LOCUS       sequence-360462-        4708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-DNaseI fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4708)
  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 4708)
  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-DNAseI
FEATURES             Location/Qualifiers
     source          1..4708
                     /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             1212..1229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1245..1289
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1327..1345)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1341..1388
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1618..2433)
                     /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(1618..2433)
                     /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     2341..2360
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2434..2525)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2541..3290)
                     /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(2551..2568)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2791..2810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3486..4568)
                     /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     4530..4549
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4569..4646)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4692..4708,1..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 ctcagatcta
      421 tggccctgaa gatagcagcc ttcaacatcc gcacctttgg ggagaccaag atgtccaatg
      481 ctacgctcgc cagctacatt gttcggatcg tgcgtcgtta cgacatcgtc ctcatccagg
      541 aggtcagaga cagccacctg gtggctgtgg ggaagctcct ggactatctc aaccaggatg
      601 acccaaacac ctaccactat gtggtcagtg agccgctggg ccgcaacagc tacaaggagc
      661 gctacctctt tctgttcaga cccaacaagg tgtccgtgct ggacacctac cagtacgacg
      721 acggctgcga gtcctgcggg aacgacagct tcagccggga gcccgctgtg gtcaagttct
      781 catcccactc caccaaggtc aaggaatttg ccattgttgc cctgcactcg gccccatcgg
      841 acgcagtggc tgagattaat tctctctacg atgtctacct ggatgtccag cagaagtggc
      901 acttgaacga tgtcatgttg atgggcgatt tcaatgctga ctgcagctac gtgacctcct
      961 cgcagtggtc atccatccgc ctgcgtacga gctccacctt ccagtggctg attcctgaca
     1021 gtgccgacac cacggctacg tccacgaact gcgcctatga caggatcgtg gtcgcagggt
     1081 ctctgctcca gagttctgtg gttcctggct cggccgctcc ctttgacttc caagctgcat
     1141 acggactgag caatgagatg gccctggcca tcagtgacca ttacccggtg gaggtgacgc
     1201 tgacaggtac ccatcatcat catcatcatt aactcgagtc tggtaaagaa accgctgctg
     1261 cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg
     1321 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1381 gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
     1441 ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga
     1501 cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc
     1561 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1621 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1681 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1741 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1801 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1861 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1921 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1981 ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     2041 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     2101 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     2161 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     2221 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     2281 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     2341 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2401 agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag
     2461 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2521 acaaataggc atgcagcgct cttccgcttc ctcgctcact gactcgctac gctcggtcgt
     2581 tcgactgcgg cgagcggtgt cagctcactc aaaagcggta atacggttat ccacagaatc
     2641 aggggataaa gccggaaaga acatgtgagc aaaaagcaaa gcaccggaag aagccaacgc
     2701 cgcaggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     2761 gccagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     2821 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     2881 cttcgggaag cgtggcgctt tctcatagct cacgctgttg gtatctcagt tcggtgtagg
     2941 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3001 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3061 cagccattgg taactgattt agaggacttt gtcttgaagt tatgcacctg ttaaggctaa
     3121 actgaaagaa cagattttgg tgagtgcggt cctccaaccc acttaccttg gttcaaagag
     3181 ttggtagctc agcgaacctt gagaaaacca ccgttggtag cggtggtttt tctttattta
     3241 tgagatgatg aatcaatcgg tctatcaagt caacgaacag ctattccgtt actctagatt
     3301 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     3361 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     3421 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     3481 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3541 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3601 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3661 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3721 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     3781 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     3841 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     3901 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     3961 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4021 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4081 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4141 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4201 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4261 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4321 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     4381 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     4441 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4501 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4561 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4621 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     4681 cattaggaaa ttaatacgac tcactata
//
