LOCUS       sequence-360473-        4960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating 
            peptide-mNeongreen-stefinA fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4960)
  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 4960)
  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-StefinA
FEATURES             Location/Qualifiers
     source          1..4960
                     /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             1464..1481
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1497..1541
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1579..1597)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1593..1640
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1870..2685)
                     /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(1870..2685)
                     /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     2593..2612
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2686..2777)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2793..3542)
                     /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(2803..2820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3043..3062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3738..4820)
                     /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     4782..4801
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4821..4898)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4944..4960,1..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 ggaatgattc ccggcggttt atcagaggca aaacccgcga
     1201 cgccagagat tcaagaaatc gtcgacaaag ttaaaccgca attggaagag aagaccaatg
     1261 aaacttatgg caagttagag gccgttcaat acaagaccca agttgttgcc ggcacgaatt
     1321 attacattaa ggttcgtgcg ggcgacaata agtacatgca cctcaaggtc tttaaatcat
     1381 tgcctggtca gaatgaggat ctcgttttaa cgggctacca agttgacaag aataaagacg
     1441 acgaactcac tggatttggt acccatcatc atcatcatca ttaactcgag tctggtaaag
     1501 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     1561 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1621 gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc
     1681 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     1741 gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat
     1801 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1861 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1921 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1981 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     2041 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2101 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2161 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2221 tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac aattacaaac
     2281 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2341 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2401 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2461 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2521 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     2581 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2641 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca
     2701 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     2761 ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca ctgactcgct
     2821 acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt
     2881 atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca aagcaccgga
     2941 agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3001 atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3061 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3121 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt tggtatctca
     3181 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3241 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3301 cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa gttatgcacc
     3361 tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac ccacttacct
     3421 tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt agcggtggtt
     3481 tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac agctattccg
     3541 ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     3601 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     3661 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     3721 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3781 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     3841 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     3901 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     3961 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg
     4021 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4081 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4141 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4201 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4261 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4321 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     4381 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     4441 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     4501 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     4561 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     4621 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     4681 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     4741 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     4801 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     4861 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     4921 atgcgactcc tgcattagga aattaatacg actcactata
//
