LOCUS       sequence-486680-        6380 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Pyrimidine deaminase/reductase from Aquifex aeolicus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6380)
  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 6380)
  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: AaRibD
FEATURES             Location/Qualifiers
     source          1..6380
                     /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             1317..1339
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1354..1378
                     /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(1378..1397)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1379..1397)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1485..1504)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1710..1787
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1788..2870
                     /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(1807..1826)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3679..3870
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3864..3886)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3972..4111
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4027..4046
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4126..4143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4297..4885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4377..4396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4996..5015)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5007..5822
                     /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             5007..5822
                     /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(5080..5099)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5914..6369)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6051..6072)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6263..6282
                     /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 aatagcgctt gaaaaagatg accaagtctt cacccaggtt ctcgaccttc ttcttacgta
      301 ccttcagaga ttcctcgatg ctgcggatgc ccagatcgcc aatgcttaga ccttcaccca
      361 taattttcgg agcgataaac accacgatac ggtcgaagaa accctccttc aagaacgaag
      421 tcagcgtacg cggaccgcct tcaaccatca ggtgcatgat ctccaactct ttcagtttct
      481 tcaagatgtt cttcaggttg aaacctttca gaatcagaac ctggacgcct ttctccttca
      541 gtttttcggc cttttcattc tcctttttgg taatcacaat tgttggggcc tcctcggtat
      601 taagcacctt tgcgcttagc ggcacctcca attccgggtc caagatgatg cgaaccggct
      661 gtttttcggt cggaacaaaa cgaaccgtca ggtgcggatc gtctttaatc acggtattaa
      721 cacccaccag caccgcggtg gcctcacgac gcagaatatg cgccacttta cggctctctt
      781 tggaggtaat ccatttgctg ctaccggtca aagtcgccag tttgccgtcc aaggtctgtg
      841 cccattttag agtaatatat gggcgttctt gcgtgatata ggtaaagaag tcctcattca
      901 gttcacgcgc ctcctcttcg cacacgccca catcgacttc aattcccgcg ttgcgcagct
      961 tctcaacgcc cttaccggac atcagcgggt tcggatcgag cgtggcaaca acaacacgct
     1021 tgataccaga tctgataatt gcatcggtgc aaggcggggt acgaccaaag tgggtacacg
     1081 gttccagggt tacgtataag gtcgcgccct tcgctttttc gcccgcttgg cccagtgcca
     1141 taacttctgc atgcggcttg ccagcctttt catggtaacc cagaccaacg atcttgccct
     1201 ctttaacaac gaccgcaccc accgtcgggt tcgggtgggt gtaacctttg cgttttttag
     1261 ccagggacag cgccagcttc atataatttt tatcaatttc ccttatcata tgtatatctc
     1321 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     1381 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg
     1441 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     1501 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     1561 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     1621 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     1681 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
     1741 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     1801 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     1861 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     1921 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     1981 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     2041 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     2101 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     2161 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     2221 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     2281 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     2341 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     2401 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     2461 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     2521 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     2581 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     2641 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     2701 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     2761 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     2821 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     2881 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     2941 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     3001 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     3061 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     3121 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     3181 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
     3241 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     3301 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     3361 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     3421 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     3481 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     3541 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     3601 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     3661 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     3721 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     3781 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     3841 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3901 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3961 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     4021 actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca
     4081 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     4141 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4201 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4261 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4321 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4381 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4441 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4501 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4561 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4621 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4681 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4741 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4801 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4861 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4921 tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt
     4981 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
     5041 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     5101 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     5161 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     5221 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     5281 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
     5341 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     5401 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     5461 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     5521 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt
     5581 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     5641 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     5701 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     5761 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     5821 aagaattaat tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5881 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5941 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6001 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     6061 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     6121 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     6181 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     6241 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     6301 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     6361 agggcgcgtc ccattcgcca
//
