LOCUS       sequence-360456-        4414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide6-DARPinOFF7 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4414)
  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 4414)
  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-DARPinOFF7
FEATURES             Location/Qualifiers
     source          1..4414
                     /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             918..935
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             951..995
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1033..1051)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1047..1094
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1324..2139)
                     /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(1324..2139)
                     /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     2047..2066
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2140..2231)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2247..2996)
                     /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(2257..2274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2497..2516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3192..4274)
                     /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     4236..4255
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4275..4352)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4398..4414,1..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 aatgggttcg gacttaggcc gcaagttact ggaagctgca cgcgcgggtc
      481 aagacgacga ggtacgcatc cttatggcca atggtgcgga cgtcaacgcc gccgataata
      541 ccggaacaac tcctttgcac ctcgcagcat attccggcca tttagagatt gtagaggtac
      601 tgcttaagca cggtgccgat gtagatgcgt cggacgtatt cgggtacacg cctctccacc
      661 tcgccgctta ttggggccac ttagaaattg tagaggtgct cttgaagaat ggtgccgacg
      721 tgaatgctat ggattcagac ggcatgacac cgctccatct ggccgcgaaa tggggttatc
      781 tggagatcgt cgaggttctc ttgaaacatg gcgccgacgt caatgcacaa gataagttcg
      841 gcaagactgc cttcgacatc agcatcgaca acggtaatga ggacctggcc gagatccttc
      901 aaaagttgaa tggtacccat catcatcatc atcattaact cgagtctggt aaagaaaccg
      961 ctgctgcgaa atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct
     1021 aggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1081 tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg
     1141 tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg
     1201 aaccgacgac aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc
     1261 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1321 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1381 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1441 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1501 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1561 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1621 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     1681 ttgcgcctga gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat
     1741 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     1801 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     1861 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     1921 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     1981 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2041 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2101 cggcctagag caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta
     2161 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     2221 aaataaacaa ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc
     2281 ggtcgttcga ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac
     2341 agaatcaggg gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc
     2401 caacgccgca ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2461 gctcaagcca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2521 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2581 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg
     2641 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2701 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2761 tggcagcagc cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa
     2821 ggctaaactg aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc
     2881 aaagagttgg tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt
     2941 tatttatgag atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc
     3001 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     3061 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     3121 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     3181 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3241 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     3301 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     3361 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     3421 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     3481 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     3541 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     3601 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     3661 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     3721 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     3781 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     3841 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     3901 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     3961 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     4021 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     4081 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     4141 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     4201 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     4261 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     4321 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     4381 ctcctgcatt aggaaattaa tacgactcac tata
//
