LOCUS       sequence-360501-        4606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide15-mNeongreen 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4606)
  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 4606)
  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-mNeongreen
FEATURES             Location/Qualifiers
     source          1..4606
                     /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             378..1085
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             1110..1127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1143..1187
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1225..1243)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1239..1286
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1516..2331)
                     /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(1516..2331)
                     /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     2239..2258
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2332..2423)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2439..3188)
                     /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(2449..2466)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2689..2708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3384..4466)
                     /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     4428..4447
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4467..4544)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4590..4606,1..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 gtgagcaagg gcgaggagga taacatggcc tctctcccag
      421 cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac atggtgggtc
      481 agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc accaagggtg
      541 acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc catcagtacc
      601 tgccctaccc tgacgggatg tcgcctttcc aggccgcgat ggtagatggc tccggatacc
      661 aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac taccgctaca
      721 cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt ttccctgctg
      781 acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt
      841 accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact ggaaatggca
      901 agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg gcggctaact
      961 atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac tccaagaccg
     1021 agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg gacgagctgt
     1081 acaaggctag cggaggaagc ggaggtaccc atcatcatca tcatcattaa ctcgagtctg
     1141 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     1201 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1261 ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca
     1321 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac
     1381 gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg
     1441 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1501 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1561 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1621 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     1681 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1741 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     1801 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     1861 attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt
     1921 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     1981 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2041 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2101 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2161 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2221 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2281 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
     2341 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2401 atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga
     2461 ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat
     2521 acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc
     2581 accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc
     2641 acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg
     2701 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2761 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt
     2821 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2881 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2941 acttatcgcc actggcagca gccattggta actgatttag aggactttgt cttgaagtta
     3001 tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac
     3061 ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg
     3121 gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct
     3181 attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     3241 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc
     3301 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3361 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3421 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3481 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3541 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3601 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3661 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3721 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     3781 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     3841 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     3901 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     3961 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4021 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4081 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4141 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4201 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4261 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4321 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4381 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4441 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4501 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4561 tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
