LOCUS       sequence-360454-        4477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-fgf21 fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4477)
  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 4477)
  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-FGF21
FEATURES             Location/Qualifiers
     source          1..4477
                     /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             981..998
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1014..1058
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      1110..1157
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1387..2202)
                     /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(1387..2202)
                     /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     2110..2129
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2203..2294)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2310..3059)
                     /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(2320..2337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2560..2579)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3255..4337)
                     /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     4299..4318
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4338..4415)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4461..4477,1..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 acatccgatt ccggatagca gtccgctgct gcagtttggt ggtcaggttc
      481 gtcagcgtta tctgtatacc gatgatgcac agcagaccga agcacatctg gaaattcgtg
      541 aagatggcac cgttggtggt gcagcagatc agagtccgga aagcctgctg cagctgaaag
      601 cactgaaacc gggtgttatt cagattctgg gtgttaaaac cagccgtttt ctgtgtcagc
      661 gtccggatgg tgcactgtat ggtagcctgc attttgatcc ggaagcatgt agctttcgtg
      721 aactgctgct ggaagatggt tataatgttt atcagagcga agcccatggt ctgccgctgc
      781 atctgcctgg taataaaagt ccgcatcgtg atccggcacc gcgtggtccg gcacgttttc
      841 tgccgttacc gggtctgcct ccggctctgc cggaaccgcc tggtattctg gcaccgcagc
      901 ctccggatgt tggtagcagc gatccgctga gcatggttgg tccgagccag ggtcgtagcc
      961 cgagctatgc aagcggtacc catcatcatc atcatcatta actcgagtct ggtaaagaaa
     1021 ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa
     1081 cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1141 tcttgagggg ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc
     1201 cggtagtcaa taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     1261 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     1321 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     1381 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1441 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1501 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1561 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1621 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     1681 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     1741 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     1801 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     1861 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     1921 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     1981 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2041 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2101 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2161 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     2221 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2281 gaaaaataaa caaataggca tgcagcgctc ttccgcttcc tcgctcactg actcgctacg
     2341 ctcggtcgtt cgactgcggc gagcggtgtc agctcactca aaagcggtaa tacggttatc
     2401 cacagaatca ggggataaag ccggaaagaa catgtgagca aaaagcaaag caccggaaga
     2461 agccaacgcc gcaggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2521 gacgctcaag ccagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2581 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2641 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgttgg tatctcagtt
     2701 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2761 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2821 cactggcagc agccattggt aactgattta gaggactttg tcttgaagtt atgcacctgt
     2881 taaggctaaa ctgaaagaac agattttggt gagtgcggtc ctccaaccca cttaccttgg
     2941 ttcaaagagt tggtagctca gcgaaccttg agaaaaccac cgttggtagc ggtggttttt
     3001 ctttatttat gagatgatga atcaatcggt ctatcaagtc aacgaacagc tattccgtta
     3061 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     3121 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3181 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3241 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3301 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3361 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3421 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3481 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     3541 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     3601 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3661 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3721 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3781 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     3841 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     3901 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     3961 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4021 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4081 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4141 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4201 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4261 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4321 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4381 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4441 cgactcctgc attaggaaat taatacgact cactata
//
