LOCUS       sequence-360663-        4501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-human growth 
            hormone fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4501)
  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 4501)
  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-hGH
FEATURES             Location/Qualifiers
     source          1..4501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      692..1441
                     /direction=RIGHT
                     /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     1172..1191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1414..1431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1457..1548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1549..2364
                     /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             1549..2364
                     /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     complement(1622..1641)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(2594..2641)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2637..2655
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2693..2737)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(2753..2770)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3518..3542
                     /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)."
     promoter        complement(3543..3561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(3675..3692)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    3748..3772
                     /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)."
     promoter        complement(3773..3791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3837..3914
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(3915..4501,1..496)
                     /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     complement(3934..3953)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
       61 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
      121 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
      181 cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat acgacgatac
      241 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
      301 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
      361 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
      421 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
      481 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
      541 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
      601 gcatgactaa catgagaatt acaacttata tcgtatgggg ctgacttcag gtgctacatt
      661 tgaagagata aattgcactg aaatctagag taacggaata gctgttcgtt gacttgatag
      721 accgattgat tcatcatctc ataaataaag aaaaaccacc gctaccaacg gtggttttct
      781 caaggttcgc tgagctacca actctttgaa ccaaggtaag tgggttggag gaccgcactc
      841 accaaaatct gttctttcag tttagcctta acaggtgcat aacttcaaga caaagtcctc
      901 taaatcagtt accaatggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      961 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1021 agcccagctt ggagcgaacg acctacaccg aactgagata ccaacagcgt gagctatgag
     1081 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1141 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1201 tcgggtttcg ccacctctgg cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1261 gcctatggaa aaacgcctgc ggcgttggct tcttccggtg ctttgctttt tgctcacatg
     1321 ttctttccgg ctttatcccc tgattctgtg gataaccgta ttaccgcttt tgagtgagct
     1381 gacaccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa
     1441 gagcgctgca tgcctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1501 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagccatatt
     1561 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     1621 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     1681 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     1741 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     1801 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     1861 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     1921 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgac
     1981 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     2041 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     2101 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     2161 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     2221 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     2281 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     2341 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     2401 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacttgcgga
     2461 gacccggtcg tcagcttgtc gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct
     2521 attgctggtt taccggttta ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg
     2581 cctgaggttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     2641 gttattgctc agcggtggca gcagcctagg ttaattaagc tgcgctagta gacgagtcca
     2701 tgtgctggcg ttcaaatttc gcagcagcgg tttctttacc agactcgagt taatgatgat
     2761 gatgatgatg ggtacctcat tagaagccgc aagaaccctc aacagagcga cactgaacaa
     2821 tacgcaggaa ggtctcaact ttgtccatgt ctttacggaa gcagtacagc agaccatagt
     2881 ttttcagcag ggcgtcatca ttatgggagt tggtgtcgaa tttagagtaa gtctgtttaa
     2941 aaatctggcc agtacgcggg ctgccgtcct ccaggcggcc catcagggtc tggataccct
     3001 cctccaggtc tttcagcaga tcgtatacgt tagagtcgga agcgccatat accagggagt
     3061 tcgcgaagac gctgcgcagg aattgcaccg gttccagcca agactggatc agcagcaggc
     3121 taatacgcag cagttccaga ttagatttct gctgggtttc ttcgcggtta gacggagtcg
     3181 ggatggattc gctgaagcac agggaggttt gcgggttctg caggaagctg tatttctgtt
     3241 ctttcgggat gtaggcctcc tcgaattctt gataagtgtc gaatgccagc tggtgcagac
     3301 gatgtgcacg cagcatagcg ttatcgaaca gacgagacag cggaatcgtc gggaaagatc
     3361 tgagaacacc ctcttttgtc tttcctgctg cttctgccac accctgtttg gttttctcag
     3421 cagctgccac aactccctcc ttggcctttg aaagtccttt catgaataca tccatatgta
     3481 tatctccttc ttatacttaa ctaatatact aagatgggga attgttatcc gctcacaatt
     3541 cccctatagt gagtcgtatt aatttcgatt atgcggccgt gtacaatacg attactttct
     3601 gttcgactta agcattatgc ggccgcaagc ttgtcgacct gcaggcgcgc cgagctcgaa
     3661 ttcggatcct ggctgtggtg atgatggtga tggctgctgc ccatggtata tctccttatt
     3721 aaagttaaac aaaattattt ctacagggga attgttatcc gctcacaatt cccctatagt
     3781 gagtcgtatt aatttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca
     3841 ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     3901 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     3961 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     4021 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     4081 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     4141 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     4201 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     4261 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     4321 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     4381 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     4441 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     4501 t
//
