LOCUS       sequence-360636-        4228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide6-stefinA fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4228)
  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 4228)
  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_VNp6-StefinA
FEATURES             Location/Qualifiers
     source          1..4228
                     /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             732..749
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             765..809
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(847..865)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      861..908
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1138..1953)
                     /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(1138..1953)
                     /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     1861..1880
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1954..2045)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2061..2810)
                     /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(2071..2088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2311..2330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3006..4088)
                     /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     4050..4069
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4089..4166)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4212..4228,1..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 aaaccgatca gggtgtgaca gaagcagcag aaaagacaaa agagggtgtt atgagatctg
      421 gaggaagcgg aatgattccc ggcggtttat cagaggcaaa acccgcgacg ccagagattc
      481 aagaaatcgt cgacaaagtt aaaccgcaat tggaagagaa gaccaatgaa acttatggca
      541 agttagaggc cgttcaatac aagacccaag ttgttgccgg cacgaattat tacattaagg
      601 ttcgtgcggg cgacaataag tacatgcacc tcaaggtctt taaatcattg cctggtcaga
      661 atgaggatct cgttttaacg ggctaccaag ttgacaagaa taaagacgac gaactcactg
      721 gatttggtac ccatcatcat catcatcatt aactcgagtc tggtaaagaa accgctgctg
      781 cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg
      841 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      901 gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
      961 ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga
     1021 cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc
     1081 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1141 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1201 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1261 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1321 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1381 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1441 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1501 ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1561 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1621 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1681 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1741 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1801 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1861 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     1921 agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag
     1981 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2041 acaaataggc atgcagcgct cttccgcttc ctcgctcact gactcgctac gctcggtcgt
     2101 tcgactgcgg cgagcggtgt cagctcactc aaaagcggta atacggttat ccacagaatc
     2161 aggggataaa gccggaaaga acatgtgagc aaaaagcaaa gcaccggaag aagccaacgc
     2221 cgcaggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2281 gccagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2341 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2401 cttcgggaag cgtggcgctt tctcatagct cacgctgttg gtatctcagt tcggtgtagg
     2461 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     2521 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     2581 cagccattgg taactgattt agaggacttt gtcttgaagt tatgcacctg ttaaggctaa
     2641 actgaaagaa cagattttgg tgagtgcggt cctccaaccc acttaccttg gttcaaagag
     2701 ttggtagctc agcgaacctt gagaaaacca ccgttggtag cggtggtttt tctttattta
     2761 tgagatgatg aatcaatcgg tctatcaagt caacgaacag ctattccgtt actctagatt
     2821 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     2881 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     2941 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     3001 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3061 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3121 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3181 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3241 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     3301 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     3361 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     3421 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     3481 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     3541 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     3601 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     3661 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     3721 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     3781 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     3841 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     3901 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     3961 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4021 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4081 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4141 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     4201 cattaggaaa ttaatacgac tcactata
//
