LOCUS       sequence-338789-        7169 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 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: pJH126
FEATURES             Location/Qualifiers
     source          1..7169
                     /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)."
     promoter        complement(28..46)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(160..177)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     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)."
     promoter        complement(258..276)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        322..399
                     /gene="lacI"
                     /label=lacI promoter
     CDS             400..1482
                     /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     complement(419..438)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        2543..2634
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2635..3426
                     /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"
     terminator      complement(3597..3644)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3640..3658
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3696..3740)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(6829..7122)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt cgattatgcg
       61 gccgtgtaca atacgattac tttctgttcg acttaagcat tatgcggccg caagcttgtc
      121 gacctgcagg cgcgccgagc tcgaattcgg atcctggctg tggtgatgat ggtgatggct
      181 gctgcccatg gtatatctcc ttattaaagt taaacaaaat tatttctaca ggggaattgt
      241 tatccgctca caattcccct atagtgagtc gtattaattt cctaatgcag gagtcgcata
      301 agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca
      361 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
      421 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
      481 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
      541 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
      601 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
      661 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
      721 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
      781 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
      841 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
      901 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
      961 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     1021 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     1081 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     1141 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac
     1201 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     1261 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     1321 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     1381 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     1441 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     1501 aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc
     1561 agtcagctcc ttccggtggg cgcggggcat gactaacatg agaattacaa cttatatcgt
     1621 atggggctga cttcaggtgc tacatttgaa gagataaatt gcactgaaat ctagagcggt
     1681 tcagtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatcttttgc
     1741 cctgtaaacg aaaaaaccac ctggggaggt ggtttgatcg aaggttaagc agttccccaa
     1801 ctgacttaac cttcgaaaaa accacctggg gaggtggttt gatcgaaggt taagtcagtt
     1861 ggggaactgc ttaaccgtgg taactggctt tcgcagagca cagcaaccaa atctgtcctt
     1921 ccagtgtagc cggactttgg cgcacacttc aagagcaacc gcgtgtttag ctaaacaaat
     1981 cctctgcgaa ctcccagtta ccaatggctg ctgccagtgg cgttttaccg tgcttttccg
     2041 ggttggactc aagtgaacag ttaccggata aggcgcagca gtcgggctga acggggagtt
     2101 cttgcttaca gcccagcttg gagcgaacga cctacaccga gccgagatac cagtgtgtga
     2161 gctatgagaa agcgccacac ttcccgtaag ggagaaaggc ggaacaggta tccggtaaac
     2221 ggcagggtcg gaacaggaga gcgcaagagg gagcgacccg ccggaaacgg tggggatctt
     2281 taagtcctgt cgggtttcgc ccgtactgtc agattcatgg ttgagcctca cggctcccac
     2341 agatgcaccg gaaaagcgtc tgtttatgtg aactctggca ggagggcgga gcctatggaa
     2401 aaacgccacc ggcgcggccc tgctgttttg cctcacatgt tagtcccctg cttatccacg
     2461 gaatctgtgg gtaactttgt atgtgtccgc agcgcccgcc gcagtctcac gcccggagcg
     2521 tagcgaccga gtgagctagc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2581 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagg
     2641 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     2701 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     2761 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     2821 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     2881 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     2941 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
     3001 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
     3061 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     3121 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     3181 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     3241 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     3301 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     3361 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     3421 aaataatgtc taacaattcg ttcaagccga ggggccgcaa gatccggcca cgatgacccg
     3481 gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggt
     3541 ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg tttcagcaaa
     3601 aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg ctcagcggtg
     3661 gcagcagcct aggttaatta agctgcgcta gtagacgagt ccatgtgctg gcgttcaaat
     3721 ttcgcagcag cggtttcttt accagactcg agggtaccga cgtcagcgat cgcgtggccg
     3781 gccgatatcc aattgagatc tgccatatgt atatttacaa aatgctatac agcatagaag
     3841 cagttccaat acccagaggt tccataatca actccacccc gtatttttct tgaagttcac
     3901 ggctgatcgc gcaagggata ccgggcgtag agatgacaca attgtcgctg atgacagact
     3961 ctggtactgt atcagcacac ggggtcgcct caaaaatgat tgagaagcga cggggattct
     4021 caggctcgaa aggtgtgatc ccaaggtctg atacagtctt ttctaacaac gccttatccg
     4081 cgtcgtaccc atacacacga aaccccttat taaccaaatg tgcagcgcct ggaaacccca
     4141 cttttccaag cccgacggca agcacatctt tggaatcggc tttcatatag cgcgaggcga
     4201 tctccgcgta gataattccg gtacacggtt gattattagc gatcttgccg ttctttaaat
     4261 tgtgtgcaag aaacgtatta tcatctgcca taagaataat ttgtgctcca ttacggaccg
     4321 cctcatagta gccactaacg tctggcatgg tcgtcacgaa tgagtcaaat ccaaagtact
     4381 cagtgatcgc acgaagactt tcagaaaagt tgccaatgat gccgttgcca ctagttaccg
     4441 ggacgatacc gaccttctca cagcatggag tagtcccata gaaatcttta caaactccct
     4501 tgatatctaa cccagtcaca cgacgtaccg tagaatcggc ttcgtgaagt tgacggttaa
     4561 tgttctccag gtcctcggga gtcaacaaag ccatttttta tctccttttt ctgttttaaa
     4621 caaattttac tttgggcaaa tttttattct tatccgattt ttgccatttt aaacggctgc
     4681 cccgaagcgg tccttcaaaa tcagcaaacc agtgcgcttt ttgttttcgg cttcctcacg
     4741 gtcctttccc caaaagacaa gcgtaaacac ggggtcctct ccgcgacata aaaagatttc
     4801 aattcctggt tcttcatagt atttcccata gtcattcccc atcgacagaa cctgctcacc
     4861 tactgggatc agggccccat tttctgcaag catcaggtgc tcgtagatac agtagcggtc
     4921 ctctggaagg gttttaactt cctccacccc ttctccaaag gcacgaaaca acagctcaat
     4981 taaattcact cccgacgaat aatacacggc tgtgggggtc tgggagggga aacgtgaatc
     5041 aatttcaatg accttcaaac cttgcgggcc cgaaattgct tcaacgtcca tgatcccctt
     5101 caaaggcagg ttggccgcta aagaatagct gatctcacgg aaagacaaat cagatggcag
     5161 gggagttacc atatggcagt cataggtgcg gtcgatgtga ataagtgtct ctttaacaac
     5221 tgcaaagtgg gtaccgtccc cgattacttc aagtgaaata acctcacctt ccacatactc
     5281 ttctaccagc atgccgggct ccaattctcc cagttcttct tcatcgtaga taatgcgagc
     5341 accaaccgac gagctttcgc acggtggctt aacgaagtat ggaggacgac caggtttgtc
     5401 ttgcggtgtc ggaatgccga tggaacggaa atactctttc gatttcttct tatcgcgcga
     5461 gatgcggtaa gcctcaaaat cgaacagtac tgggcaagaa aattcctctt ttacactact
     5521 taagaattta atacattcta agttttcgtt gacgggcaag atagcgtcta catttttgct
     5581 gatttcaaga agtttctctg gttccttgat aacatcaaaa caatgaaact cgtctgcgta
     5641 attgcggata agagcttggg ggttcttgtc gataaccact actttcatat cggctttttt
     5701 agacagatac gcggcctcaa aaccctgcaa tttaccacca atcaggcaga tggttttcat
     5761 ttttcctcct tattagcagc gcagtacagt ctcgaattcc gcctgtggcg ctggttccat
     5821 gcccattccc tcaaggcgct tgatgacgct tttaatgtcg cggtcgcgct cttccaggtc
     5881 acgatcgtag ttagcaactc cctcaagttt tgaatcacta ggaaggatgc tagtcacgat
     5941 attagcacct gcgttaagac ggtgtaccat cccgtcgatc ccttccaggt ccaacgacgc
     6001 ggggattaag cacttcggaa agatcagacg caagatcgca atgattttca gttcgctcag
     6061 gttactattt tccttaaaac gctcaagggg agttccttcc tggggtaaga aagtcatcac
     6121 gcgaaccatg tcgggatcat ttgatttcat gccgcgtaaa gatttgatcg ttgactcgat
     6181 atcgttcccg actcccgtta agataccgtc ttcaatgcaa tacccttgtt ctttagcaaa
     6241 acgacgagta ttgtagcgcc ccttgaacga ctgacctacg cgcaacttct cgtacaactc
     6301 ttgatcatag gtttcttgat ataaggcgaa gaaattagct cccttttcgc gtgcttttaa
     6361 aagggtagca ttatccatta ctcctggcga aatcatgatt ggaagtccaa gctcattctt
     6421 aaccatttgg accagctcca cgaagcgatc aggttcctcg taataatagg gatcctcccc
     6481 catagtcaga tcaatcatat ggaaacccgc acctttaagg gccttgcaag tctctttgat
     6541 ttcttcgcgg gacaaacgat agcggttaat ctcgtttttg cagttataat agcaaaacgc
     6601 acattggttt ttgcagtatg tgctgaagta gataaaacag ttcaggaata cgcgatttcc
     6661 aaagtaatgg ttgcgtactt tacgtgcgac gtaatacagc ttttccaatt cttcttccga
     6721 ttccaggcta agcaaagcgc gtaaatcgtc gtcagataac tgaaatcctt caataatttt
     6781 ttcgccaaaa cgatccagat cttcagtcgc catcttctta atcatatgac caccaatctg
     6841 ttctctgtga gcctcaataa tatcgttatc ctccatgtcc aaatcttcag gggtctgatc
     6901 agcttgaatt ctaataccgt cgtacaagaa tcttaaggag tccatttcct taccctgtct
     6961 tttagcgaac gcttccatca gccttcttaa aggagtggtc tttttgatct tgaagaagat
     7021 ctctgaagac ccatcggaca cctttaaatt gatgtgagtc tcaggcttga cttctggctt
     7081 gacctctggc ttagcttctt gattgacttc tgagtccgac atgctgctgc ccatcatctc
     7141 cttcttatac ttaactaata tactaagat
//
