LOCUS       sequence-486681-        5778 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Lumazine synthase from Aquifex aeolicus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5778)
  AUTHORS   Hoffpauir ZA, Lamb AL
  TITLE     Identification of the phosphatase essential for riboflavin 
            biosynthesis in Aquifex aeolicus.
  JOURNAL   J Biol Chem. 2025 May;301(5):108443. doi: 10.1016/j.jbc.2025.108443.
            Epub 2025 Mar 25.
  PUBMED    40147773
REFERENCE   2  (bases 1 to 5778)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem."; date: "2025-05"; volume: "301(5):108443. doi"; pages: " 
            10.1016/j.jbc.2025.108443. Epub 2025 Mar 25"
COMMENT     Sequence Label: AaLS
FEATURES             Location/Qualifiers
     source          1..5778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(215..232)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     RBS             711..733
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    748..772
                     /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)."
     primer_bind     complement(772..791)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(773..791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(879..898)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1104..1181
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1182..2264
                     /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     complement(1201..1220)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3073..3264
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3258..3280)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3366..3508
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3421..3440
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3523..3540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3694..4282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3774..3793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4393..4412)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4404..5219
                     /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             4404..5219
                     /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     complement(4477..4496)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5312..5767)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5449..5470)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5661..5680
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgatatcg ccatggaacc gcgtggcacc agggtacctt
      241 agcgcaggct tttaaacaag ttggccatct cgattgcgct cagcgctgcc tcccaacctt
      301 tgttcccgtg cttggtgccc gcacgttcga tagcctgctc caaggtatca gcggtgatga
      361 cgccgaacgt aatcggcttg cgcagttcca aggaaagatt cgccaggcct ttcgaaactt
      421 cagaggcaat gtagtcaaaa tgtggggtcg caccacgaat cagcacgcca atcgcaataa
      481 ctgcgtcgat gtcctcctta cgagccagtt cacccgcagc aaccgggatc tcccagctac
      541 ccggaacacg taccagagtg atgtcctctt ctctaccgcc atgacgcacg atgcaatcga
      601 tcgcaccttc caccagacga tcgacgagcg cgtggttgaa acggctggcc acaataccga
      661 agcgcaggcc ttcagctgtt agttttccct catatatttg catatgtata tctccttctt
      721 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
      781 gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt
      841 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
      901 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
      961 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     1021 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     1081 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     1141 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     1201 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     1261 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     1321 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     1381 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     1441 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     1501 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     1561 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     1621 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     1681 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     1741 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     1801 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     1861 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     1921 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     1981 acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     2041 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     2101 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     2161 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     2221 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2281 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     2341 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     2401 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     2461 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     2521 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     2581 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     2641 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     2701 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     2761 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     2821 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     2881 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     2941 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     3001 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     3061 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     3121 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     3181 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     3241 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     3301 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     3361 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     3421 cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag
     3481 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     3541 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3601 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3661 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3721 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3781 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3841 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3901 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3961 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4021 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4081 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4141 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4201 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4261 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4321 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
     4381 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
     4441 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     4501 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     4561 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     4621 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     4681 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     4741 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     4801 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     4861 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     4921 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     4981 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     5041 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     5101 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     5161 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     5221 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5281 ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa acgttaatat tttgttaaaa
     5341 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5401 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5461 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5521 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5581 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5641 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5701 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5761 ggcgcgtccc attcgcca
//
