LOCUS       sequence-451212-        7926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Olsenella uli indoleacetate decarboxylase (IAD) R226M 
            variant in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7926)
  AUTHORS   Fu B, Nazemi A, Levin BJ, Yang Z, Kulik HJ, Balskus EP
  TITLE     Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme 
            Suggest Reaction Initiation via Hydrogen Atom Transfer.
  JOURNAL   J Am Chem Soc. 2022 Jun 29;144(25):11110-11119. doi: 
            10.1021/jacs.1c13580. Epub 2022 Jun 15.
  PUBMED    35704859
REFERENCE   2  (bases 1 to 7926)
  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"; doi: 
            "10.1021/jacs.1c13580"; journalName: "J Am Chem Soc"; date: 
            "2022-06-29- 29"; volume: "144"; issue: "25"; pages: "11110-11119"
COMMENT     Sequence Label: pET28a-OuIAD(R226M)
FEATURES             Location/Qualifiers
     source          1..7926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     10..29
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     94..113
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      224..812
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     713..732
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     966..983
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    998..1137
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1063..1082)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1223..1245
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1239..1430)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2239..3321)
                     /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     3283..3302
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3322..3399)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3605..3624
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3708..3727
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3708..3726
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3727..3751
                     /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)."
     RBS             3766..3788
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3807..3824
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3834..3851
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6498..6515
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6568..6586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6582..6629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6666..7121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6753..6772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6963..6984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(7213..7926,1..102))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(7213..7926,1..102))
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
       61 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
      121 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
      181 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
      241 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
      301 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
      361 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
      421 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
      481 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
      541 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
      601 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
      661 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
      721 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
      781 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
      841 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
      901 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
      961 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     1021 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     1081 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     1141 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     1201 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     1261 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     1321 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     1381 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     1441 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     1501 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     1561 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     1621 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     1681 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     1741 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     1801 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     1861 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     1921 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     1981 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     2041 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     2101 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     2161 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     2221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     2341 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     2401 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     2461 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     2521 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     2581 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     2641 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     2701 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     2761 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     2821 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     2881 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     2941 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     3001 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     3061 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     3121 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     3181 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     3241 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     3301 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     3361 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     3421 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     3481 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     3541 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     3601 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     3661 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     3721 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     3781 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc
     3841 cgcgcggcag ccatatggag gagtctctgg ttctcgagat gctgcagacc ggtaagacgg
     3901 ccacgtggcc ggcaaagaat cagctgcagg aaagcggcga ggtcgtcgac aaggaggtca
     3961 agggcactcc ttcgaccgac cgcaccaggc gcatgaagga acgcttcatg aatgccaagt
     4021 gcaagatgga catggaggcg cccatcgcat ataccaaggc atggcgcgag catgaaggca
     4081 agccgcttta tgtccgcagg gggcttgcct ataagtatat gctcgagcac ctcacgccgg
     4141 ctatccgtga ggacgagctc atcaccatgt ccaagacgcg ctatgaccgt ggtgccaccc
     4201 aggtccccca attcgccact gacttcatga tctccttcct cacccaggcc gaagaccaga
     4261 aggaagaggc aaagctgtat tccgtcgagg gcaaggatga ggcccatacg gtcgaagagg
     4321 aagggtggac caaggtcggc cagctgttct ccatccgcga ggaggaggtc aagccgatgc
     4381 ttgaggtcct cgagtattgg aagacgcgct gcgttgagaa cgtgtccgac gattggatga
     4441 agacctcctt cccctactac caggactacg tcaacgcaaa gaaggtcggc ctcttcccgg
     4501 ggtcgggcct gcatgccggc tgtgacggca tgtggattcc cgcctatgac gttgccctcg
     4561 ggggcctcaa cagggtaatc gaggagtgcc gcgagaagat cgagaagacc gtggtgacca
     4621 ccaaggaggt cgccgacaag gtcttcttct ggcaaggctg catctatgcc tgcgagggcg
     4681 cgatcgcctg ggcccacaac tatgcggtcg aggcccggcg ccttgccgag actgcggtcg
     4741 agccacgcaa gaccgagctt ctcgagatgg cagaacgcct cgatcgcgtc cctgccgaag
     4801 caccccgcaa cttcatggaa gccgtccagg ccctttggac cacccagatc ctcgtcatct
     4861 ccgactccct tgccctcggc gtctccccgg gccgttgggg caagttcctt gagccttatt
     4921 acgagaagga tcttgccgag gggcgcatca ccaagggaca ggcccttgag gtcatggagc
     4981 tgctccgtat caaattctcg actgaggagt acatcacccc gtccctctgg gcggccatgg
     5041 cctcctcgaa ctcgttcatg aaccttgccg ttggcggcct cgatcccaag acgggcaagt
     5101 gcaccgacaa cgagatcgag gacctcatcc tcgaggccgg catcaacatg cccaccccgc
     5161 aaccgaccct ctcgatcctt ctctccgaca agacgacaga ccatctcgcc atcaaggccg
     5221 ccgagtgcac caaggccggc aacgggtatc ccgcgtggtt taactatgac atgatggtcc
     5281 aacacaacct cgctatgtat cgcgacgagg gcatcaccct tgaggacgcc cgcaactgtg
     5341 cgctctccgg ctgcgtcgag aacggcctcg ccggtaccgg tcaccccatc gcccacccgg
     5401 ccttctacaa cgagggcaag accatcgagc ttgcctgcaa cgaaggcgtc gacccccgca
     5461 cgggcatcaa ggtcatggat ggcatcgtcc ccatcaagac gtacgaggac ctttgggaca
     5521 acttcatcaa gatccgcgag cacttcatgc atgtctacat gcagtactgg aatgaggtcg
     5581 tcgcctgcca gcgcgacatc caccccaaga tcatgggctc cgtcctcatg catgactgca
     5641 tcgagtccgg ccgcccggtt gacaatctcg gctgccgcta caacggttcc gtcacgctgc
     5701 tcgactccgg tacagttaac gtggtcaacg gtctcgcggc catcaagaag ctcgtcttcg
     5761 aggaccacaa gtacacttgg gatgagttca aggaggccat ggacaataac ttcggtttcg
     5821 tgcttggtgc cgagaagggc aacttctcca tgctcaacca ggagatcgac cctgaaaagc
     5881 acatgaagta cgcccatatc caccgagatg tcctcaacgc acccaagttt ggcaacgacg
     5941 atgacttcgt tgacgacatc tttgtcgacc tctggcatga ctacgaccgc gttaccgcgt
     6001 ccgaaaccac ctacaacggc taccgctgga tcaccgccgc gctttccata tccgcccatg
     6061 gcccgcatgg tcgtgtgacc ggcgctaccc ctgacggtcg tctctccgga gtgacgctgt
     6121 gcgacggcat cctgtccgcc tcgcctggca ccgacgtgaa cggtccgatc gccctcatcc
     6181 gctccggcgt gaagctggat ccgaccgagt tcgcctcggt gcagctcaat atgaagttcc
     6241 atccgactgc catccgcggt gacgagggct cgaggaactt cgtggacttc atccacagct
     6301 acttccagat gggcggctat cacgtccagt tcaacatcgt ggattcgaag atgctgcgag
     6361 acgcccagga tcatccgcag aactatcgcg acctgatggt ccgtgtcgcc ggcttctcag
     6421 cctactggaa cgagctcggc aaacccatcc aggacgaggt cattgctcgt accgagtacg
     6481 acgctctctg actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     6541 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     6601 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     6661 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6721 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6781 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6841 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     6901 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6961 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7021 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7081 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     7141 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7201 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     7261 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     7321 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     7381 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     7441 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     7501 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     7561 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     7621 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     7681 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     7741 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     7801 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     7861 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     7921 tgattg
//
