LOCUS       sequence-447349-        6381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses multinuclear non-heme iron dependent oxidative enzyme ChrH
            in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6381)
  AUTHORS   Ayikpoe RS, Zhu L, Chen JY, Ting CP, van der Donk WA
  TITLE     Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis
            by a SAM-Dependent Domain-of-Unknown-Function 692.
  JOURNAL   ACS Cent Sci. 2023 Apr 24;9(5):1008-1018. doi: 
            10.1021/acscentsci.3c00160. eCollection 2023 May 24.
  PUBMED    37252350
REFERENCE   2  (bases 1 to 6381)
  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/acscentsci.3c00160"; journalName: "ACS Cent Sci"; date: 
            "2023-04-24- 24"; volume: "9"; issue: "5"; pages: "1008-1018"
COMMENT     Sequence Label: pET28a:ChrH
FEATURES             Location/Qualifiers
     source          1..6381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             13..30
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             40..57
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1159..1176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1229..1247)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1243..1290
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1327..1782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1414..1433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1624..1645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1874..2689)
                     /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(1874..2689)
                     /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     2597..2616
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2681..2700
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2811..3399
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3300..3319
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3553..3570
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3585..3724
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3650..3669)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3810..3832
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3826..4017)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4826..5908)
                     /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     5870..5889
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5909..5986)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6192..6211
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6295..6314
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6295..6313
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6314..6338
                     /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             6353..6375
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
       61 atgaagaaac ctctccttgg attagccatg atgccggaag ctgactttgt ttccgctatt
      121 cttccgcttt tgcaaacgca aagtgttgac gtactcgagt ggagcttcga tacgctgtat
      181 gacgtcgaag agccggaatg gctgtccgga ctgcttgact tctattcaga caattcacgc
      241 ctgttgggac acggggtgta ctattcatta ttcgacgccc gctggatgga gcgccaagag
      301 atttggctca agaagcttaa agaagaggtg aagcgccgca agtacaatca tatcaccgag
      361 cattttggct tcatgaacac tgagaatttc caccaaggcg tacctcttcc tgtaccactt
      421 ctgccgaaga cgctgcaaat tgggaaggat cgcctgtctc gtctccagga cgccgtggag
      481 atcccggtgg gtgtcgagaa tctggcattc tctttcagca tggatgacgt caaggagcaa
      541 ggtgaattct tggaccgttt ggtggaagac atcgacgggt tcctcatcct cgatctccac
      601 aatatttact gccagagctg caacttcaag gtggacatga tggagatcat caatttatat
      661 ccgctcgaga aggtcaacga gatccacctc tccggcggct cttggcaaga gtcggcatac
      721 ggcaagaaac ctgtgcgccg tgatacgcat gacgaccgta tccctgaaga gattctgaat
      781 ttattacccg aggttttaat tcagtgccat cctgagtacg taattattga acgcttaggc
      841 cacacactta atacagaggt ggccaagcaa attttcttcg atgactttaa ccgcgtaaag
      901 aaaatcttgg agttatcaga ttacccagtc ggagaagaga aaatttggaa tcgccagaat
      961 accgtccact cgaagcctgt tgaagatctc ctcttgtatg ctgagcaaac tgaattaacc
     1021 cgtttacttt tcgatggtaa tacgacggaa tctattaaga atcgcgattt tcattatttc
     1081 aagcctgaat cctgggacga ggagatgatc acaactgcgc aacagatcat taagaagtgg
     1141 aacccgtact aactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1201 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1261 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1321 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1381 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1441 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1501 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1561 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1621 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1681 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1741 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1801 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1861 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     1921 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     1981 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     2041 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     2101 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     2161 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     2221 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     2281 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     2341 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     2401 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2461 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2521 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2581 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     2641 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     2701 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     2761 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2821 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2881 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2941 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3001 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3061 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3121 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3181 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3241 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3301 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3361 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3421 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3481 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3541 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3601 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3661 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3721 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3781 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3841 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3901 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3961 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4021 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4081 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4141 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4201 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4261 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4321 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4381 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4441 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4501 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4561 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4621 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4681 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4741 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4801 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4861 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4921 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4981 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5041 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5101 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5161 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5221 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5281 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5341 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5401 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5461 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5521 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5581 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5641 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5701 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5761 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     5821 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5881 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5941 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6001 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6061 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6121 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6181 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6241 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     6301 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     6361 actttaagaa ggagatatac c
//
