LOCUS       sequence-360468-        5519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine Zipper 
            and Vesicle Nucleating peptide-Leucine Zipper-mNeongreen-human 
            growth hormone fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5519)
  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 5519)
  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-hGH
FEATURES             Location/Qualifiers
     source          1..5519
                     /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             2023..2040
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2056..2100
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2138..2156)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2152..2199
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2429..3244)
                     /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(2429..3244)
                     /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     3152..3171
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3245..3336)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3352..4101)
                     /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(3362..3379)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3602..3621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4297..5379)
                     /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     5341..5360
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5380..5457)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5503..5519,1..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 cagatctttc
     1441 ccgacgattc cgctgtctcg tctgttcgat aacgctatgc tgcgtgcaca tcgtctgcac
     1501 cagctggcat tcgacactta tcaagaattc gaggaggcct acatcccgaa agaacagaaa
     1561 tacagcttcc tgcagaaccc gcaaacctcc ctgtgcttca gcgaatccat cccgactccg
     1621 tctaaccgcg aagaaaccca gcagaaatct aatctggaac tgctgcgtat tagcctgctg
     1681 ctgatccagt cttggctgga accggtgcaa ttcctgcgca gcgtcttcgc gaactccctg
     1741 gtatatggcg cttccgactc taacgtatac gatctgctga aagacctgga ggagggtatc
     1801 cagaccctga tgggccgcct ggaggacggc agcccgcgta ctggccagat ttttaaacag
     1861 acttactcta aattcgacac caactcccat aatgatgacg ccctgctgaa aaactatggt
     1921 ctgctgtact gcttccgtaa agacatggac aaagttgaga ccttcctgcg tattgttcag
     1981 tgtcgctctg ttgagggttc ttgcggcttc taatgaggta cccatcatca tcatcatcat
     2041 taactcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg
     2101 tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa
     2161 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag
     2221 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     2281 gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag
     2341 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2401 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2461 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2521 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2581 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2641 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2701 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2761 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt
     2821 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2881 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2941 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3001 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3061 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3121 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3181 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3241 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3301 gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgcagcgc tcttccgctt
     3361 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact
     3421 caaaagcggt aatacggtta tccacagaat caggggataa agccggaaag aacatgtgag
     3481 caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt ttttccatag gctccgcccc
     3541 cctgacgagc atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc gacaggacta
     3601 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3661 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3721 tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3781 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3841 ccggtaagac acgacttatc gccactggca gcagccattg gtaactgatt tagaggactt
     3901 tgtcttgaag ttatgcacct gttaaggcta aactgaaaga acagattttg gtgagtgcgg
     3961 tcctccaacc cacttacctt ggttcaaaga gttggtagct cagcgaacct tgagaaaacc
     4021 accgttggta gcggtggttt ttctttattt atgagatgat gaatcaatcg gtctatcaag
     4081 tcaacgaaca gctattccgt tactctagat ttcagtgcaa tttatctctt caaatgtagc
     4141 acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc
     4201 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4261 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4381 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4441 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4501 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4561 gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4621 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4681 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4741 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4801 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4861 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4921 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4981 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5041 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5101 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5161 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5221 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5281 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5341 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5401 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5461 gatctcgacg ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
