LOCUS       sequence-451205-        6237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Olsenella uli indoleacetate decarboxylase activating 
            enzyme (IAD-AE) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6237)
  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 6237)
  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-AE
FEATURES             Location/Qualifiers
     source          1..6237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2..20
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    21..45
                     /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             60..82
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             128..145
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1103..1120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1173..1191)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1187..1234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1271..1726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1358..1377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1568..1589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1818..2633)
                     /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(1818..2633)
                     /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     2541..2560
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2625..2644
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2755..3343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3244..3263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3497..3514
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3529..3668
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3594..3613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3754..3776
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3770..3961)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4770..5852)
                     /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     5814..5833
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5853..5930)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6136..6155
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
       61 ttgtttaact ttaagaagga gatataccat gggcagcagc catcatcatc atcatcacag
      121 cagcggcctg gtgccgcgcg gcagccatat ggacggcaag ggcaaagagc tcagggccgt
      181 gattttcgac gtgcagagct tttctacgca tgatggacct gggatcagga cgaacatctt
      241 cttcaagggg tgtgcgctca ggtgcccttg gtgcgccaac cccgagtcgc agaagttcag
      301 cccgcagctc ctttacacaa agatgaagtg catagggtgc atgtgctgtg ccagggcatg
      361 cccccatggg gccgtgacgg cctatactgc gccagaggac atcgagcgtt atggacacgt
      421 acggtatgac cgtggcaagt gtgataagtg caccacgcat gagtgcgtgg acgcatgctt
      481 ccaggaagcc ctcgccgtct ccgggaagga aatgaccgtc gatgacgtaa tggataagat
      541 tcgtcgcgac tctcctgtct tccggggtaa gggaggcgtc accgtatccg gtggggatcc
      601 gctgctctat cctgagttct tggccgagct gcttggtagg tgccgagatg aggggtatag
      661 cgttgccctt gagtccgagc tctgcgttcc cacgaggaac ctcgagacgg tcatgccgtt
      721 cgtaagctac taccttaccg actgcaaaat tatcgactcc gccgagcacc gcagaatcac
      781 tggtgtggac aatgagatca tcctcaggaa cctgcgcctg attggcgatg tatgcccaga
      841 gcgcatgtgc ctgcgcatac cgatcatacc tgggtacaca gactccgacg agaacgttgg
      901 cggtatcgcc gcgtttgccg cgtcgtgtca attcacaaag ataaacatcc tgccgtatca
      961 caagcttggc gtgcccaagc acgagcgcct tggcaccgtc tatcagctac ctcatgtcca
     1021 gccacccgac gatgggcata tgaggcactt ggctgacgtc atagaagccc atgggatcga
     1081 gtgcgttatc aactgactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     1141 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     1201 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     1261 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     1321 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     1381 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     1441 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     1501 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1561 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1621 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1681 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1741 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1801 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     1861 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1921 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1981 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2041 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2101 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2161 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2221 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2281 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     2341 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2401 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2461 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     2521 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2581 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     2641 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     2701 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2761 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2821 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2881 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2941 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3001 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3061 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3121 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3181 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3241 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3301 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3361 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3421 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3481 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3541 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3601 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3661 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3721 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3781 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3841 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3901 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     3961 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4021 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4081 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4141 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4201 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4261 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4321 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4381 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     4441 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4501 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4561 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4621 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4681 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4741 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4801 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4861 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     4921 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4981 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5041 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5101 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5161 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5221 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5281 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5341 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5401 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5461 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5521 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5581 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5641 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5701 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5761 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     5821 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     5881 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     5941 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6001 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6061 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6121 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6181 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaa
//
