LOCUS       sequence-451213-        7926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Olsenella uli indoleacetate decarboxylase (IAD) R226K 
            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(R226K)
FEATURES             Location/Qualifiers
     source          1..7926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..64
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(58..249)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1058..2140)
                     /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     2102..2121
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2141..2218)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2424..2443
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2527..2546
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2527..2545
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2546..2570
                     /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             2585..2607
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2626..2643
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2653..2670
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5317..5334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5387..5405)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5401..5448
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5485..5940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5572..5591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5782..5803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6032..6847)
                     /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(6032..6847)
                     /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"
     primer_bind     6755..6774
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6839..6858
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6969..7557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7458..7477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7711..7728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7743..7882
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7808..7827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
       61 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
      121 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
      181 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
      241 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
      301 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
      361 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
      421 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
      481 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
      541 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
      601 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
      661 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
      721 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
      781 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
      841 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
      901 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
      961 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     1021 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1141 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1201 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1261 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1321 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1381 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1441 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1501 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1561 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1621 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1681 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1741 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     1801 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     1861 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     1921 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     1981 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2041 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2101 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2161 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     2221 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     2281 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     2341 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     2401 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     2461 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     2521 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     2581 taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat
     2641 cacagcagcg gcctggtgcc gcgcggcagc catatggagg agtctctggt tctcgagatg
     2701 ctgcagaccg gtaagacggc cacgtggccg gcaaagaatc agctgcagga aagcggcgag
     2761 gtcgtcgaca aggaggtcaa gggcactcct tcgaccgacc gcaccaggcg catgaaggaa
     2821 cgcttcatga atgccaagtg caagatggac atggaggcgc ccatcgcata taccaaggca
     2881 tggcgcgagc atgaaggcaa gccgctttat gtccgcaggg ggcttgccta taagtatatg
     2941 ctcgagcacc tcacgccggc tatccgtgag gacgagctca tcaccatgtc caagacgcgc
     3001 tatgaccgtg gtgccaccca ggtcccccaa ttcgccactg acttcatgat ctccttcctc
     3061 acccaggccg aagaccagaa ggaagaggca aagctgtatt ccgtcgaggg caaggatgag
     3121 gcccatacgg tcgaagagga agggtggacc aaggtcggcc agctgttctc catccgcgag
     3181 gaggaggtca agccgatgct tgaggtcctc gagtattgga agacgcgctg cgttgagaac
     3241 gtgtccgacg attggatgaa gacctccttc ccctactacc aggactacgt caacgcaaag
     3301 aaggtcggcc tcttcccggg gtcgggcctg catgccggct gtgacggcaa atggattccc
     3361 gcctatgacg ttgccctcgg gggcctcaac agggtaatcg aggagtgccg cgagaagatc
     3421 gagaagaccg tggtgaccac caaggaggtc gccgacaagg tcttcttctg gcaaggctgc
     3481 atctatgcct gcgagggcgc gatcgcctgg gcccacaact atgcggtcga ggcccggcgc
     3541 cttgccgaga ctgcggtcga gccacgcaag accgagcttc tcgagatggc agaacgcctc
     3601 gatcgcgtcc ctgccgaagc accccgcaac ttcatggaag ccgtccaggc cctttggacc
     3661 acccagatcc tcgtcatctc cgactccctt gccctcggcg tctccccggg ccgttggggc
     3721 aagttccttg agccttatta cgagaaggat cttgccgagg ggcgcatcac caagggacag
     3781 gcccttgagg tcatggagct gctccgtatc aaattctcga ctgaggagta catcaccccg
     3841 tccctctggg cggccatggc ctcctcgaac tcgttcatga accttgccgt tggcggcctc
     3901 gatcccaaga cgggcaagtg caccgacaac gagatcgagg acctcatcct cgaggccggc
     3961 atcaacatgc ccaccccgca accgaccctc tcgatccttc tctccgacaa gacgacagac
     4021 catctcgcca tcaaggccgc cgagtgcacc aaggccggca acgggtatcc cgcgtggttt
     4081 aactatgaca tgatggtcca acacaacctc gctatgtatc gcgacgaggg catcaccctt
     4141 gaggacgccc gcaactgtgc gctctccggc tgcgtcgaga acggcctcgc cggtaccggt
     4201 caccccatcg cccacccggc cttctacaac gagggcaaga ccatcgagct tgcctgcaac
     4261 gaaggcgtcg acccccgcac gggcatcaag gtcatggatg gcatcgtccc catcaagacg
     4321 tacgaggacc tttgggacaa cttcatcaag atccgcgagc acttcatgca tgtctacatg
     4381 cagtactgga atgaggtcgt cgcctgccag cgcgacatcc accccaagat catgggctcc
     4441 gtcctcatgc atgactgcat cgagtccggc cgcccggttg acaatctcgg ctgccgctac
     4501 aacggttccg tcacgctgct cgactccggt acagttaacg tggtcaacgg tctcgcggcc
     4561 atcaagaagc tcgtcttcga ggaccacaag tacacttggg atgagttcaa ggaggccatg
     4621 gacaataact tcggtttcgt gcttggtgcc gagaagggca acttctccat gctcaaccag
     4681 gagatcgacc ctgaaaagca catgaagtac gcccatatcc accgagatgt cctcaacgca
     4741 cccaagtttg gcaacgacga tgacttcgtt gacgacatct ttgtcgacct ctggcatgac
     4801 tacgaccgcg ttaccgcgtc cgaaaccacc tacaacggct accgctggat caccgccgcg
     4861 ctttccatat ccgcccatgg cccgcatggt cgtgtgaccg gcgctacccc tgacggtcgt
     4921 ctctccggag tgacgctgtg cgacggcatc ctgtccgcct cgcctggcac cgacgtgaac
     4981 ggtccgatcg ccctcatccg ctccggcgtg aagctggatc cgaccgagtt cgcctcggtg
     5041 cagctcaata tgaagttcca tccgactgcc atccgcggtg acgagggctc gaggaacttc
     5101 gtggacttca tccacagcta cttccagatg ggcggctatc acgtccagtt caacatcgtg
     5161 gattcgaaga tgctgcgaga cgcccaggat catccgcaga actatcgcga cctgatggtc
     5221 cgtgtcgccg gcttctcagc ctactggaac gagctcggca aacccatcca ggacgaggtc
     5281 attgctcgta ccgagtacga cgctctctga ctcgagcacc accaccacca ccactgagat
     5341 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     5401 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     5461 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5521 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5581 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5641 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5701 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5761 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5821 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5881 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     5941 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6001 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     6061 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     6121 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     6181 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     6241 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     6301 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     6361 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     6421 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     6481 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     6541 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     6601 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     6661 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     6721 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     6781 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     6841 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     6901 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6961 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7021 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7081 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7141 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7201 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7261 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7321 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7381 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7441 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7501 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7561 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7621 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7681 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7741 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     7801 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     7861 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     7921 ggcttg
//
