LOCUS       sequence-338823-        6857 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 6857)
  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 6857)
  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: pJH750
FEATURES             Location/Qualifiers
     source          1..6857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1230..1321
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1322..2113
                     /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(2284..2331)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2327..2345
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2383..2427)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    5547..5571
                     /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(5572..5590)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(5704..5721)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    5777..5801
                     /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(5802..5820)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5866..5943
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5944..6857,1..169)
                     /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(5963..5982)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
       61 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
      121 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
      181 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
      241 tcaacccagt cagctccttc cggtgggcgc ggggcatgac taacatgaga attacaactt
      301 atatcgtatg gggctgactt caggtgctac atttgaagag ataaattgca ctgaaatcta
      361 gagcggttca gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      421 cttttgccct gtaaacgaaa aaaccacctg gggaggtggt ttgatcgaag gttaagcagt
      481 tccccaactg acttaacctt cgaaaaaacc acctggggag gtggtttgat cgaaggttaa
      541 gtcagttggg gaactgctta accgtggtaa ctggctttcg cagagcacag caaccaaatc
      601 tgtccttcca gtgtagccgg actttggcgc acacttcaag agcaaccgcg tgtttagcta
      661 aacaaatcct ctgcgaactc ccagttacca atggctgctg ccagtggcgt tttaccgtgc
      721 ttttccgggt tggactcaag tgaacagtta ccggataagg cgcagcagtc gggctgaacg
      781 gggagttctt gcttacagcc cagcttggag cgaacgacct acaccgagcc gagataccag
      841 tgtgtgagct atgagaaagc gccacacttc ccgtaaggga gaaaggcgga acaggtatcc
      901 ggtaaacggc agggtcggaa caggagagcg caagagggag cgacccgccg gaaacggtgg
      961 ggatctttaa gtcctgtcgg gtttcgcccg tactgtcaga ttcatggttg agcctcacgg
     1021 ctcccacaga tgcaccggaa aagcgtctgt ttatgtgaac tctggcagga gggcggagcc
     1081 tatggaaaaa cgccaccggc gcggccctgc tgttttgcct cacatgttag tcccctgctt
     1141 atccacggaa tctgtgggta actttgtatg tgtccgcagc gcccgccgca gtctcacgcc
     1201 cggagcgtag cgaccgagtg agctagctat ttgtttattt ttctaaatac attcaaatat
     1261 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1321 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     1381 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     1441 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     1501 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     1561 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     1621 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     1681 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     1741 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     1801 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     1861 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     1921 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     1981 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     2041 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     2101 agtcggcaaa taatgtctaa caattcgttc aagccgaggg gccgcaagat ccggccacga
     2161 tgacccggtc gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt
     2221 taccggttta ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg cctgaggttt
     2281 cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc
     2341 agcggtggca gcagcctagg ttaattaagc tgcgctagta gacgagtcca tgtgctggcg
     2401 ttcaaatttc gcagcagcgg tttctttacc agactcgagg gtaccgacgt cagcgatcgc
     2461 gtggccggcc gatatccaat tgagatctgc catatgtata tttacaaaat gctatacagc
     2521 atagaaacag ttccaatacc cagaggttcc ataatcaact ccaccccgta tttttcttga
     2581 agttcacggc tgatcgcgca agggataccg ggcgtagaga tgacacaatt gtcgctgatg
     2641 acagactctg gtactgtatc agcacacggg gtcgcctcaa aaatgattga gaagcgacgg
     2701 ggattctcag gctcgaaagg tgtgatccca aggtctgata cagtcttttc taacaacgcc
     2761 ttatccgcgt cgtacccata cacacgaaac cccttattaa ccaaatgtgc agcgcctgga
     2821 aaccccactt ttccaagccc gacggcaagc acatctttgg aatcggcttt catatagcgc
     2881 gaggcgatct ccgcgtagat aattccggta cacggttgat tattagcgat cttgccgttc
     2941 tttaaattgt gtgcaagaaa cgtattatca tctgccataa gaataatttg tgctccatta
     3001 cggaccgcct catagtagcc actaacgtct ggcatggtcg tcacgaatga gtcaaatcca
     3061 aagtactcag tgatcgcacg aagactttca gaaaagttgc caatgatgcc gttgccacta
     3121 gttaccggga cgataccgac cttctcacag catggagtag tcccatagaa atctttacaa
     3181 actcccttga tatctaaccc agtcacacga cgtaccgtag aatcggcttc gtgaagttga
     3241 cggttaatgt tctccaggtc ctcgggagtc aacaaagcca ttttttatct cctttttctg
     3301 ttttaaacaa attttacttt gggcaaattt ttattcttat ccgatttttg ccattttaaa
     3361 cggctgcccc gaagcggtcc ttcaaaatca gcaaaccagt gcgctttttg ttttcggctt
     3421 cctcacggtc ctttccccaa aagacaagcg taaacacggg gtcctctccg cgacataaaa
     3481 agatttcaat tcctggttct tcatagtatt tcccatagtc attccccatc gacagaacct
     3541 gctcacctac tgggatcagg gccccatttt ctgcaagcat caggtgctcg tagatacagt
     3601 agcggtcctc tggaagggtt ttaacttcct ccaccccttc tccaaaggca cgaaacaaca
     3661 gctcaattaa attcactccc gacgaataat acacggctgt gggggtctgg gaggggaaac
     3721 gtgaatcaat ttcaatgacc ttcaaacctt gcgggcccga aattgcttca acgtccatga
     3781 tccccttcaa aggcaggttg gccgctaaag aatagctgat ctcacggaaa gacaaatcag
     3841 atggcagggg agttaccata tggcagtcat aggtgcggtc gatgtgaata agtgtctctt
     3901 taacaactgc aaagtgggta ccgtccccga ttacttcaag tgaaataacc tcaccttcca
     3961 catactcttc taccagcatg ccgggctcca attctcccag ttcttcttca tcgtagataa
     4021 tgcgagcacc aaccgacgag ctttcgcacg gtggcttaac gaagtatgga ggacgaccag
     4081 gtttgtcttg cggtgtcgga atgccgatgg aacggaaata ctctttcgat ttcttcttat
     4141 cgcgcgagat gcggtaagcc tcaaaatcga acagtactgg gcaagaaaat tcctctttta
     4201 cactacttaa gaatttaata cattctaagt tttcgttgac gggcaagata gcgtctacat
     4261 ttttgctgat ttcaagaagt ttctctggtt ccttgataac atcaaaacaa tgaaactcgt
     4321 ctgcgtaatt gcggataaga gcttgggggt tcttgtcgat aaccactact ttcatatcgg
     4381 cttttttaga cagatacgcg gcctcaaaac cctgcaattt accaccaatc aggcagatgg
     4441 ttttcatttt tcctccttat tagcagcgca gtacagtctc gaattccgcc tgtggcgctg
     4501 gttccatgcc cattccctca aggcgcttga tgacgctttt aatgtcgcgg tcgcgctctt
     4561 ccaggtcacg atcgtagtta gcaactccct caagttttga atcactagga aggatgctag
     4621 tcacgatatt agcacctgcg ttaagacggt gtaccatccc gtcgatccct tccaggtcca
     4681 acgacgcggg gattaagcac ttcggaaaga tcagacgcaa gatcgcaatg attttcagtt
     4741 cgctcaggtt actattgtcc ttaaaacgct caaggggagt tccttcctgg ggtaagaaag
     4801 tcatcacgcg aaccatgtcg ggatcatttg atttcatgcc gcgtaaagat ttgatcgttg
     4861 actcgatatc gttcccgact cccgttaaga taccgtcttc aatgcaatac ccttgttctt
     4921 tagcaaaacg acgagtattg tagcgcctct tgaacgactg acctacgcgc aacttcttgt
     4981 acaactcttg atcataggtt tcttgatata aggcgaagaa attagctccc ttttcgcgtg
     5041 cttttaaaag ggtagcatta tccattactc ctggcgaaat catgattgga agtccaagct
     5101 cattcttaac catttggacc agctccacga agcgatcagg ttccttgtaa taatagggat
     5161 cctcccccat agtcagatca atcatatgga aacccgcacc tttaagggcc ttgcaagtct
     5221 ctttgatttc ttcgcgggac aaacgatagc ggttaatctt gtttttgcag ttataatagc
     5281 aaaacgcaca ttggtttttg cagtatgtgc tgaagtagat aaaacagctc aggaatacgc
     5341 gatttccaaa gtaatggttg cgtactttac gtgcgacgta atacagcttt tccaattctt
     5401 cttccgattc caggctaagc aaagcgcgta aatcgtcgtc agataactga aatccttcaa
     5461 taattttttc gccaaaacga tccagatctt cagtcgccat cttcttaatc atctccttct
     5521 tatacttaac taatatacta agatggggaa ttgttatccg ctcacaattc ccctatagtg
     5581 agtcgtatta atttcgatta tgcggccgtg tacaatacga ttactttctg ttcgacttaa
     5641 gcattatgcg gccgcaagct tgtcgacctg caggcgcgcc gagctcgaat tcggatcctg
     5701 gctgtggtga tgatggtgat ggctgctgcc catggtatat ctccttatta aagttaaaca
     5761 aaattatttc tacaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta
     5821 atttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     5881 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     5941 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     6001 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     6061 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     6121 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     6181 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     6241 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     6301 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     6361 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     6421 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     6481 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     6541 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     6601 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     6661 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     6721 tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac cgaagacagc
     6781 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     6841 agcgtggacc gcttgct
//
