LOCUS       sequence-360743-        3949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3949)
  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 3949)
  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-His6
FEATURES             Location/Qualifiers
     source          1..3949
                     /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             453..470
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             486..530
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(568..586)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      582..629
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(859..1674)
                     /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(859..1674)
                     /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     1582..1601
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1675..1766)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1782..2531)
                     /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(1792..1809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2032..2051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2727..3809)
                     /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     3771..3790
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3810..3887)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(3933..3949,1..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 gaggaggagc tagcggagga agcggaggta cccatcatca tcatcatcat taactcgagt
      481 ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg
      541 cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg
      601 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg
      661 tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt
      721 aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt
      781 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      841 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      901 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      961 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1021 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1081 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1141 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1201 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca
     1261 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1321 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1381 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1441 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1501 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1561 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1621 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt
     1681 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1741 tgaatgtatt tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac
     1801 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt
     1861 aatacggtta tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa
     1921 agcaccggaa gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc
     1981 atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc
     2041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt
     2161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2281 acgacttatc gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag
     2341 ttatgcacct gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc
     2401 cacttacctt ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta
     2461 gcggtggttt ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca
     2521 gctattccgt tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     2581 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     2641 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     2701 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2761 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     2821 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     2881 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     2941 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3001 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     3061 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     3121 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     3181 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     3241 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     3301 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     3361 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     3421 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     3481 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     3541 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     3601 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     3661 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     3721 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     3781 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     3841 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     3901 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
