LOCUS       sequence-360465-        4360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide15-DARPinOFF7 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4360)
  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 4360)
  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-DARPinOFF7
FEATURES             Location/Qualifiers
     source          1..4360
                     /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             864..881
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             897..941
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(979..997)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      993..1040
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1270..2085)
                     /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(1270..2085)
                     /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     1993..2012
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2086..2177)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2193..2942)
                     /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(2203..2220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2443..2462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3138..4220)
                     /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     4182..4201
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4221..4298)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4344..4360,1..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 ggttcggact taggccgcaa gttactggaa gctgcacgcg
      421 cgggtcaaga cgacgaggta cgcatcctta tggccaatgg tgcggacgtc aacgccgccg
      481 ataataccgg aacaactcct ttgcacctcg cagcatattc cggccattta gagattgtag
      541 aggtactgct taagcacggt gccgatgtag atgcgtcgga cgtattcggg tacacgcctc
      601 tccacctcgc cgcttattgg ggccacttag aaattgtaga ggtgctcttg aagaatggtg
      661 ccgacgtgaa tgctatggat tcagacggca tgacaccgct ccatctggcc gcgaaatggg
      721 gttatctgga gatcgtcgag gttctcttga aacatggcgc cgacgtcaat gcacaagata
      781 agttcggcaa gactgccttc gacatcagca tcgacaacgg taatgaggac ctggccgaga
      841 tccttcaaaa gttgaatggt acccatcatc atcatcatca ttaactcgag tctggtaaag
      901 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
      961 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1021 gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc
     1081 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     1141 gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat
     1201 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1261 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1321 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1381 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1441 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     1501 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     1561 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     1621 tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac aattacaaac
     1681 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     1741 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     1801 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     1861 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     1921 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     1981 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2041 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca
     2101 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2161 ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca ctgactcgct
     2221 acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt
     2281 atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca aagcaccgga
     2341 agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2401 atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2461 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2521 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt tggtatctca
     2581 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2641 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2701 cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa gttatgcacc
     2761 tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac ccacttacct
     2821 tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt agcggtggtt
     2881 tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac agctattccg
     2941 ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     3001 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     3061 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     3121 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3181 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     3241 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     3301 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     3361 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     3421 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     3481 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     3541 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     3601 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     3661 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     3721 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     3781 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     3841 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     3901 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     3961 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4021 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4081 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     4141 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     4201 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     4261 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     4321 atgcgactcc tgcattagga aattaatacg actcactata
//
