LOCUS       sequence-360650-        4787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine Zipper 
            and Vesicle Nucleating peptide-Leucine zipper-human growth hormone 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4787)
  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 4787)
  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-hGH
FEATURES             Location/Qualifiers
     source          1..4787
                     /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             1291..1308
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1324..1368
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1406..1424)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1420..1467
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1697..2512)
                     /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(1697..2512)
                     /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     2420..2439
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2513..2604)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2620..3369)
                     /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(2630..2647)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2870..2889)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3565..4647)
                     /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     4609..4628
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4648..4725)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4771..4787,1..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 gatctttccc gacgattccg
      721 ctgtctcgtc tgttcgataa cgctatgctg cgtgcacatc gtctgcacca gctggcattc
      781 gacacttatc aagaattcga ggaggcctac atcccgaaag aacagaaata cagcttcctg
      841 cagaacccgc aaacctccct gtgcttcagc gaatccatcc cgactccgtc taaccgcgaa
      901 gaaacccagc agaaatctaa tctggaactg ctgcgtatta gcctgctgct gatccagtct
      961 tggctggaac cggtgcaatt cctgcgcagc gtcttcgcga actccctggt atatggcgct
     1021 tccgactcta acgtatacga tctgctgaaa gacctggagg agggtatcca gaccctgatg
     1081 ggccgcctgg aggacggcag cccgcgtact ggccagattt ttaaacagac ttactctaaa
     1141 ttcgacacca actcccataa tgatgacgcc ctgctgaaaa actatggtct gctgtactgc
     1201 ttccgtaaag acatggacaa agttgagacc ttcctgcgta ttgttcagtg tcgctctgtt
     1261 gagggttctt gcggcttcta atgaggtacc catcatcatc atcatcatta actcgagtct
     1321 ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca
     1381 gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1441 tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc
     1501 acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa
     1561 cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg
     1621 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1681 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1741 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1801 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1861 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1921 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     1981 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2041 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat
     2101 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2161 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2221 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2281 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2341 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2401 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2461 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc
     2521 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2581 aatgtattta gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg
     2641 actcgctacg ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa
     2701 tacggttatc cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag
     2761 caccggaaga agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat
     2821 cacaaaaatc gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag
     2881 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2941 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg
     3001 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3061 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3121 gacttatcgc cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt
     3181 atgcacctgt taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca
     3241 cttaccttgg ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc
     3301 ggtggttttt ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc
     3361 tattccgtta ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
     3421 ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag
     3481 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     3541 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3601 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     3661 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     3721 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     3781 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     3841 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     3901 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     3961 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4021 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4081 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4141 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4201 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4261 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4321 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4381 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4441 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4501 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     4561 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     4621 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     4681 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     4741 ctcccttatg cgactcctgc attaggaaat taatacgact cactata
//
