LOCUS       sequence-360630-        4726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of uricase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4726)
  AUTHORS   Eastwood TA, Baker K, Streather BR, Allen N, Wang L, Botchway SW, 
            Brown IR, Hiscock JR, Lennon C, Mulvihill DP
  TITLE     High-yield vesicle-packaged recombinant protein production from E. 
            coli.
  JOURNAL   Cell Rep Methods. 2023 Feb 2;3(2):100396. doi: 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27.
  PUBMED    36936078
REFERENCE   2  (bases 1 to 4726)
  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"; journalName: "Cell Rep 
            Methods."; date: "2023-02-2"; volume: "3(2):100396. doi"; pages: " 
            10.1016/j.crmeth.2023.100396. eCollection 2023 Feb 27"
COMMENT     Sequence Label: pRSFDuet-1_Uricase
FEATURES             Location/Qualifiers
     source          1..4726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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)."
     CDS             1230..1247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1263..1307
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1345..1363)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1359..1406
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1636..2451)
                     /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(1636..2451)
                     /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     2359..2378
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2452..2543)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2559..3308)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2569..2586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2809..2828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3504..4586)
                     /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     4548..4567
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4587..4664)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4710..4726,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgggaggaag cggaatgagc actacactgt cttcatcgac gtacgggaag gataacgtta
      361 agtttctcaa ggttaagaag gaccctcaga atccgaagaa gcaggaagtt atggaagcca
      421 cggttacgtg cttgttagag ggtggattcg ataccagtta cactgaggcg gacaactcgt
      481 cgattgtgcc cacagatacc gtgaagaaca ctatcctcgt tcttgcgaaa acgaccgaga
      541 tttggcctat cgagcgcttt gccgcgaagc ttgccactca ttttgtagag aaatactccc
      601 atgtcagcgg cgtttctgtg aaaatcgttc aggatcgttg ggtcaagtac gccgtagacg
      661 gcaagcccca tgatcacagc ttcatccacg agggcggaga aaagcgtatt actgaccttt
      721 actataagcg cagcggcgat tataagttgt ccagtgcgat caaagacctg accgtgctga
      781 agtcaaccgg cagtatgttc tatgggtata ataaatgtga ttttacgacg ttacagccaa
      841 ccactgatcg tatccttagt acggacgtcg acgcaacatg ggtgtgggac aacaagaaaa
      901 tcggaagcgt ttatgacatc gctaaagccg cggacaaggg catctttgat aatgtctaca
      961 atcaggcgcg cgagattaca ctgacaacgt tcgctctgga aaactccccg agcgtccaag
     1021 ctacaatgtt taatatggcg acgcaaatcc tggagaaagc atgttcggtt tactcggttt
     1081 cgtacgcgtt accgaataag cattatttcc tgattgactt aaaatggaag ggtctcgaaa
     1141 acgataatga actgttttac ccgtctccgc atccgaatgg cctgatcaag tgtaccgtgg
     1201 tacgtaagga aaagacgaag ttaggtaccc atcatcatca tcatcattaa ctcgagtctg
     1261 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     1321 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1381 ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca
     1441 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac
     1501 gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg
     1561 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1621 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1681 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1741 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1801 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1861 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1921 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1981 attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt
     2041 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2101 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2161 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2221 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2281 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2341 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2401 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
     2461 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2521 atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga
     2581 ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat
     2641 acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc
     2701 accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc
     2761 acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg
     2821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt
     2941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3061 acttatcgcc actggcagca gccattggta actgatttag aggactttgt cttgaagtta
     3121 tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac
     3181 ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg
     3241 gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct
     3301 attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     3361 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc
     3421 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3481 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3541 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3601 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3661 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3721 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3781 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3841 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     3901 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     3961 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4021 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4081 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4141 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4201 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4261 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4321 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4381 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4441 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4501 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4561 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4621 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4681 tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
