LOCUS       sequence-364909-        7818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Vesicle Nucleating peptide-mNeongreen and 
            ompA-mCherry fusions.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7818)
  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 7818)
  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-mNeongreen_OmpA-mCherry
FEATURES             Location/Qualifiers
     source          1..7818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             182..892
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(327..345)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     595..614
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1001..1019
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1020..1044
                     /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             1219..1926
                     /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             2269..2313
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2351..2369)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2365..2412
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2449..2904
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2536..2555)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2746..2767
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3019..3810,3811..3879))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3642..3661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3880..3972)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4053..4641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4542..4561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4795..4812
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4827..4966
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4892..4911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5052..5074
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5068..5259)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5831..6913)
                     /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     6875..6894
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6914..6991)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7197..7216
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7304..7322
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7323..7347
                     /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)."
     RBS             7362..7384
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     sig_peptide     7391..7453
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
ORIGIN
        1 cttcgctggc ggtgttgagt acgcgatcac tcctgaaatc gctacccgtc tggaatacca
       61 gtggaccaac aacatcggtg acgcacacac catcggcact cgtccggaca acggcatgct
      121 gagcctgggt gtttcctacc gtttcggtca gggcgaagga tccggtggtg gtggttcggg
      181 tatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
      241 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      301 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
      361 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
      421 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
      481 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
      541 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
      601 aatgcagaag aagactatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
      661 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
      721 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
      781 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
      841 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aaagatccga
      901 attcgagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa
      961 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
     1021 gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat
     1081 atacatatgg atgtattcat gaaaggactt tcaaaggcca aggagggagt tgtggcagct
     1141 gctgagaaaa ccaaacaggg tgtggcagaa gcagcaggaa agacaaaaga gggtgttctc
     1201 agatctggag gaagcggaat ggtgagcaag ggcgaggagg ataacatggc ctctctccca
     1261 gcgacacatg agttacacat ctttggctcc atcaacggtg tggactttga catggtgggt
     1321 cagggcaccg gcaatccaaa tgatggttat gaggagttaa acctgaagtc caccaagggt
     1381 gacctccagt tctccccctg gattctggtc cctcatatcg ggtatggctt ccatcagtac
     1441 ctgccctacc ctgacgggat gtcgcctttc caggccgcga tggtagatgg ctccggatac
     1501 caagtccatc gcacaatgca gtttgaagat ggtgcctccc ttactgttaa ctaccgctac
     1561 acctacgagg gaagccacat caaaggagag gcccaggtga aggggactgg tttccctgct
     1621 gacggtcctg tgatgaccaa ctcgctgacc gctgcggact ggtgcaggtc gaagaagact
     1681 taccccaacg acaaaaccat catcagtacc tttaagtgga gttacaccac tggaaatggc
     1741 aagcgctacc ggagcactgc gcggaccacc tacacctttg ccaagccaat ggcggctaac
     1801 tatctgaaga accagccgat gtacgtgttc cgtaagacgg agctcaagca ctccaagacc
     1861 gagctcaact tcaaggagtg gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg
     1921 tacaaggcta gcggaggaag cggaatgatg ttcttaccaa ccgattattg ttgcaggtta
     1981 agcgatcaag agtatatgga gcttgtgttt gagaatggcc agattcttgc aaagggccaa
     2041 agatccaacg tttctctgca taatcaacgt accaaatcga tcatggattt gtatgaggca
     2101 gagtataacg aggatttcat gaagagtatc attcatggtg gtggtggtgc catcacaaat
     2161 ctcggggaca cgcaggttgt tccacaaagt catgttgctg ctgcccatga aacaaacatg
     2221 ttggaaagca ataaacatgt tgactaaact agttaactcg agtctggtaa agaaaccgct
     2281 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     2341 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2401 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2461 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2521 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2581 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2641 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2701 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2761 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2821 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2881 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     2941 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3001 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3061 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3121 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3181 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3241 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3301 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3361 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3421 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3481 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3541 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3601 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3661 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3721 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3781 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3841 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     3901 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3961 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4021 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4081 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4141 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4201 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4261 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4321 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4381 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4441 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4501 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4561 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4621 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4681 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4741 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4801 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4861 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4921 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4981 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5041 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5101 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5161 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5221 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5281 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5341 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5401 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5461 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5521 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5581 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5641 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     5701 caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     5761 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     5821 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5881 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     5941 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     6001 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     6061 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac
     6121 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     6181 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     6241 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     6301 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     6361 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     6421 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     6481 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     6541 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     6601 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     6661 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     6721 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     6781 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     6841 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     6901 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     6961 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     7021 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     7081 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     7141 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     7201 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     7261 cgtccggcgt agaggatcga gatcgatctc gatcccgcga aattaatacg actcactata
     7321 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     7381 gagatatacc atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac
     7441 cgtagcgcag gccgctccga aagataacac ctggtacact ggtgctaaac tgggctggtc
     7501 ccagtaccat gacactggtt tcatcaacaa caatggcccg acccatgaaa accaactggg
     7561 cgctggtgct tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggttacga
     7621 ctggttaggt cgtatgccgt acaaaggcag cgttgaaaac ggtgcataca aagctcaggg
     7681 cgttcaactg accgctaaac tgggttaccc aatcactgac gacctggaca tctacactcg
     7741 tctgggtggc atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga
     7801 caccggcgtt tctccggt
//
