LOCUS       sequence-383717-        5722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an evolved rotundine biosensor in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5722)
  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 5722)
  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 Chem 
            Biol. 2022 Jul 7. pii: 10.1038/s41589-022-01072-w. doi: 
            10.1038/s41589-022-01072-w."
COMMENT     Sequence Label: pReg-ROTU4
FEATURES             Location/Qualifiers
     source          1..5722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        78..112
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     misc_RNA        133..182
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             1870..2061
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2055..2077)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2218..2237
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2312..2329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2483..3071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2563..2582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3242..4033,4034..4102))
                     /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"
     promoter        complement(4103..4174)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(4362..5444)
                     /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     5406..5425
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5445..5522)
                     /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."
ORIGIN
        1 acttcatctt cattacctcg tatcattgta cacctgccga acgcaagggc atgggctgtc
       61 gacctttgaa aagtaccttt atggctagct cagtcctagg tacaatgcta gcaatcaaga
      121 tactgagcac agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac
      181 agcctctaca aataattttg tttaaactag tgaaccacga ggcctacata tggttgctcg
      241 cccaaagtct gaggacaaaa agcaggcatt gcttgaagcg gcaactcaag ccatcgcgca
      301 atcaggcatt gccgctagta ccgctgtaat tgcacgcaat gcgggagttg cggaagggac
      361 gttgttccgc tatttcgcaa cgaaagatga gttgatcaac accctttact tacatttgta
      421 ccaggaccac tgccaatcac tgatcatgga attggatcgt tctattactg acgctaagat
      481 gatgatccgt tttacctgga acagttacat tagctgggga ttgaaccacc cagctcgcca
      541 tcgtgccatt cgtcagttgg cggtttctga aaagttgacg aaggaaacca agcaacgcat
      601 cgaggatatg ttcccggagt tacgcgacat cctgcaccgt agtgttctta tggtgtttat
      661 gtccgacgag tactccgcct tcggcaaggg gttgttctac gcgcttgctg agacgactat
      721 ggatttcgct gcgcgcgacc cggctcgcgc tggtgagtac attgcgttgg gcttcgaggc
      781 tatgtggcgc gcacttacgc gcgaagagca gtaataatcc taagaattcg aggagtgcag
      841 gctcggtaac atacggtcta gctatctgac tatcgccgct gtgagctcgg taccaaattc
      901 cagaaaagag gccgcgaaag cggccttttt tcgttttggt ccaagcccga tgcgccagag
      961 ttgtttctga aacatggcaa aggtatcact agtcttcgcg gccgccatgc tgtccaggca
     1021 ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac
     1081 ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa
     1141 cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg
     1201 cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga
     1261 ttcaccactc caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc
     1321 aacccttggc agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc
     1381 tcgggcagcg ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
     1441 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
     1501 acgtgaagtg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcatc
     1561 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
     1621 cggatctgca ttgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
     1681 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
     1741 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
     1801 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
     1861 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
     1921 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
     1981 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
     2041 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     2101 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     2161 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
     2221 catcagagca gattgtactg agagtgcacc ggtgtgaaat accgcacaga tgcgtaagga
     2281 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2401 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2461 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2521 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2581 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2641 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2701 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2761 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2821 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2881 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     2941 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3001 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3061 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3121 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3181 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctaaca
     3241 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3301 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3361 ccagtgctgc aatgataccg cgcgatccac gctcaccggc tccagattta tcagcaataa
     3421 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3481 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3541 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3601 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     3661 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3721 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3781 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3841 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3901 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3961 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4021 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4081 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4141 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaatatg cgccttgagc
     4201 gacacgaatt atgcagtgat ttacgacctg cacagccata ccacagcttc cgatggctgc
     4261 ctgacgccag aagcattggt gcaccgtgca gtcgatgata agctgtcaaa catgagaatt
     4321 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4381 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4441 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4501 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4561 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta
     4621 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     4681 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4741 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4801 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4861 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4921 accagatgct ccacgcccag tcgcgtacca tcttcatggg agaaaataat actgttgatg
     4981 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     5041 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     5101 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     5161 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     5221 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     5281 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     5341 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     5401 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5461 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg
     5521 tcgggacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgttgcggcc
     5581 gcgaagacag cgttatcaga gatgagacac cgtgcgataa tgtcgggcaa tcaggtgcga
     5641 ctcggtacca aattccagaa aagaggggag cgggaaaccg ctcccctttt ttcgttttgg
     5701 tcccaatcta tcgattgtat gg
//
