cgccaaagtc
     4141 cggctacact ggaaggacag atttggttgc tgtgctctgc gaaagccagt taccacggtt
     4201 aagcagttcc ccaactgact taaccttcga tcaaaccacc tccccaggtg gttttttcga
     4261 aggttaagtc agttggggaa ctgcttaacc ttcgatcaaa ccacctcccc aggtggtttt
     4321 ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4381 cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     4441 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     4501 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4561 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4621 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4681 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4741 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4801 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4861 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4921 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4981 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5041 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5101 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5161 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5221 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5281 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5341 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5401 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5461 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5521 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5581 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5641 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5701 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5761 gacgctctcc cttatgcgac tcctgcatta gg
//
