LOCUS       sequence-451218-        8253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Clostridium scatologenes hydroxyphenylacetate 
            decarboxylase (HPAD) beta/gamma subunits in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8253)
  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 8253)
  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-CsHPAD_beta-gamma
FEATURES             Location/Qualifiers
     source          1..8253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..88
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(82..273)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1082..2164)
                     /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     2126..2145
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2165..2242)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2448..2467
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2551..2570
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2551..2569
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2570..2594
                     /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             2609..2631
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2650..2667
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2677..2694
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5668..5685
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5738..5756)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5752..5799
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5836..6291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5923..5942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6133..6154
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6383..7198)
                     /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(6383..7198)
                     /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     7106..7125
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7190..7209
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7320..7908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7809..7828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8062..8079
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8094..8233
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8159..8178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
       61 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      121 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      181 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      241 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
      301 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
      361 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
      421 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
      481 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
      541 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
      601 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
      661 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
      721 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
      781 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
      841 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
      901 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
      961 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     1021 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     1081 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1141 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     1201 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     1261 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     1321 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     1381 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1441 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1501 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1561 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1621 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1681 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1741 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1801 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1861 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1921 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1981 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     2041 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     2101 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     2161 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     2221 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     2281 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     2341 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     2401 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     2461 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     2521 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     2581 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
     2641 ggcagcagcc atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg
     2701 aacgttaaag aaactaaact tgaagatgtt ttaaaaagtc gtggtattga catgaaagat
     2761 gcttacaata tttcagaagc agatattcca gaagcaaagg aaagtactca aaagcttatg
     2821 gatatctatt atactttaaa ggttactgca gatatggaag ctgcatattg gtataacaga
     2881 acttggtggg aaaatgatgg tgaagtaatt gaagtaagaa gagctaaagc tgtagcagct
     2941 tctttatcac acatgacacc taccatttta ccttatgaga agcttgttat gaacaagaca
     3001 aaaaatgtta gaggtgcatt tccttttcct tgggtttgtg caagtttttt taatgctcaa
     3061 gcagaagctt taatgaacga agttgatgct ccagcagaga atgaagctga ctcagtaagt
     3121 gttgttggtg ctggtggagg taatgttact gaaagttatg gaaatgttat ttctattgct
     3181 aaaaagtttg gtatgagaaa agaagaaatt cctgttttag ttaaaacctc aaagccttgg
     3241 gaaggaattt cagtagaaga attaagtaat aaatactcaa agatgacacc aggatatgat
     3301 cagtttaaaa atatcatgga aagtgttatc tgtatgtttg actcttttgc gattccacag
     3361 gggcgtgaag taattaacta ttatatgcct ttgcagtatg gttttgatgg aattatcaaa
     3421 ttatgtgatg aaaaaattgc tgaagtcatg ggtgaagctg gtgacgatgg tgattttggt
     3481 atgagcagag gttattacta tgcagcaatg aaggaaatta ctaagggttt aagtgcatgg
     3541 tgtgaaaatt attcaaagag agcaaaatat ttagcttcca ttgaaacaga ttcagaaatt
     3601 aaagccaact atgaaaaaat tgaagaagtt atgggaaaca ttgctcataa gaaacctgca
     3661 aacttctggg aagcaattca gatgacactt tgctgtcatt ttggtgttgt taatgaagat
     3721 cctcaatctg gtctttcaat tggaagatta ggacaagtat tacagccatt ctatgaaaaa
     3781 gatgttgagg atggtattat gacagatgaa gaagtaattg aacttcttga attatacaga
     3841 attaaaatta cttgtattga atgttttgca tcagctggtg tatctggtgg tgttctttct
     3901 ggtaatacct ttaacaatct ttcattagga ggacagaatt atgatggact ttcagcagtt
     3961 actccactag aatatttaat tgttgaagca ggtatgagaa atcaaactcc tcaacctacg
     4021 ttgagtgtat tatacgatga aaaaacgcca gaagatttcc ttatgaaggc tgcttcctgt
     4081 acaaaacttg gtcttggata tcctgcatgg atgaacaatc aaacaggtat gaactttatg
     4141 atgagaaact atggaccaga gggaatggat cttcatgatg caagagcatg gtgtcttgga
     4201 ggatgtctag aatctgcacc tggatgtttc ttgccacttg aatataatgg aaaagtaaca
     4261 atgattccag gaggtgcatc acctacatgt ggtacaggtg ttcattttat tggtatgcct
     4321 aaagtacttg agcttgtttt aacaaacggt ttagataaaa gaacaggaaa acaagtatac
     4381 ccacctcaca acaagaagct tgattcttat gaaacaatgg tgaatcaatg gaaagagtac
     4441 atggaactta ctacagatgt tgtaaatagg tgtaacaata ttcaaatgga tatttggaga
     4501 aaatacaata tgccagctgt taattccttg ttaaaacctg attgtttcaa aaaaggcaaa
     4561 cacattggca ctatgggagc aagatataat tcatgtatta attttgaatc ctgtggaact
     4621 attacttttg taaattcatt gagttctatt aagaagaacg tttttgatga tagtaaattt
     4681 acaattgaag aaatgacaga tgcaatgctt aataattttg gatttaagac tgcatatgaa
     4741 acagaagtat tttcacctga ttttagagaa agtacagata agagcactaa atatgagaaa
     4801 atatttgcag catgtgtaaa tgcaccaaaa tacggtaatg ctgataagta tgcagatgaa
     4861 atttttaagg catatcatta ctatatttat gatatgacac ataaattccg ttcatattat
     4921 ggcaaacctt tatatttgtg tcagatttca gtatcaacac atggacctca aggctttgta
     4981 actctagcta cagctgatgg aagacttgca ggaacaactt attcagatgg ttctgtatct
     5041 gcagcagcag gaacagataa gaatggtatt tatgctatat ttgaatctgc aacagtttat
     5101 gatcactcaa tgcatcaaaa tgcacagatg aatttaaaat tacatccaac agcggttaaa
     5161 ggtatcaatg gaacaagaaa gcttcttgat cttgtacgtg cttacatgag aaaaggtgga
     5221 ttccatgttc agtttaatgt tgttgattct aaaacattga gagatgccca gcttacacca
     5281 gagaaatata gagaacttat ggttcgtgta gctggattta ctcaatactg gtgtgaaatc
     5341 ggtaagccta ttcaagatga agttatttat agaactgaat atgataaata gggtaatgat
     5401 atgcgccact atgattgtaa aaattatata aatttagatt gtgaaaaagg tctttgtgca
     5461 ctaacaaagg gaatggttcc tattgatggt gaaggaagtg aagcttgccc taatttcaaa
     5521 ccagctgaaa aatgcggaaa ttgcaaaaat ttttgtaatc cggataaata cggattggga
     5581 acatgtacag gcttagaaaa agaaaattgg gcatatgcta cttgtggtgc ttctgcatgt
     5641 cctagttata aagcagaata gctcgagcac caccaccacc accactgaga tccggctgct
     5701 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     5761 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     5821 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5881 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5941 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     6001 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6061 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6121 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6181 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6241 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     6301 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6361 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     6421 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     6481 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     6541 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     6601 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     6661 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     6721 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     6781 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     6841 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     6901 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     6961 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     7021 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     7081 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     7141 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     7201 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     7261 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7321 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7381 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7441 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     7501 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7561 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7621 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7681 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7741 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7801 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7861 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7921 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7981 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     8041 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     8101 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     8161 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     8221 ggtcatggct gcgccccgac acccgccaac acc
//
