LOCUS       sequence-451219-        6228 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expressing Clostridium scatologenes hydroxyphenylacetate 
            decarboxylase activating enzyme (HPAD-AE) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6228)
  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 6228)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-AE
FEATURES             Location/Qualifiers
     source          1..6228
                     /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             3643..3660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3713..3731)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3727..3774
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3811..4266
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3898..3917)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4108..4129
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4358..5173)
                     /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(4358..5173)
                     /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     5081..5100
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5165..5184
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5295..5883
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5784..5803
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6037..6054
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6069..6208
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6134..6153)
                     /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 aaggaaaaag gtttaatatt tgatatacaa agcttttctg tacatgatgg accaggttgt
     2761 agaaccagtg tattttttat agggtgtcca cttcagtgta aatggtgtgc caatccagaa
     2821 agttggacta agaaaaaaca tattatggtt gcagaaaatg tatgcaagtg gaaaaatgga
     2881 tgtcgatcat gtataaatgc atgttcacat gattcaatta aattcagtga ggatggcaag
     2941 cttaaaatat cctgggatac ttgtgaaaaa tgtgagactt ttgactgtgt taatatgtgt
     3001 cctaataatg ctttgaagca gtgtgttaag gaatatacag tagatgagct tatgacaatt
     3061 ttaaagcgtg atttcaataa ttggggttct gatggaggag taacctttac aggaggagat
     3121 cctcttatgc atcatgaatt tttggtggaa gtactgaaaa aatgttatga tagccagatt
     3181 cataaagcaa ttgaaactag tggatatgca aaacaggaag tgttcttaga agtattaaag
     3241 tacattgatt ttgcatttat tgatgtaaaa aatatggata gagaaaagca taagcaagga
     3301 actggagttt ataatgattt aatactttca aatattgaag cacttaaaaa gtccaattgg
     3361 aatggaagac ttgttttaag acaacctact atagcaggat ataatgacag tgatgaaaat
     3421 gcatataagc taattgaatt tatgaataaa aattctctat atgaaatcaa ccttttgaaa
     3481 ttccacaggt tgggagaaac aaagtggaat cagctaggaa aagagtacga atatagtaag
     3541 tatggagata tgacaaatga aaaaatggag catcttcaac agttatattt agataataat
     3601 atagcttgct atataggtga taatacgcct ttttaactcg agcaccacca ccaccaccac
     3661 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     3721 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     3781 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     3841 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3901 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3961 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4021 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4081 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4141 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4201 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     4261 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4321 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     4381 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     4441 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     4501 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     4561 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     4621 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     4681 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     4741 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4801 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4861 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4921 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     4981 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     5041 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     5101 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     5161 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     5221 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5281 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5341 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5401 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5461 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5521 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5581 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5641 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5701 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5761 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5821 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5881 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5941 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     6001 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     6061 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     6121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     6181 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacc
//
