LOCUS       sequence-360639-        6067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating 
            peptide-mNeongreen-Etanercept fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  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 6067)
  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-Etanercept
FEATURES             Location/Qualifiers
     source          1..6067
                     /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             2571..2588
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2604..2648
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2686..2704)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2700..2747
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2977..3792)
                     /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(2977..3792)
                     /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     3700..3719
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3793..3884)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3900..4649)
                     /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(3910..3927)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4150..4169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4845..5927)
                     /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     5889..5908
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5928..6005)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6051..6067,1..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 ggactgcctg cacaggttgc atttaccccg tatgcaccgg
     1201 aaccgggtag cacctgtcgt ctgcgtgaat attatgatca gaccgcacag atgtgttgta
     1261 gcaaatgtag tccgggtcag catgcaaaag ttttttgtac caaaaccagc gataccgttt
     1321 gtgatagctg tgaagatagc acctatacgc agctgtggaa ttgggttcct gaatgtctga
     1381 gctgtggtag ccgttgtagc agcgatcagg ttgaaaccca ggcatgtacc cgtgaacaga
     1441 atcgtatttg tacctgccgt cctggttggt attgtgcact gagcaaacaa gagggttgtc
     1501 gtctgtgtgc accgctgcgt aaatgtcgtc caggttttgg tgttgcacgt ccgggtacag
     1561 aaaccagtga tgttgtttgt aaaccgtgcg caccgggtac atttagcaat accaccagta
     1621 gcaccgatat ttgtcgtccg catcagattt gtaatgttgt tgcaattccg ggtaatgcaa
     1681 gcatggatgc agtttgtacc agcaccagtc cgacacgtag catggcaccg ggtgcagttc
     1741 atctgccgca gccggttagc acccgtagcc agcataccca gccgactccg gaaccgagca
     1801 ccgcaccgag taccagcttt ctgctgccga tgggtccgag tccgcctgcc gaaggtagca
     1861 ccggtgatga accgaaaagc tgtgataaaa cccatacctg tccgccttgt ccggctccgg
     1921 aactgttagg tggtccgagc gtttttctgt ttccgcctaa accgaaagat accctgatga
     1981 ttagccgtac accggaagtt acctgtgttg ttgttgatgt tagccatgaa gatccggaag
     2041 tgaaatttaa ctggtatgtt gatggtgtgg aagtgcataa tgcaaaaacc aaaccgcgtg
     2101 aagaacagta taattcaacc tatcgtgttg ttagcgttct gaccgttctg catcaggatt
     2161 ggctgaatgg taaagaatac aaatgcaagg tgagcaataa agcactgcca gcaccgattg
     2221 aaaaaaccat tagcaaagca aaaggtcagc ctcgtgaacc gcaggtttat accctgcctc
     2281 cgagccgtga agaaatgacc aaaaatcagg ttagcctgac ctgtctggtg aaaggttttt
     2341 atccgagcga tattgcagtt gaatgggaaa gcaatggtca gccggaaaat aactataaaa
     2401 ccacaccgcc tgttctggat agtgatggta gcttttttct gtatagcaaa ctgaccgttg
     2461 ataaaagccg ttggcagcag ggtaatgttt ttagctgtag cgttatgcat gaagccctgc
     2521 ataatcatta tacccagaaa agcctgagcc tgagtccggg taaaggtacc catcatcatc
     2581 atcatcatta actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca
     2641 tggactcgtc tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac
     2701 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg
     2761 catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat
     2821 agacataagc ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc
     2881 tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2941 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     3001 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     3061 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     3121 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     3181 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     3241 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     3301 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     3361 gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     3421 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     3481 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     3541 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3601 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     3661 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     3721 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     3781 aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     3841 catgagcgga tacatatttg aatgtattta gaaaaataaa caaataggca tgcagcgctc
     3901 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggtgtc
     3961 agctcactca aaagcggtaa tacggttatc cacagaatca ggggataaag ccggaaagaa
     4021 catgtgagca aaaagcaaag caccggaaga agccaacgcc gcaggcgttt ttccataggc
     4081 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag ccagaggtgg cgaaacccga
     4141 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4201 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4261 ctcatagctc acgctgttgg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4321 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4381 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccattggt aactgattta
     4441 gaggactttg tcttgaagtt atgcacctgt taaggctaaa ctgaaagaac agattttggt
     4501 gagtgcggtc ctccaaccca cttaccttgg ttcaaagagt tggtagctca gcgaaccttg
     4561 agaaaaccac cgttggtagc ggtggttttt ctttatttat gagatgatga atcaatcggt
     4621 ctatcaagtc aacgaacagc tattccgtta ctctagattt cagtgcaatt tatctcttca
     4681 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     4741 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4801 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4921 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4981 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5041 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5101 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     5161 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5221 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5281 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5341 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5401 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5461 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5521 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5581 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5641 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5701 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5761 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5821 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5881 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5941 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6001 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat taatacgact
     6061 cactata
//
