LOCUS       sequence-360564-        5575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating 
            peptide-mNeongreen-uricase fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5575)
  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 5575)
  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-mNeongreen-Uricase
FEATURES             Location/Qualifiers
     source          1..5575
                     /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             432..1139
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             2079..2096
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2112..2156
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2194..2212)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2208..2255
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2485..3300)
                     /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(2485..3300)
                     /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     3208..3227
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3301..3392)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3408..4157)
                     /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(3418..3435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3658..3677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4353..5435)
                     /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     5397..5416
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5436..5513)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5559..5575,1..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 aatggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      481 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      541 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      601 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      661 accctgacgg gatgtcgcct ttccaggccg cgatggtaga tggctccgga taccaagtcc
      721 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      781 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      841 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
      901 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
      961 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1021 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1081 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagg
     1141 ctagcagatc tggaggaagc ggaatgagca ctacactgtc ttcatcgacg tacgggaagg
     1201 ataacgttaa gtttctcaag gttaagaagg accctcagaa tccgaagaag caggaagtta
     1261 tggaagccac ggttacgtgc ttgttagagg gtggattcga taccagttac actgaggcgg
     1321 acaactcgtc gattgtgccc acagataccg tgaagaacac tatcctcgtt cttgcgaaaa
     1381 cgaccgagat ttggcctatc gagcgctttg ccgcgaagct tgccactcat tttgtagaga
     1441 aatactccca tgtcagcggc gtttctgtga aaatcgttca ggatcgttgg gtcaagtacg
     1501 ccgtagacgg caagccccat gatcacagct tcatccacga gggcggagaa aagcgtatta
     1561 ctgaccttta ctataagcgc agcggcgatt ataagttgtc cagtgcgatc aaagacctga
     1621 ccgtgctgaa gtcaaccggc agtatgttct atgggtataa taaatgtgat tttacgacgt
     1681 tacagccaac cactgatcgt atccttagta cggacgtcga cgcaacatgg gtgtgggaca
     1741 acaagaaaat cggaagcgtt tatgacatcg ctaaagccgc ggacaagggc atctttgata
     1801 atgtctacaa tcaggcgcgc gagattacac tgacaacgtt cgctctggaa aactccccga
     1861 gcgtccaagc tacaatgttt aatatggcga cgcaaatcct ggagaaagca tgttcggttt
     1921 actcggtttc gtacgcgtta ccgaataagc attatttcct gattgactta aaatggaagg
     1981 gtctcgaaaa cgataatgaa ctgttttacc cgtctccgca tccgaatggc ctgatcaagt
     2041 gtaccgtggt acgtaaggaa aagacgaagt taggtaccca tcatcatcat catcattaac
     2101 tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta
     2161 ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc
     2221 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc
     2281 acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg
     2341 ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc cgcaagtggc
     2401 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2461 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     2521 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     2581 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2641 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2701 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2761 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2821 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt cgctgttaaa
     2881 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2941 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     3001 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3061 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     3121 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     3181 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     3241 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     3301 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     3361 catatttgaa tgtatttaga aaaataaaca aataggcatg cagcgctctt ccgcttcctc
     3421 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggtgtcag ctcactcaaa
     3481 agcggtaata cggttatcca cagaatcagg ggataaagcc ggaaagaaca tgtgagcaaa
     3541 aagcaaagca ccggaagaag ccaacgccgc aggcgttttt ccataggctc cgcccccctg
     3601 acgagcatca caaaaatcga cgctcaagcc agaggtggcg aaacccgaca ggactataaa
     3661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3781 gctgttggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3901 taagacacga cttatcgcca ctggcagcag ccattggtaa ctgatttaga ggactttgtc
     3961 ttgaagttat gcacctgtta aggctaaact gaaagaacag attttggtga gtgcggtcct
     4021 ccaacccact taccttggtt caaagagttg gtagctcagc gaaccttgag aaaaccaccg
     4081 ttggtagcgg tggtttttct ttatttatga gatgatgaat caatcggtct atcaagtcaa
     4141 cgaacagcta ttccgttact ctagatttca gtgcaattta tctcttcaaa tgtagcacct
     4201 gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc
     4261 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4321 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4381 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4441 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     4501 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     4561 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     4621 cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     4681 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     4741 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     4801 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4861 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4921 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4981 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5041 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5101 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5161 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5221 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5281 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     5341 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     5401 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     5461 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     5521 tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca ctata
//
