LOCUS       sequence-360633-        4759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine 
            Zipper-mNeongreen fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4759)
  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 4759)
  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-mNeongreen
FEATURES             Location/Qualifiers
     source          1..4759
                     /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             447..503
                     /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             531..1238
                     /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             1263..1280
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1296..1340
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1378..1396)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1392..1439
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1669..2484)
                     /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(1669..2484)
                     /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     2392..2411
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2485..2576)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2592..3341)
                     /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(2602..2619)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2842..2861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3537..4619)
                     /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     4581..4600
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4620..4697)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(4743..4759,1..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 ctcagatcac
      421 gtatgaaaca gctggaagat aaagttgaag aactgctgag caaaaactac cacctggaaa
      481 atgaagttgc ccgtctgaaa aaactggttg gtagatctgg aggaagcgga atggtgagca
      541 agggcgagga ggataacatg gcctctctcc cagcgacaca tgagttacac atctttggct
      601 ccatcaacgg tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt
      661 atgaggagtt aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg
      721 tccctcatat cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt
      781 tccaggccgc gatggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag
      841 atggtgcctc ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag
      901 aggcccaggt gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga
      961 ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta
     1021 cctttaagtg gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca
     1081 cctacacctt tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt
     1141 tccgtaagac ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg
     1201 cctttaccga tgtgatgggc atggacgagc tgtacaaggc tagcggagga agcggaggta
     1261 cccatcatca tcatcatcat taactcgagt ctggtaaaga aaccgctgct gcgaaatttg
     1321 aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg
     1381 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1441 tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     1501 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgacaagct
     1561 gacgaccggg tctccgcaag tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1621 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     1681 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1741 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1801 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1861 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1921 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1981 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     2041 acgaaatacg cggtcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     2101 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     2161 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     2221 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     2281 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     2341 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     2401 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     2461 cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca
     2521 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2581 catgcagcgc tcttccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg
     2641 gcgagcggtg tcagctcact caaaagcggt aatacggtta tccacagaat caggggataa
     2701 agccggaaag aacatgtgag caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt
     2761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agccagaggt
     2821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2941 gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct
     3001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccattg
     3121 gtaactgatt tagaggactt tgtcttgaag ttatgcacct gttaaggcta aactgaaaga
     3181 acagattttg gtgagtgcgg tcctccaacc cacttacctt ggttcaaaga gttggtagct
     3241 cagcgaacct tgagaaaacc accgttggta gcggtggttt ttctttattt atgagatgat
     3301 gaatcaatcg gtctatcaag tcaacgaaca gctattccgt tactctagat ttcagtgcaa
     3361 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
     3421 tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     3481 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     3541 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3601 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     3661 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     3721 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     3781 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     3841 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     3901 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     3961 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4021 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4081 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4141 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4201 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4261 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4321 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4381 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4441 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     4501 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     4561 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     4621 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     4681 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     4741 attaatacga ctcactata
//
