LOCUS       sequence-381133-        6199 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses an evolved methyltransferase for tetrahydropapaverine 
            biosynthesis in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6199)
  AUTHORS   d'Oelsnitz S, Kim W, Burkholder NT, Javanmardi K, Thyer R, Zhang Y, 
            Alper HS, Ellington AD
  TITLE     Using fungible biosensors to evolve improved alkaloid biosyntheses.
  JOURNAL   Nat Chem Biol. 2022 Jul 7. pii: 10.1038/s41589-022-01072-w. doi: 
            10.1038/s41589-022-01072-w.
  PUBMED    35799063
REFERENCE   2  (bases 1 to 6199)
  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 Chem 
            Biol. 2022 Jul 7. pii: 10.1038/s41589-022-01072-w. doi: 
            10.1038/s41589-022-01072-w."
COMMENT     Sequence Label: pGfOMT1-GEN4
FEATURES             Location/Qualifiers
     source          1..6199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(86..105)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      899..1487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1388..1407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1641..1658
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1733..1752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1893..1915
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1909..2100)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_RNA        complement(4265..4314)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(4335..4369)
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     promoter        4647..4724
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4725..5807
                     /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(4744..4763)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        5995..6066
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6067..6135,6136..6199,1..728)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
       61 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
      121 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
      181 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
      241 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
      301 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      361 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
      421 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      481 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      541 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggatcgcgc ggtatcattg
      601 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      661 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      721 attggtaact gttagaccaa gtttactcat atatacttta gattgattta aaacttcatt
      781 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      841 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      901 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      961 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1021 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     1081 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1141 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1201 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1261 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1321 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1381 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1441 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1501 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1561 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1621 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     1681 ggtattttct ccttacgcat ctgtgcggta tttcacaccg gtgcactctc agtacaatct
     1741 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     1801 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     1861 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     1921 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     1981 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2041 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2101 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2161 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     2221 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     2281 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     2341 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     2401 acggaaaccg atgaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     2461 acttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     2521 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     2581 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     2641 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     2701 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     2761 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     2821 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     2881 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     2941 tcatctacct gcctggacag catggcggcc gcgaagacta gtgatacctt tgccatgttt
     3001 cagaaacaac tctggcgcat cgggcttgga ccaaaacgaa aaaaggccgc tttcgcggcc
     3061 tcttttctgg aatttggtac cgagctcaca gcggcgatag tcagatagct agaccgtatg
     3121 ttaccgagcc tgcactcctc gaattcttag gattattacg gaaaggcttc gattacactt
     3181 tggatggccg cgatatgacg aatcttatgc ccacgaaaac cggcacgctt taacagcttc
     3241 tcccagtcat cttcggaacg ttcacgacct ccagtattaa ccagcatatc aatatcaagg
     3301 ataagacgag tcttggtcaa ctcatgttga ctctcttcct caagaaccac gtccacaata
     3361 atgactttac cggtatcctt gggtacagct tcgcggcatt tgatcaagat gttgacgcat
     3421 tcttcgtccg tccagtcatg caaaaccagt ttcaataaga tggcctgggc acttggaact
     3481 gacttgaaca tgtcacctgg aatgcgttcg atgttcgggt gctcggggga actagcgatt
     3541 acatgtggca aatcatagac ggaacacttg atctgaggga atgcgtcgta gatgccctta
     3601 attgttgtcc cattaccccc gccgacatcg caaaggcttg tcaggccctc gaaggtatca
     3661 cgacagccat cgattaaaga cttagccata aggcgagtct caccagccat tccttcgtta
     3721 aacaaattgc tttgctcggg atgtccttcc aagtattgcc agattgtcat gcccatcccc
     3781 ctttcgaagg ccgttgtgtc attccccaaa ccttccttca tgaaatgcca gggaatcata
     3841 aagtccttct gggtcatccc caggattatt gggaccatgg aacgctcagc atcacgaagc
     3901 agcagagttg cgactggctg aagagaatac agtttgactt catttgaatc actgacttcc
     3961 ttcaaaattc ccatatgcac aaggtagcga agtacgcggt aaaggttgtc gctatttacg
     4021 ttagtgatgg gcagcttaga agcaagttcc gttacgctaa tactcccatt attactctta
     4081 atgatatctg caatgccaat ctcaacagca cagcgtaata ctaatgaatc tgcaaacccg
     4141 taaatgatgt tcaacagatg ggcctgcgct ttgatgtcaa cctcttggct ttctggttta
     4201 ttatctgaaa ctcccatatg taggcctcgt ggttcactag tttaaacaaa attatttgta
     4261 gaggctgttt cgtcctcacg gactcatcag accggaaagc acatccggtg acagctgtgc
     4321 tcagtatctt gattgctagc attgtaccta ggactgagct agccataaag gtacttttca
     4381 aaggtcgaca gcccatgccc ttgcgttcgg caggtgtaca atgatacgag gtaatgaaga
     4441 tgaagtccat acaatcgata gattgggacc aaaacgaaaa aaggggagcg gtttcccgct
     4501 cccctctttt ctggaatttg gtaccgagtc gcacctgatt gcccgacatt atcgcacggt
     4561 gtctcatctc tgataacgct gtcttcgcgg ccgcaacaaa taggggttcc gcgcacattt
     4621 ccccgaaaag tgccacctga cgtcccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4681 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4741 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4801 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4861 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4921 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4981 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5041 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5101 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5161 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagatg
     5221 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5281 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5341 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5401 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5461 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5521 cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat atcccgccgt
     5581 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5641 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5701 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5761 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5821 aatgtaagtt agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg
     5881 cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca
     5941 ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca tatttttcta aatacattca
     6001 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6061 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6121 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6181 ggtgcacgag tgggttaca
//
