LOCUS       sequence-338833-        6792 bp    DNA     circular SYN 15-APR-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 Apr 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: pJH751
FEATURES             Location/Qualifiers
     source          1..6792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     935..954
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(974..1051)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1097..1115
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1116..1140
                     /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             1196..1213
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1327..1345
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1346..1370
                     /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             4490..4534
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4572..4590)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4586..4633
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4804..5595)
                     /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(5596..5687)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5735..6473)
                     /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(join(6683..6792,1..973))
                     /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"
ORIGIN
        1 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
       61 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
      121 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
      181 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
      241 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
      301 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
      361 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
      421 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
      481 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
      541 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
      601 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
      661 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
      721 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
      781 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
      841 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
      901 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
      961 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     1021 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     1081 tcctgcatta ggaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     1141 cctgtagaaa taattttgtt taactttaat aaggagatat accatgggca gcagccatca
     1201 ccatcatcac cacagccagg atccgaattc gagctcggcg cgcctgcagg tcgacaagct
     1261 tgcggccgca taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat
     1321 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt
     1381 atattagtta agtataagaa ggagatgatt aagaagatgg cgactgaaga tctggatcgt
     1441 tttggcgaaa aaattattga aggatttcag ttatctgacg acgatttacg cgctttgctt
     1501 agcctggaat cggaagaaga attggaaaag ctgtattacg tcgcacgtaa agtacgcaac
     1561 cattactttg gaaatcgcgt attcctgagc tgttttatct acttcagcac atactgcaaa
     1621 aaccaatgtg cgttttgcta ttataactgc aaaaacaaga ttaaccgcta tcgtttgtcc
     1681 cgcgaagaaa tcaaagagac ttgcaaggcc cttaaaggtg cgggtttcca tatgattgat
     1741 ctgactatgg gggaggatcc ctattattac aaggaacctg atcgcttcgt ggagctggtc
     1801 caaatggtta agaatgagct tggacttcca atcatgattt cgccaggagt aatggataat
     1861 gctacccttt taaaagcacg cgaaaaggga gctaatttct tcgccttata tcaagaaacc
     1921 tatgatcaaa agttgtacaa gaagttgcgc gtaggtcagt cgttcaagag gcgctacaat
     1981 actcgtcgtt ttgctaaaga acaagggtat tgcattgaag acggtatctt aacgggagtc
     2041 gggaacgata tcgagtcaac gatcaaatct ttacgcggca tgaaatcaaa tgatcccgac
     2101 atggttcgcg tgatgacttt cttaccccag gaaggaactc cccttgagcg ttttaaggac
     2161 aatagtaacc tgagcgaact gaaaatcatt gcgatcttgc gtctgatctt tccgaagtgc
     2221 ttaatccccg cgtcgttgga cctggaaggg atcgacggga tggtacaccg tcttaacgca
     2281 ggtgctaata tcgtgactag catccttcct agtgattcaa aacttgaggg agttgctaac
     2341 tacgatcgtg acctggaaga gcgcgaccgc gacattaaaa gcgtcatcaa gcgccttgag
     2401 ggaatgggca tggaaccagc gccacaggcg gaattcgaga ctgtactgcg ctgctaataa
     2461 ggaggaaaaa tgaaaaccat ctgcctgatt ggtggtaaat tgcagggttt tgaggccgcg
     2521 tatctgtcta aaaaagccga tatgaaagta gtggttatcg acaagaaccc ccaagctctt
     2581 atccgcaatt acgcagacga gtttcattgt tttgatgtta tcaaggaacc agagaaactt
     2641 cttgaaatca gcaaaaatgt agacgctatc ttgcccgtca acgaaaactt agaatgtatt
     2701 aaattcttaa gtagtgtaaa agaggaattt tcttgcccag tactgttcga ttttgaggct
     2761 taccgcatct cgcgcgataa gaagaaatcg aaagagtatt tccgttccat cggcattccg
     2821 acaccgcaag acaaacctgg tcgtcctcca tacttcgtta agccaccgtg cgaaagctcg
     2881 tcggttggtg ctcgcattat ctacgatgaa gaagaactgg gagaattgga gcccggcatg
     2941 ctggtagaag agtatgtgga aggtgaggtt atttcacttg aagtaatcgg ggacggtacc
     3001 cactttgcag ttgttaaaga gacacttatt cacatcgacc gcacctatga ctgccatatg
     3061 gtaactcccc tgccatctga tttgtctttc cgtgagatca gctattcttt agcggccaac
     3121 ctgcctttga aggggatcat ggacgttgaa gcaatttcgg gcccgcaagg tttgaaggtc
     3181 attgaaattg attcacgttt cccctcccag acccccacag ccgtgtatta ttcgtcggga
     3241 gtgaatttaa ttgagctgtt gtttcgtgcc tttggagaag gggtggagga agttaaaacc
     3301 cttccagagg accgctactg tatctacgag cacctgatgc ttgcagaaaa tggggccctg
     3361 atcccagtag gtgagcaggt tctgtcgatg gggaatgact atgggaaata ctatgaagaa
     3421 ccaggaattg aaatcttttt atgtcgcgga gaggaccccg tgtttacgct tgtcttttgg
     3481 ggaaaggacc gtgaggaagc cgaaaacaaa aagcgcactg gtttgctgat tttgaaggac
     3541 cgcttcgggg cagccgttta aaatggcaaa aatcggataa gaataaaaat ttgcccaaag
     3601 taaaatttgt ttaaaacaga aaaaggagat aaaaaatggc tttgttgact cccgaggacc
     3661 tggagaacat taaccgtcaa cttcacgaag ccgattctac ggtacgtcgt gtgactgggt
     3721 tagatatcaa gggagtttgt aaagatttct atgggactac tccatgctgt gagaaggtcg
     3781 gtatcgtccc ggtaactagt ggcaacggca tcattggcaa cttttctgaa agtcttcgtg
     3841 cgatcactga gtactttgga tttgactcat tcgtgacgac catgccagac gttagtggct
     3901 actatgaggc ggtccgtaat ggagcacaaa ttattcttat ggcagatgat aatacgtttc
     3961 ttgcacacaa tttaaagaac ggcaagatcg ctaataatca accgtgtacc ggaattatct
     4021 acgcggagat cgcctcgcgc tatatgaaag ccgattccaa agatgtgctt gccgtcgggc
     4081 ttggaaaagt ggggtttcca ggcgctgcac atttggttaa taaggggttt cgtgtgtatg
     4141 ggtacgacgc ggataaggcg ttgttagaaa agactgtatc agaccttggg atcacacctt
     4201 tcgagcctga gaatccccgt cgcttctcaa tcatttttga ggcgaccccg tgtgctgata
     4261 cagtaccaga gtctgtcatc agcgacaatt gtgtcatctc tacgcccggt atcccttgcg
     4321 cgatcagccg tgaacttcaa gaaaaatacg gggtggagtt gattatggaa cctctgggta
     4381 ttggaactgt ttctatgctg tatagcattt tgtaaatata catatggcag atctcaattg
     4441 gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta aagaaaccgc
     4501 tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt aattaaccta
     4561 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     4621 gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac tgcttccggt
     4681 agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     4741 accgacgacc gggtcatcgt ggccggatct tgcggcccct cggcttgaac gaattgttag
     4801 acattatttg ccgactacct tggtgatctc gcctttcacg tagtggacaa attcttccaa
     4861 ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca agataagcct gtctagcttc
     4921 aagtatgacg ggctgatact gggccggcag gcgctccatt gcccagtcgg cagcgacatc
     4981 cttcggcgcg attttgccgg ttactgcgct gtaccaaatg cgggacaacg taagcactac
     5041 atttcgctca tcgccagccc agtcgggcgg cgagttccat agcgttaagg tttcatttag
     5101 cgcctcaaat agatcctgtt caggaaccgg atcaaagagt tcctccgccg ctggacctac
     5161 caaggcaacg ctatgttctc ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt
     5221 ggctggctcg aagatacctg caagaatgtc attgcgctgc cattctccaa attgcagttc
     5281 gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc acaacaatgg tgacttctac
     5341 agcgcggaga atctcgctct ctccagggga agccgaagtt tccaaaaggt cgttgatcaa
     5401 agctcgccgc gttgtttcat caagccttac ggtcaccgta accagcaaat caatatcact
     5461 gtgtggcttc aggccgccat ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg
     5521 ttcgagatgg cgctcgatga cgccaactac ctctgatagt tgagtcgata cttcggcgat
     5581 caccgcttcc ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5641 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag ctagctcact
     5701 cggtcgctac gctccgggcg tgagactgcg gcgggcgctg cggacacata caaagttacc
     5761 cacagattcc gtggataagc aggggactaa catgtgaggc aaaacagcag ggccgcgccg
     5821 gtggcgtttt tccataggct ccgccctcct gccagagttc acataaacag acgcttttcc
     5881 ggtgcatctg tgggagccgt gaggctcaac catgaatctg acagtacggg cgaaacccga
     5941 caggacttaa agatccccac cgtttccggc gggtcgctcc ctcttgcgct ctcctgttcc
     6001 gaccctgccg tttaccggat acctgttccg cctttctccc ttacgggaag tgtggcgctt
     6061 tctcatagct cacacactgg tatctcggct cggtgtaggt cgttcgctcc aagctgggct
     6121 gtaagcaaga actccccgtt cagcccgact gctgcgcctt atccggtaac tgttcacttg
     6181 agtccaaccc ggaaaagcac ggtaaaacgc cactggcagc agccattggt aactgggagt
     6241 tcgcagagga tttgtttagc taaacacgcg gttgctcttg aagtgtgcgc caaagtccgg
     6301 ctacactgga aggacagatt tggttgctgt gctctgcgaa agccagttac cacggttaag
     6361 cagttcccca actgacttaa ccttcgatca aaccacctgg ggaggtggtt ttttcgttta
     6421 cagggcaaaa gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6481 ctgaaccgct ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc
     6541 ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct
     6601 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6661 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6721 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6781 tggtttttct tt
//
