LOCUS       sequence-360559-        7204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-Leucine Zipper 
            and cydAB--mNeongreen fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7204)
  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 7204)
  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_CydAB-mNeongreen
FEATURES             Location/Qualifiers
     source          1..7204
                     /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             2594..3304
                     /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"
     promoter        3370..3388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3389..3413
                     /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             3603..3659
                     /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             3708..3725
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3741..3785
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3823..3841)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3837..3884
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4114..4929)
                     /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(4114..4929)
                     /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     4837..4856
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4930..5021)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5037..5786)
                     /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(5047..5064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5287..5306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5982..7064)
                     /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     7026..7045
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7065..7142)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7188..7204,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggagttcc agttcgggac taactggtct tactattccc actatgtagg
      121 ggatatcttc ggtgcgccgc tggcaatcga aggtctgatg gccttcttcc tcgaatccac
      181 ctttgtaggt ctgttcttct tcggttggga tcgtctgggt aaagttcagc atatgtgtgt
      241 cacctggctg gtggcgctcg gttcaaacct gtccgcactg tggattctgg ttgcgaacgg
      301 ctggatgcaa aacccaatcg cgtccgattt caactttgaa actatgcgta tggagatggt
      361 gagcttctcc gagctggtgc ttaacccggt tgctcaggtg aaattcgttc acactgtagc
      421 gtctggttat gtgactggcg cgatgttcat cctcggtatc agcgcatggt atatgctgaa
      481 aggtcgtgac ttcgccttcg ctaaacgctc ctttgctatc gctgccagct tcggtatggc
      541 tgctgttctg tctgttattg ttctgggtga tgaatccggc tacgaaatgg gcgacgtgca
      601 gaaaaccaaa ctggctgcta ttgaagccga gtgggaaacg caacctgcgc ctgctgcctt
      661 tactctgttc ggcattcctg atcaggaaga ggagacgaac aaatttgcga ttcagatccc
      721 ttacgcactg ggcatcattg caacgcgttc cgtggatacc ccggttatcg gcctgaaaga
      781 gctgatggtg cagcatgaag aacgcattcg taacgggatg aaggcgtact ctctgctcga
      841 acaactgcgt tctggttcta ccgaccaggc ggttcgtgac cagttcaata gcatgaagaa
      901 agacctcggt tacggtctgc tgctgaaacg ctatacgcca aacgtggctg atgcgactga
      961 agcgcagatt caacaggcaa ccaaagactc catcccgcgt gtagcgccgc tgtactttgc
     1021 gttccgtatc atggtggcgt gtggcttcct gcttctggca atcatcgcgc tctctttctg
     1081 gagtgtcatc cgcaaccgca ttggcgagaa aaaatggctt ctgcgcgccg cgctgtacgg
     1141 tattccgctg ccgtggattg ctgtagaagc gggctggttc gtggctgaat atggccgcca
     1201 accgtgggct atcggtgaag tgctgccgac agctgtggcg aactcgtcac tgaccgcagg
     1261 cgatctcatc ttctcaatgg tgctgatttg cggcctgtat accctgttcc tggtggcaga
     1321 attgttctta atgttcaagt ttgcacgcct cggcccaagc agcctgaaaa ccggtcgcta
     1381 tcactttgag cagtcttcca cgactactca gccggcacgc taagacagga gtcgtcaaat
     1441 gatcgattat gaagtattgc gttttatctg gtggctgctg gttggcgttc tgctgattgg
     1501 ttttgcagtc actgacggtt tcgacatggg ggtgggcatg ctcacccgtt tcctcggtcg
     1561 taacgacacc gagcgtcgaa ttatgattaa ctccattgca ccacactggg acggtaacca
     1621 ggtttggctg atcaccgcgg gcggcgcact ctttgctgcc tggccgatgg tctatgccgc
     1681 tgcgttctcc ggcttctatg tggcgatgat cctcgtgctg gcgtctttgt tcttccgtcc
     1741 ggtcggtttt gactaccgct ccaagattga agaaacccgc tggcgtaaca tgtgggactg
     1801 gggcatcttc attggtagct tcgttccgcc gctggtaatt ggtgtagcgt tcggtaacct
     1861 gttgcagggc gtaccgttca acgttgatga atatctgcgt ctgtactaca ccggtaactt
     1921 cttccagttg cttaacccgt tcggcctgct ggcaggcgtg gtgagcgtag ggatgatcat
     1981 tactcagggc gcaacctatc tgcaaatgcg taccgtgggc gaactgcacc tgcgtacccg
     2041 tgcaacggct caggtggctg cgctggtgac actggtctgt ttcgcactgg ctggcgtatg
     2101 ggtgatgtac ggtatcgatg gttatgtcgt gaaatcgaca atggaccatt acgcagcctc
     2161 taacccactg aataaagaag tggttcgtga agctggcgca tggctggtta acttcaacaa
     2221 cacgccaatt ctgtgggcta ttccggcact gggtgtggtt ctgccgctgc tgaccatcct
     2281 gactgcacgt atggataaag ccgcgtgggc gtttgtgttc tcctccctga cgctggcctg
     2341 catcatcctg acagccggta tcgcaatgtt cccgtttgtg atgccgtcca gcaccatgat
     2401 gaacgcaagt ctgacaatgt gggatgcaac ttccagccag ctgacgctta acgtcatgac
     2461 ctgggttgcg gtggttctgg taccgatcat tctgctctac accgcctggt gttactggaa
     2521 aatgttcggt cgtatcacca aagaagatat tgaacgtaac acccactctc tgtacgtcga
     2581 cggaggaagc ggaatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac
     2641 acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg
     2701 caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct
     2761 ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc
     2821 ctaccctgac gggatgtcgc ctttccaggc cgcgatggta gatggctccg gataccaagt
     2881 ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta
     2941 cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg
     3001 tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc
     3061 caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg
     3121 ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct
     3181 gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct
     3241 caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa
     3301 gtaagcggcc gcataatgct taagtcgaac agaaagtaat cgtattgtac acggccgcat
     3361 aatcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tccccatctt
     3421 agtatattag ttaagtataa gaaggagata tacatatgga tgtattcatg aaaggacttt
     3481 caaaggccaa ggagggagtt gtggcagctg ctgagaaaac caaacagggt gtggcagaag
     3541 cagcaggaaa gacaaaagag ggtgttctca gatcacgtat gaaacagctg gaagataaag
     3601 ttgaagaact gctgagcaaa aactaccacc tggaaaatga agttgcccgt ctgaaaaaac
     3661 tggttggtag atctggagga ggagctagcg gaggaagcgg aggtacccat catcatcatc
     3721 atcattaact cgagtctggt aaagaaaccg ctgctgcgaa atttgaacgc cagcacatgg
     3781 actcgtctac tagcgcagct taattaacct aggctgctgc caccgctgag caataactag
     3841 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat
     3901 ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga
     3961 cataagcggc tatttaacga ccctgccctg aaccgacgac aagctgacga ccgggtctcc
     4021 gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4081 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     4141 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     4201 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     4261 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     4321 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     4381 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     4441 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcggtc
     4501 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     4561 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     4621 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     4681 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     4741 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     4801 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     4861 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     4921 atggctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4981 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggcatgc agcgctcttc
     5041 cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggtgtcagc
     5101 tcactcaaaa gcggtaatac ggttatccac agaatcaggg gataaagccg gaaagaacat
     5161 gtgagcaaaa agcaaagcac cggaagaagc caacgccgca ggcgtttttc cataggctcc
     5221 gcccccctga cgagcatcac aaaaatcgac gctcaagcca gaggtggcga aacccgacag
     5281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5401 atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cattggtaac tgatttagag
     5581 gactttgtct tgaagttatg cacctgttaa ggctaaactg aaagaacaga ttttggtgag
     5641 tgcggtcctc caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga
     5701 aaaccaccgt tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta
     5761 tcaagtcaac gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat
     5821 gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc
     5881 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5941 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     6001 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     6061 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     6121 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6181 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6241 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     6301 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6361 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6421 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6481 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6541 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6601 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6661 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6721 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6781 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6841 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6901 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6961 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     7021 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     7081 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     7141 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
     7201 tata
//
