LOCUS       sequence-372572-        5949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3,4-dihydroxy-2-butanone 4-phosphate synthase RibB from 
            Vibrio cholerae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5949)
  AUTHORS   Kenjic N, Meneely KM, Wherritt DJ, Denler MC, Jackson TA, Moran GR, 
            Lamb AL
  TITLE     Evidence for the Chemical Mechanism of RibB 
            (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin 
            Biosynthesis.
  JOURNAL   J Am Chem Soc. 2022 Jul 20;144(28):12769-12780. doi: 
            10.1021/jacs.2c03376. Epub 2022 Jul 8.
  PUBMED    35802469
REFERENCE   2  (bases 1 to 5949)
  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.2c03376"; journalName: "J Am Chem Soc"; date: 
            "2022-07-20- 20"; volume: "144"; issue: "28"; pages: "12769-12780"
COMMENT     Sequence Label: pET28a-VcRibB
FEATURES             Location/Qualifiers
     source          1..5949
                     /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(827..844)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(854..871)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             890..912
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    927..951
                     /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(951..970)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(952..970)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1054..1073)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1279..1356
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1357..2439
                     /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(1376..1395)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3248..3439
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3433..3455)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3541..3680
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3596..3615
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3695..3712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3866..4454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3946..3965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4565..4584)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4576..5391
                     /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             4576..5391
                     /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(4649..4668)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5483..5938)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5620..5641)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5832..5851
                     /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 gagttattaa gccagtttta
      181 gatcaaactg aatgcggtat tgcaccatat cttcaatggt caatacgggc atattgtgca
      241 gtttgccaaa ctcgatgatt tctggcgttt ttgccatgct gccatctggg ttggtcagtt
      301 cacacaatac accggctggc tgtaagcccg ccatctgcat caaatcaacc gtaccttcgg
      361 tatgaccacg acgcgccaat accccacccg cacgagcacg tagtgggaat acatggccgg
      421 gacgcgccaa atcttctggc tttgcttgtg ggttggcggc ggttttgatg gtggtgacgc
      481 gatcttgcgc cgaaaccccc gtggttacgc catgtttggc ttcaatcgat accgtaaacg
      541 ccgtttggtt cgcactgttg ttgttgacca ccataggcgg tagggctaag cgatcggctt
      601 gcgcttcagt cagacacaaa cagacgatgc cgctgcattc acggatcatc aatgccattt
      661 gcgctgtagt aagagattct actgcgtaga taatatcgcc ttcgttttcg cgatcttcat
      721 catcgagcag cagcacacca cgcccctcac gtaaggcttg cagtgcgttt tctacacgag
      781 taatggggtc gccgaattcg gcaagtaatg aggactgatt catatggctg ccgcgcggca
      841 ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat ctccttctta
      901 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
      961 agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg
     1021 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     1081 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
     1141 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
     1201 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
     1261 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     1321 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     1381 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     1441 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     1501 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     1561 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     1621 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     1681 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     1741 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     1801 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     1861 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     1921 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     1981 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     2041 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     2101 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     2161 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     2221 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     2281 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     2341 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     2401 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     2461 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     2521 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     2581 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     2641 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     2701 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     2761 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     2821 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     2881 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     2941 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     3001 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     3061 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     3121 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     3181 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     3241 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     3301 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     3361 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     3421 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3481 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3541 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     3601 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     3661 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3721 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3781 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3841 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3901 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3961 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4021 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4081 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4141 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4201 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4261 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4321 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4381 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4441 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4501 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     4561 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     4621 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     4681 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     4741 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     4801 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     4861 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     4921 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     4981 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     5041 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     5101 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     5161 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     5221 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     5281 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     5341 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     5401 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5461 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     5521 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5581 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     5641 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     5701 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     5761 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     5821 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     5881 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     5941 cattcgcca
//
