LOCUS       sequence-360498-        4843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-uricase fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4843)
  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 4843)
  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_VNp-Uricase
FEATURES             Location/Qualifiers
     source          1..4843
                     /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             1347..1364
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1380..1424
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1462..1480)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1476..1523
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1753..2568)
                     /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(1753..2568)
                     /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     2476..2495
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2569..2660)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2676..3425)
                     /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(2686..2703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2926..2945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3621..4703)
                     /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     4665..4684
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4704..4781)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4827..4843,1..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 tggatgtatt catgaaagga ctttcaaagg ccaaggaggg agttgtggca gctgctgaga
      361 aaaccaaaca gggtgtggca gaagcagcag gaaagacaaa agagggtgtt ctcagatctg
      421 gaggaagcgg aatgagcact acactgtctt catcgacgta cgggaaggat aacgttaagt
      481 ttctcaaggt taagaaggac cctcagaatc cgaagaagca ggaagttatg gaagccacgg
      541 ttacgtgctt gttagagggt ggattcgata ccagttacac tgaggcggac aactcgtcga
      601 ttgtgcccac agataccgtg aagaacacta tcctcgttct tgcgaaaacg accgagattt
      661 ggcctatcga gcgctttgcc gcgaagcttg ccactcattt tgtagagaaa tactcccatg
      721 tcagcggcgt ttctgtgaaa atcgttcagg atcgttgggt caagtacgcc gtagacggca
      781 agccccatga tcacagcttc atccacgagg gcggagaaaa gcgtattact gacctttact
      841 ataagcgcag cggcgattat aagttgtcca gtgcgatcaa agacctgacc gtgctgaagt
      901 caaccggcag tatgttctat gggtataata aatgtgattt tacgacgtta cagccaacca
      961 ctgatcgtat ccttagtacg gacgtcgacg caacatgggt gtgggacaac aagaaaatcg
     1021 gaagcgttta tgacatcgct aaagccgcgg acaagggcat ctttgataat gtctacaatc
     1081 aggcgcgcga gattacactg acaacgttcg ctctggaaaa ctccccgagc gtccaagcta
     1141 caatgtttaa tatggcgacg caaatcctgg agaaagcatg ttcggtttac tcggtttcgt
     1201 acgcgttacc gaataagcat tatttcctga ttgacttaaa atggaagggt ctcgaaaacg
     1261 ataatgaact gttttacccg tctccgcatc cgaatggcct gatcaagtgt accgtggtac
     1321 gtaaggaaaa gacgaagtta ggtacccatc atcatcatca tcattaactc gagtctggta
     1381 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     1441 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1501 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
     1561 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     1621 cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga
     1681 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1741 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1801 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1861 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1921 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     1981 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2041 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2101 cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca
     2161 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2221 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2281 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2341 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2401 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2461 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2521 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt
     2581 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     2641 tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc
     2701 gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg
     2761 gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc
     2821 ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2881 aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2941 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3001 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc
     3061 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3121 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3181 tatcgccact ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc
     3241 acctgttaag gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta
     3301 ccttggttca aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg
     3361 gtttttcttt atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt
     3421 ccgttactct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     3481 atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga
     3541 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     3601 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3661 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     3721 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     3781 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     3841 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     3901 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     3961 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4021 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4081 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     4141 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     4201 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     4261 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     4321 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     4381 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     4441 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     4501 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     4561 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     4621 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     4681 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     4741 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     4801 cttatgcgac tcctgcatta ggaaattaat acgactcact ata
//
