LOCUS       sequence-360651-        5146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating 
            peptide-mNeongreen-DARPinOFF7 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5146)
  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 5146)
  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-mNeongreen-DARPinOFF7
FEATURES             Location/Qualifiers
     source          1..5146
                     /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             432..1139
                     /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             1650..1667
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1683..1727
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1765..1783)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1779..1826
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2056..2871)
                     /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(2056..2871)
                     /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     2779..2798
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2872..2963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2979..3728)
                     /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(2989..3006)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3229..3248)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3924..5006)
                     /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     4968..4987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5007..5084)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5130..5146,1..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 gaggaagcgg aatggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      481 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      541 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      601 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      661 accctgacgg gatgtcgcct ttccaggccg cgatggtaga tggctccgga taccaagtcc
      721 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      781 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      841 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
      901 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
      961 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1021 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1081 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagg
     1141 ctagcagatc tggaggaagc ggaatgggtt cggacttagg ccgcaagtta ctggaagctg
     1201 cacgcgcggg tcaagacgac gaggtacgca tccttatggc caatggtgcg gacgtcaacg
     1261 ccgccgataa taccggaaca actcctttgc acctcgcagc atattccggc catttagaga
     1321 ttgtagaggt actgcttaag cacggtgccg atgtagatgc gtcggacgta ttcgggtaca
     1381 cgcctctcca cctcgccgct tattggggcc acttagaaat tgtagaggtg ctcttgaaga
     1441 atggtgccga cgtgaatgct atggattcag acggcatgac accgctccat ctggccgcga
     1501 aatggggtta tctggagatc gtcgaggttc tcttgaaaca tggcgccgac gtcaatgcac
     1561 aagataagtt cggcaagact gccttcgaca tcagcatcga caacggtaat gaggacctgg
     1621 ccgagatcct tcaaaagttg aatggtaccc atcatcatca tcatcattaa ctcgagtctg
     1681 gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag
     1741 cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1801 ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca
     1861 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac
     1921 gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg
     1981 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2041 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     2101 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2161 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2221 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2281 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2341 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2401 attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt
     2461 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2521 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2581 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2641 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2701 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2761 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2821 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct
     2881 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2941 atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga
     3001 ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat
     3061 acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc
     3121 accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc
     3181 acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg
     3241 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3301 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt
     3361 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3421 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3481 acttatcgcc actggcagca gccattggta actgatttag aggactttgt cttgaagtta
     3541 tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac
     3601 ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg
     3661 gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct
     3721 attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     3781 cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc
     3841 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3901 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3961 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4021 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4081 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4141 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4201 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4261 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4321 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4381 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4441 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4501 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4561 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4621 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4681 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4741 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4801 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4861 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4921 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4981 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5041 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5101 tcccttatgc gactcctgca ttaggaaatt aatacgactc actata
//
