LOCUS       sequence-338791-        6792 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 6792)
  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 6792)
  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: pJH123
FEATURES             Location/Qualifiers
     source          1..6792
                     /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             3377..3421
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3459..3477)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3473..3520
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3691..4482)
                     /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(4483..4574)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4622..5360)
                     /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(5570..6652)
                     /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     6614..6633
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6653..6730)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6776..6792,1..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 gatgattaag
      301 aagatggcga ctgaagatct ggatcgtttt ggcgaaaaaa ttattgaagg atttcagtta
      361 tctgacgacg atttacgcgc tttgcttagc ctggaatcgg aagaagaatt ggaaaagctg
      421 tattacgtcg cacgtaaagt acgcaaccat tactttggaa atcgcgtatt cctgaactgt
      481 tttatctact tcagcacata ctgcaaaaac caatgtgcgt tttgctatta taactgcaaa
      541 aacgagatta accgctatcg tttgtcccgc gaagaaatca aagagacttg caaggccctt
      601 aaaggtgcgg gtttccatat gattgatctg actatggggg aggatcccta ttattacgag
      661 gaacctgatc gcttcgtgga gctggtccaa atggttaaga atgagcttgg acttccaatc
      721 atgatttcgc caggagtaat ggataatgct acccttttaa aagcacgcga aaagggagct
      781 aatttcttcg ccttatatca agaaacctat gatcaagagt tgtacgagaa gttgcgcgta
      841 ggtcagtcgt tcaaggggcg ctacaatact cgtcgttttg ctaaagaaca agggtattgc
      901 attgaagacg gtatcttaac gggagtcggg aacgatatcg agtcaacgat caaatcttta
      961 cgcggcatga aatcaaatga tcccgacatg gttcgcgtga tgactttctt accccaggaa
     1021 ggaactcccc ttgagcgttt taaggaaaat agtaacctga gcgaactgaa aatcattgcg
     1081 atcttgcgtc tgatctttcc gaagtgctta atccccgcgt cgttggacct ggaagggatc
     1141 gacgggatgg tacaccgtct taacgcaggt gctaatatcg tgactagcat ccttcctagt
     1201 gattcaaaac ttgagggagt tgctaactac gatcgtgacc tggaagagcg cgaccgcgac
     1261 attaaaagcg tcatcaagcg ccttgaggga atgggcatgg aaccagcgcc acaggcggaa
     1321 ttcgagactg tactgcgctg ctaataagga ggaaaaatga aaaccatctg cctgattggt
     1381 ggtaaattgc agggttttga ggccgcgtat ctgtctaaaa aagccgatat gaaagtagtg
     1441 gttatcgaca agaaccccca agctcttatc cgcaattacg cagacgagtt tcattgtttt
     1501 gatgttatca aggaaccaga gaaacttctt gaaatcagca aaaatgtaga cgctatcttg
     1561 cccgtcaacg aaaacttaga atgtattaaa ttcttaagta gtgtaaaaga ggaattttct
     1621 tgcccagtac tgttcgattt tgaggcttac cgcatctcgc gcgataagaa gaaatcgaaa
     1681 gagtatttcc gttccatcgg cattccgaca ccgcaagaca aacctggtcg tcctccatac
     1741 ttcgttaagc caccgtgcga aagctcgtcg gttggtgctc gcattatcta cgatgaagaa
     1801 gaactgggag aattggagcc cggcatgctg gtagaagagt atgtggaagg tgaggttatt
     1861 tcacttgaag taatcgggga cggtacccac tttgcagttg ttaaagagac acttattcac
     1921 atcgaccgca cctatgactg ccatatggta actcccctgc catctgattt gtctttccgt
     1981 gagatcagct attctttagc ggccaacctg cctttgaagg ggatcatgga cgttgaagca
     2041 atttcgggcc cgcaaggttt gaaggtcatt gaaattgatt cacgtttccc ctcccagacc
     2101 cccacagccg tgtattattc gtcgggagtg aatttaattg agctgttgtt tcgtgccttt
     2161 ggagaagggg tggaggaagt taaaaccctt ccagaggacc gctactgtat ctacgagcac
     2221 ctgatgcttg cagaaaatgg ggccctgatc ccagtaggtg agcaggttct gtcgatgggg
     2281 aatgactatg ggaaatacta tgaagaacca ggaattgaaa tctttttatg tcgcggagag
     2341 gaccccgtgt ttacgcttgt cttttgggga aaggaccgtg aggaagccga aaacaaaaag
     2401 cgcactggtt tgctgatttt gaaggaccgc ttcggggcag ccgtttaaaa tggcaaaaat
     2461 cggataagaa taaaaatttg cccaaagtaa aatttgttta aaacagaaaa aggagataaa
     2521 aaatggcttt gttgactccc gaggacctgg agaacattaa ccgtcaactt cacgaagccg
     2581 attctacggt acgtcgtgtg actgggttag atatcaaggg agtttgtaaa gatttctatg
     2641 ggactactcc atgctgtgag aaggtcggta tcgtcccggt aactagtggc aacggcatca
     2701 ttggcaactt ttctgaaagt cttcgtgcga tcactgagta ctttggattt gactcattcg
     2761 tgacgaccat gccagacgtt agtggctact atgaggcggt ccgtaatgga gcacaaatta
     2821 ttcttatggc agatgataat acgtttcttg cacacaattt aaagaacggc aagatcgcta
     2881 ataatcaacc gtgtaccgga attatctacg cggagatcgc ctcgcgctat atgaaagccg
     2941 attccaaaga tgtgcttgcc gtcgggcttg gaaaagtggg gtttccaggc gctgcacatt
     3001 tggttaataa ggggtttcgt gtgtatgggt acgacgcgga taaggcgttg ttagaaaaga
     3061 ctgtatcaga ccttgggatc acacctttcg agcctgagaa tccccgtcgc ttctcaatca
     3121 tttttgaggc gaccccgtgt gctgatacag taccagagtc tgtcatcagc gacaattgtg
     3181 tcatctctac gcccggtatc ccttgcgcga tcagccgtga acttcaagaa aaatacgggg
     3241 tggagttgat tatggaacct ctgggtattg gaactgcttc tatgctgtat agcattttgt
     3301 aaatatacat atggcagatc tcaattggat atcggccggc cacgcgatcg ctgacgtcgg
     3361 taccctcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc
     3421 gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata
     3481 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaacctc aggcatttga
     3541 gaagcacacg gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata
     3601 agcggctatt taacgaccct gccctgaacc gacgaccggg tcatcgtggc cggatcttgc
     3661 ggcccctcgg cttgaacgaa ttgttagaca ttatttgccg actaccttgg tgatctcgcc
     3721 tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
     3781 ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
     3841 ctccattgcc cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
     3901 ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
     3961 gttccatagc gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
     4021 aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
     4081 caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
     4141 gcgctgccat tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
     4201 gtcgtgcaca acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
     4261 cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
     4321 caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
     4381 gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
     4441 tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata
     4501 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4561 gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg
     4621 ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat
     4681 gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc
     4741 agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat
     4801 gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg
     4861 tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct
     4921 ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct
     5041 gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac
     5101 tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt
     5161 gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct
     5221 ctgcgaaagc cagttaccac ggttaagcag ttccccaact gncttaacct tcgatcaaac
     5281 cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat
     5341 ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct
     5401 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt
     5461 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5521 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5581 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5641 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5701 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5761 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5821 cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg
     5881 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5941 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6001 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6061 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6121 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6181 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6241 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6301 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6361 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6421 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6481 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6541 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6601 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6661 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6721 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata
     6781 cgactcacta ta
//
