LOCUS       sequence-333830-        5727 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 5727)
  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 5727)
  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..5727
                     /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             2312..2356
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2394..2412)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2408..2455
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2626..3417)
                     /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(3418..3509)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3557..4295)
                     /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(4505..5587)
                     /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     5549..5568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5588..5665)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5711..5727,1..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 gatgaaaacc
      301 atctgcctga ttggtggtaa attgcagggt tttgaggccg cgtatctgtc taaaaaagcc
      361 gatatgaaag tagtggttat cgacaagaac ccccaagctc ttatccgcaa ttacgcagac
      421 gagtttcatt gttttgatgt tatcaaggaa ccagagaaac ttcttgaaat cagcaaaaat
      481 gtagacgcta tcttgcccgt caacgaaaac ttagaatgta ttaaattctt aagtagtgta
      541 aaagaggaat tttcttgccc agtactgttc gattttgagg cttaccgcat ctcgcgcgat
      601 aagaagaaat cgaaagagta tttccgttcc atcggcattc cgacaccgca agacaaacct
      661 ggtcgtcctc catacttcgt taagccaccg tgcgaaagct cgtcggttgg tgctcgcatt
      721 atctacgatg aagaagaact gggagaattg gagcccggca tgctggtaga agagtatgtg
      781 gaaggtgagg ttatttcact tgaagtaatc ggggacggta cccactttgc agttgttaaa
      841 gagacactta ttcacatcga ccgcacctat gactgccata tggtaactcc cctgccatct
      901 gatttgtctt tccgtgagat cagctattct ttagcggcca acctgccttt gaaggggatc
      961 atggacgttg aagcaatttc gggcccgcaa ggtttgaagg tcattgaaat tgattcacgt
     1021 ttcccctccc agacccccac agccgtgtat tattcgtcgg gagtgaattt aattgagctg
     1081 ttgtttcgtg cctttggaga aggggtggag gaagttaaaa cccttccaga ggaccgctac
     1141 tgtatctacg agcacctgat gcttgcagaa aatggggccc tgatcccagt aggtgagcag
     1201 gttctgtcga tggggaatga ctatgggaaa tactatgaag aaccaggaat tgaaatcttt
     1261 ttatgtcgcg gagaggaccc cgtgtttacg cttgtctttt ggggaaagga ccgtgaggaa
     1321 gccgaaaaca aaaagcgcac tggtttgctg attttgaagg accgcttcgg ggcagccgtt
     1381 taaaatggca aaaatcggat aagaataaaa atttgcccaa agtaaaattt gtttaaaaca
     1441 gaaaaaggag ataaaaaatg gctttgttga ctcccgagga cctggagaac attaaccgtc
     1501 aacttcacga agccgattct acggtacgtc gtgtgactgg gttagatatc aagggagttt
     1561 gtaaagattt ctatgggact actccatgct gtgagaaggt cggtatcgtc ccggtaacta
     1621 gtggcaacgg catcattggc aacttttctg aaagtcttcg tgcgatcact gagtactttg
     1681 gatttgactc attcgtgacg accatgccag acgttagtgg ctactatgag gcggtccgta
     1741 atggagcaca aattattctt atggcagatg ataatacgtt tcttgcacac aatttaaaga
     1801 acggcaagat cgctaataat caaccgtgta ccggaattat ctacgcggag atcgcctcgc
     1861 gctatatgaa agccgattcc aaagatgtgc ttgccgtcgg gcttggaaaa gtggggtttc
     1921 caggcgctgc acatttggtt aataaggggt ttcgtgtgta tgggtacgac gcggataagg
     1981 cgttgttaga aaagactgta tcagaccttg ggatcacacc tttcgagcct gagaatcccc
     2041 gtcgcttctc aatcattttt gaggcgaccc cgtgtgctga tacagtacca gagtctgtca
     2101 tcagcgacaa ttgtgtcatc tctacgcccg gtatcccttg cgcgatcagc cgtgaacttc
     2161 aagaaaaata cggggtggag ttgattatgg aacctctggg tattggaact gcttctatgc
     2221 tgtatagcat tttgtaaata tacatatggc agatctcaat tggatatcgg ccggccacgc
     2281 gatcgctgac gtcggtaccc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg
     2341 ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga
     2401 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     2461 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     2521 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcatc
     2581 gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt tgccgactac
     2641 cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc
     2701 caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata
     2761 ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc
     2821 ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc
     2881 ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg
     2941 ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc
     3001 tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc
     3061 tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg
     3121 ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct
     3181 ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc
     3241 atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc
     3301 atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat
     3361 gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact
     3421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3481 atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct acgctccggg
     3541 cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt ccgtggataa
     3601 gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt tttccatagg
     3661 ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc tgtgggagcc
     3721 gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt aaagatcccc
     3781 accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg
     3841 atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag ctcacacact
     3901 ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg
     3961 ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac ccggaaaagc
     4021 acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag gatttgttta
     4081 gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga
     4141 tttggttgct gtgctctgcg aaagccagtt accacggtta agcagttccc caactgactt
     4201 aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa aagattacgc
     4261 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg ctctagattt
     4321 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
     4381 aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4441 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4501 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4621 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4681 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4741 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     4801 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4861 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4921 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4981 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5041 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5101 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5161 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5221 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5281 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5341 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5401 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5461 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5521 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5581 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5641 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5701 attaggaaat taatacgact cactata
//
