LOCUS       sequence-360455-        4174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide15-stefinA fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4174)
  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 4174)
  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_VNp15-StefinA
FEATURES             Location/Qualifiers
     source          1..4174
                     /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             678..695
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             711..755
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(793..811)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      807..854
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1084..1899)
                     /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(1084..1899)
                     /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     1807..1826
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1900..1991)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2007..2756)
                     /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(2017..2034)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2257..2276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2952..4034)
                     /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     3996..4015
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4035..4112)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4158..4174,1..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 caagaaagga ttttcaatag ccgatgaggg agttgtggga gctgttgaga
      361 gatctggagg aagcggaatg attcccggcg gtttatcaga ggcaaaaccc gcgacgccag
      421 agattcaaga aatcgtcgac aaagttaaac cgcaattgga agagaagacc aatgaaactt
      481 atggcaagtt agaggccgtt caatacaaga cccaagttgt tgccggcacg aattattaca
      541 ttaaggttcg tgcgggcgac aataagtaca tgcacctcaa ggtctttaaa tcattgcctg
      601 gtcagaatga ggatctcgtt ttaacgggct accaagttga caagaataaa gacgacgaac
      661 tcactggatt tggtacccat catcatcatc atcattaact cgagtctggt aaagaaaccg
      721 ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct
      781 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
      841 tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg
      901 tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg
      961 aaccgacgac aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc
     1021 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1081 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1141 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1201 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1261 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1321 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1381 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     1441 ttgcgcctga gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat
     1501 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1561 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     1621 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1681 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     1741 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     1801 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     1861 cggcctagag caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta
     1921 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     1981 aaataaacaa ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc
     2041 ggtcgttcga ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac
     2101 agaatcaggg gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc
     2161 caacgccgca ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2221 gctcaagcca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2281 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2341 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg
     2401 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2461 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2521 tggcagcagc cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa
     2581 ggctaaactg aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc
     2641 aaagagttgg tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt
     2701 tatttatgag atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc
     2761 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     2821 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     2881 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     2941 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3001 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     3061 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     3121 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     3181 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     3241 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     3301 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     3361 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     3421 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     3481 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     3541 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     3601 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     3661 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     3721 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     3781 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     3841 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     3901 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     3961 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     4021 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     4081 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     4141 ctcctgcatt aggaaattaa tacgactcac tata
//
