LOCUS       sequence-360472-        6353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine Zipper 
            and Vesicle Nucleating peptide-Leucine Zipper-mNeongreen-Etanercept 
            fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6353)
  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 6353)
  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-LZ_VNp-LZ-mNeongreen-Etanercept
FEATURES             Location/Qualifiers
     source          1..6353
                     /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             218..274
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     promoter        401..419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    420..444
                     /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             634..690
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             718..1425
                     /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             2857..2874
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2890..2934
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2972..2990)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2986..3033
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3263..4078)
                     /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(3263..4078)
                     /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     3986..4005
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4079..4170)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4186..4935)
                     /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(4196..4213)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4436..4455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5131..6213)
                     /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     6175..6194
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6214..6291)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(6337..6353,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggatgtat tcatgaaagg actttcaaag gccaaggagg gagttgtggc
      121 agctgctgag aaaaccaaac agggtgtggc agaagcagca ggaaagacaa aagagggtgt
      181 tctcagatca cgtatgaaac agctggaaga taaagttgaa gaactgctga gcaaaaacta
      241 ccacctggaa aatgaagttg cccgtctgaa aaaactggtt ggtggatcct agagatccga
      301 attcgagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa
      361 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
      421 gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat
      481 atacatatgg atgtattcat gaaaggactt tcaaaggcca aggagggagt tgtggcagct
      541 gctgagaaaa ccaaacaggg tgtggcagaa gcagcaggaa agacaaaaga gggtgttctc
      601 agatcacgta tgaaacagct ggaagataaa gttgaagaac tgctgagcaa aaactaccac
      661 ctggaaaatg aagttgcccg tctgaaaaaa ctggttggta gatctggagg aagcggaatg
      721 gtgagcaagg gcgaggagga taacatggcc tctctcccag cgacacatga gttacacatc
      781 tttggctcca tcaacggtgt ggactttgac atggtgggtc agggcaccgg caatccaaat
      841 gatggttatg aggagttaaa cctgaagtcc accaagggtg acctccagtt ctccccctgg
      901 attctggtcc ctcatatcgg gtatggcttc catcagtacc tgccctaccc tgacgggatg
      961 tcgcctttcc aggccgcgat ggtagatggc tccggatacc aagtccatcg cacaatgcag
     1021 tttgaagatg gtgcctccct tactgttaac taccgctaca cctacgaggg aagccacatc
     1081 aaaggagagg cccaggtgaa ggggactggt ttccctgctg acggtcctgt gatgaccaac
     1141 tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt accccaacga caaaaccatc
     1201 atcagtacct ttaagtggag ttacaccact ggaaatggca agcgctaccg gagcactgcg
     1261 cggaccacct acacctttgc caagccaatg gcggctaact atctgaagaa ccagccgatg
     1321 tacgtgttcc gtaagacgga gctcaagcac tccaagaccg agctcaactt caaggagtgg
     1381 caaaaggcct ttaccgatgt gatgggcatg gacgagctgt acaaggctag cagatctgga
     1441 ggaagcggac tgcctgcaca ggttgcattt accccgtatg caccggaacc gggtagcacc
     1501 tgtcgtctgc gtgaatatta tgatcagacc gcacagatgt gttgtagcaa atgtagtccg
     1561 ggtcagcatg caaaagtttt ttgtaccaaa accagcgata ccgtttgtga tagctgtgaa
     1621 gatagcacct atacgcagct gtggaattgg gttcctgaat gtctgagctg tggtagccgt
     1681 tgtagcagcg atcaggttga aacccaggca tgtacccgtg aacagaatcg tatttgtacc
     1741 tgccgtcctg gttggtattg tgcactgagc aaacaagagg gttgtcgtct gtgtgcaccg
     1801 ctgcgtaaat gtcgtccagg ttttggtgtt gcacgtccgg gtacagaaac cagtgatgtt
     1861 gtttgtaaac cgtgcgcacc gggtacattt agcaatacca ccagtagcac cgatatttgt
     1921 cgtccgcatc agatttgtaa tgttgttgca attccgggta atgcaagcat ggatgcagtt
     1981 tgtaccagca ccagtccgac acgtagcatg gcaccgggtg cagttcatct gccgcagccg
     2041 gttagcaccc gtagccagca tacccagccg actccggaac cgagcaccgc accgagtacc
     2101 agctttctgc tgccgatggg tccgagtccg cctgccgaag gtagcaccgg tgatgaaccg
     2161 aaaagctgtg ataaaaccca tacctgtccg ccttgtccgg ctccggaact gttaggtggt
     2221 ccgagcgttt ttctgtttcc gcctaaaccg aaagataccc tgatgattag ccgtacaccg
     2281 gaagttacct gtgttgttgt tgatgttagc catgaagatc cggaagtgaa atttaactgg
     2341 tatgttgatg gtgtggaagt gcataatgca aaaaccaaac cgcgtgaaga acagtataat
     2401 tcaacctatc gtgttgttag cgttctgacc gttctgcatc aggattggct gaatggtaaa
     2461 gaatacaaat gcaaggtgag caataaagca ctgccagcac cgattgaaaa aaccattagc
     2521 aaagcaaaag gtcagcctcg tgaaccgcag gtttataccc tgcctccgag ccgtgaagaa
     2581 atgaccaaaa atcaggttag cctgacctgt ctggtgaaag gtttttatcc gagcgatatt
     2641 gcagttgaat gggaaagcaa tggtcagccg gaaaataact ataaaaccac accgcctgtt
     2701 ctggatagtg atggtagctt ttttctgtat agcaaactga ccgttgataa aagccgttgg
     2761 cagcagggta atgtttttag ctgtagcgtt atgcatgaag ccctgcataa tcattatacc
     2821 cagaaaagcc tgagcctgag tccgggtaaa ggtacccatc atcatcatca tcattaactc
     2881 gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact
     2941 agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     3001 ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac
     3061 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     3121 atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac
     3181 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3241 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3301 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3361 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3421 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3481 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3541 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3601 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag
     3661 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3721 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3781 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3841 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3901 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3961 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     4021 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac
     4081 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4141 tatttgaatg tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc
     4201 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag
     4261 cggtaatacg gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa
     4321 gcaaagcacc ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac
     4381 gagcatcaca aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga
     4441 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4501 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4561 tgttggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4621 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4681 agacacgact tatcgccact ggcagcagcc attggtaact gatttagagg actttgtctt
     4741 gaagttatgc acctgttaag gctaaactga aagaacagat tttggtgagt gcggtcctcc
     4801 aacccactta ccttggttca aagagttggt agctcagcga accttgagaa aaccaccgtt
     4861 ggtagcggtg gtttttcttt atttatgaga tgatgaatca atcggtctat caagtcaacg
     4921 aacagctatt ccgttactct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     4981 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     5041 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5101 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5161 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5221 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5281 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5341 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5401 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5461 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5521 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5581 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5641 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5701 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5761 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5821 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5881 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5941 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6001 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6061 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6121 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6181 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6241 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6301 gacgctctcc cttatgcgac tcctgcatta ggaaattaat acgactcact ata
//
