LOCUS       sequence-486678-        6530 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP 
            cyclohydrolase II from Aquifex aeolicus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6530)
  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 6530)
  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: AaRibBA
FEATURES             Location/Qualifiers
     source          1..6530
                     /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             1467..1489
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1504..1528
                     /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(1528..1547)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1529..1547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1635..1654)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1860..1937
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1938..3020
                     /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(1957..1976)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3829..4020
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4014..4036)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4122..4261
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4177..4196
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4276..4293)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4447..5035)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4527..4546)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5146..5165)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5157..5972
                     /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             5157..5972
                     /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(5230..5249)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6064..6519)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6201..6222)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6413..6432
                     /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 agaacatgtg gcccagttta tcctttttga cttgcaggta gatcttgtta tacggattcg
      301 gttcaatctt aatcggcacg cgctcaacca cctccagacc aaaaccctcc agggccacaa
      361 ttttacgcgg gttattggtc atcagtttca ttttacgaac acccaggtcg cgcaggatct
      421 gtgcaccgat gccataatcc cgaaggtcag gcgggtagcc caacttctcg ttcgcctcaa
      481 cggtgtcgta gcccttctct tgcagttcat aggccttgat tttattcgca atgccgatgc
      541 ctctgccttc gtgccccaag atataaacca gtacgccttt gccctctttc gcgatcatct
      601 ccagagcctt ctcgagctgc ggacgacaat cacaacgaaa ggaacggaac acgtcaccgg
      661 tcaggcactc ggaatgaaca cgaaccaaga ccggttcgtc ttctttccac tcacccatgg
      721 tcaacgcaac ctgctcttca ccagtaagtt tatgacgata ggcgtggatt ttgaaaacac
      781 cccacggggt cggcaggtgt gcagacgcca ccttttcaac cagggtttca cgacgcaggc
      841 ggtacttgat cagatccgca atggtgatga ttttcagatt gtgcttcttt gcgaattcca
      901 tcaggtccgg cacgcgcgcc atggtgccgt cgtccttcat aatttcgcag atcacgcctg
      961 ccggatacaa acccgccaaa cgagccagat cgacgcttgc ttcggtgtgg ccagcgcgct
     1021 ctaacacacc acccggacgc gctttcagcg ggaacacgtg acctgggcga acaaaatcag
     1081 acggtttcgc gtccggcgaa atcgctagct tgatcgtcag agcacggtcg tacgcgctga
     1141 tacccgtggt cgtaccatgc ttcggatgcg catcgatgct aacgcagaag tacgtacctt
     1201 ttggatcggt gttcatcgga gtcatcggat gcagatcaag ttgctcgcaa cgctcggggg
     1261 tcaagcttaa gcaaatcaga ccacggccgt acttggccat aaagttaatg gcttctgggg
     1321 tgaccttttc tgcggccatc accaggtctc cttcgttctc gcggtccggg tcatccacga
     1381 caataaccat tttaccctga cgaatatcct cgatcgcttc ttccactgta ttgaatttaa
     1441 attcctccct ttctgacata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1501 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     1561 gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg
     1621 tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt
     1681 cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact
     1741 gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
     1801 cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gagatcccgg
     1861 acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     1921 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     1981 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     2041 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     2101 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     2161 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     2221 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     2281 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     2341 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     2401 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     2461 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     2521 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     2581 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     2641 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     2701 gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg ggatacgacg
     2761 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     2821 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     2881 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     2941 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3001 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     3061 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     3121 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     3181 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
     3241 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
     3301 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
     3361 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
     3421 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
     3481 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
     3541 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
     3601 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
     3661 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
     3721 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
     3781 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
     3841 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
     3901 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
     3961 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     4021 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4081 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     4141 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     4201 gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     4261 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4321 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4381 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4441 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4501 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4561 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4621 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4681 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4741 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4801 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4861 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     4921 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4981 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5041 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5101 tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     5161 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg
     5221 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     5281 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     5341 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     5401 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     5461 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     5521 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     5581 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     5641 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     5701 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     5761 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     5821 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     5881 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     5941 agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt
     6001 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6061 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6121 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6181 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6241 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6301 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6361 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6421 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6481 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
