LOCUS       sequence-272413-        9587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""Burdensome"" GFP expressing plasmid carrying microcin-V 
            bacteriocin for plasmid stabilisation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9587)
  AUTHORS   Fedorec AJH, Ozdemir T, Doshi A, Ho YK, Rosa L, Rutter J, Velazquez 
            O, Pinheiro VB, Danino T, Barnes CP
  TITLE     Two New Plasmid Post-segregational Killing Mechanisms for the 
            Implementation of Synthetic Gene Networks in Escherichia coli.
  JOURNAL   iScience. 2019 Apr 26;14:323-334. doi: 10.1016/j.isci.2019.03.019. 
            Epub 2019 Mar 22.
  PUBMED    30954530
REFERENCE   2  (bases 1 to 9587)
  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: "iScience.";
            date: "2019-04-26"; volume: "14:323-334. doi"; pages: " 
            10.1016/j.isci.2019.03.019. Epub 2019 Mar 22"
COMMENT     Sequence Label: pUC-GFP-MCC
FEATURES             Location/Qualifiers
     source          1..9587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(36..624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(116..135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1244..1312
                     /label=recA (Delta-LexA) promoter
                     /note="constitutively active E. coli recA promoter lacking 
                     the LexA binding site (Brent and Ptashne, 1981)"
     CDS             1329..2039
                     /codon_start=1
                     /product="green fluorescent protein with peak excitation at
                     510 nm and peak emission at 521 nm"
                     /label=daGFP
                     /translation="MTALTEGAKLFEKEIPYITELEGDVEGMKFIIKGEGTGDATTGTI
                     KAKYICTTGDLPVPWATLVSTLSYGVQCFAKYPSHIKDFFKSAMPEGYTQERTISFEGD
                     GVYKTRAMVTYERGSIYNRVTLTGENFKKDGHILRKNVAFQCPPSILYILPDTVNNGIR
                     VEFNQAYDIEGVTEKLVTKCSQMNRPLAGSAAVHIPRYHHITYHTKLSKDRDERRDHMC
                     LVEVVKAVDLDTYQ"
     terminator      2046..2181
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             2438..3232
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2438..3232
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2492..2511)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3102..3121
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      3245..3381
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3526..3597
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(3602..3648)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3666..3714
                     /note="synthetic polyadenylation signal"
     polyA_signal    8833..8954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8844..8863)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8898..8917
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      9069..9115
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      9145..9281
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    9326..9397
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
ORIGIN
        1 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
       61 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
      121 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
      181 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
      241 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
      301 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
      361 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
      421 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
      481 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
      541 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
      601 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
      661 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
      721 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
      781 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
      841 tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc gtcggaaaat
      901 cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact atcgcaagtc
      961 tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag gtattactcc
     1021 aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat cctcaatccc
     1081 gatctatccg agatccgagg aatatcgaaa tcggggcgcg ccaagctgtt gtgaccgctt
     1141 gctctagcca gctatcgagt tgtgaaccga tccatctagc aattggtctc gatctagcga
     1201 taggcttcga tctagctatg tagaaacgcc gtgtgctcga tcgcttgata aggtccacgt
     1261 agctgctata attgcttcaa cagaacatat tgactatccg gtattacccg gcaaaggagg
     1321 tagccaacat gacagctctg acagagggag cgaaactgtt cgagaaggag atcccgtaca
     1381 tcacagagtt ggaaggagat gtcgaaggga tgaaattcat catcaaggga gaaggaacgg
     1441 gtgacgcaac gaccggaacg atcaaggcca aatacatttg taccacgggc gatttgcctg
     1501 tcccctgggc gacgcttgta agcacgctct cgtatggtgt ccagtgcttc gcgaaatatc
     1561 catcgcacat taaggacttt ttcaagtcgg ccatgccaga aggttacaca caagaacgaa
     1621 ccatctcctt tgagggggac ggagtgtata agacacgcgc gatggtaacg tacgagcgcg
     1681 ggtccatcta caacagggta actcttactg gggagaactt taagaaggac gggcatatct
     1741 tgcggaaaaa cgtggcattt caatgtccgc cctcgattct gtatattctc ccggacacgg
     1801 tgaataatgg gatcagagtg gagttcaacc aggcatacga tattgagggt gtgactgaaa
     1861 agctcgtcac caaatgcagc cagatgaatc gcccccttgc gggatcagca gccgtccata
     1921 tcccccggta tcaccatatt acctaccaca caaaactctc aaaagacaga gatgagagaa
     1981 gggaccatat gtgcctggtc gaagtagtga aggcggtgga tcttgataca taccagtagg
     2041 ctagccattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     2101 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     2161 ttaccagaaa tttatcctta agggcgcgcc tggtgtaccg agaacgatcc tctcagtgcg
     2221 agtctcgacg atccatatcg ttgcttggca gtcagccagt cggaatccag cttgggaccc
     2281 aggaagtcca atcgtcagat attgtactca agcctggtca cggcagcgta ccgatctgtt
     2341 taaacctaga tattgatagt ctgatcggtc aacgtataat cgagtcctag cttttgcaaa
     2401 catctatcaa gagacaggat cagcaggagg ctttcgcatg attgaacaag atggattgca
     2461 cgcaggttct ccggcggctt gggtggagag gctattcggc tatgactggg cacaacagac
     2521 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgtc cggttctttt
     2581 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     2641 gtggctggcg acgacgggcg ttccttgcgc ggctgtgctc gacgttgtca ctgaagcggg
     2701 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     2761 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     2821 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     2881 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     2941 cgaactgttc gccaggctca aggcgtctat gcccgacggc gaggatctcg tcgtgaccca
     3001 cggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3061 ctgtggccgt ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3121 tgctgaagag cttggcggcg aatgggctga ccgcttcctt gtgctttacg gtatcgccgc
     3181 gcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaccgattct
     3241 aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     3301 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     3361 ttaccagaaa tttatcctta aggtcgttta aactcgactc tggctctatc gaatctccgt
     3421 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt
     3481 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta
     3541 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat
     3601 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg
     3661 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattatctaa
     3721 ctgctgatcg agtgtagcca gatctttgtc gatcctacca tccactcgac acacccgcca
     3781 gcggccgctg ccaagcttcc gagctctgcc cttccctaga gaatcctgcc aggcttgcca
     3841 cactgatata tcttgacttt atgtaaacga tatgacactt taacatgata atgattacca
     3901 ttctctttta atatacagag aaactaggaa atagatgaat gagttatgtt actttaatat
     3961 tctctgacaa taacctaaat cagttagatt attgtcattt aataaataat gacattcttt
     4021 catcataaat aaaaagacta ttgtttataa tattgttctc agcattatat gattatttat
     4081 cctgataact ctcctatgtt gtatgtttat atgattttcc ttgaaacata taatgcaaat
     4141 tttcgattta ttttccatca ttaatccaga taaacaacaa actaatagta tgcaaggaga
     4201 cattatttgt ttcgccatga tgctttagaa aacagaaaaa tgaagtggca gggacgggca
     4261 atattacttc ccggaatacc actgtggtta atcatgctgg gaagcattgt gtttattacg
     4321 gcatttctga tgttcattat tgttggtacc tatagccgcc gtgttaatgt cagtggtgag
     4381 gtcacaacct ggccaagagc tgtcaatata tattcaggtg tacagggatt tgttgtcagg
     4441 cagtttgttc atgaagggca gttgataaaa aaaggggatc ctgtttatct gattgacatc
     4501 agtaaaagta cacgcaatgg tattgtcact gataatcatc gccgggatat agaaaaccag
     4561 ctggttcgtg tggacaacat tatttcccgt ctggaagaaa gtaaaaaaat aacgctagat
     4621 accctggaaa aacaacgtct gcaatacaca gatgcgttcc gtcgctcatc agacattata
     4681 cagcgtgcag aggaagggat aaaaataatg aaaaataata tggagaatta cagatactat
     4741 cagtcaaaag gactgattaa taaagatcaa ttaactaacc aagttgcatt atattatcaa
     4801 caacaaaaca accttctcag tctgagcgga caaaatgaac aaaatgccct gcagataacc
     4861 actctggaga gtcagattca gactcaggca gcagattttg ataatcgtat ctatcagatg
     4921 gaactgcaac gactcgaatt gcagaaagaa ctggttaaca ctgatgtgga aggcgaaatc
     4981 attatccggg cgttgtctga cgggaaagtt gactccctga gtgtcactgt agggcaaatg
     5041 gtcaataccg gagacagcct tctgcaggtt attcctgaga acattgaaaa ctattatctt
     5101 attctctggg tcccgaatga tgctgttcct tatatttcgg ctggtgacaa agtgaatatt
     5161 cgttatgaag ccttcccctc agaaaaattt gggcagttct ctgctacggt taaaactata
     5221 tccaggactc ctgcgtcaac acaggaaatg ttgacctata agggagcacc tcaaaatacg
     5281 ccgggtgcct ctgttccctg gtataaagtc attgcgacgc ctgaaaagca gataatcagg
     5341 tatgacgaaa aatacctccc tctggaaaat ggaatgaaag ccgaaagtac actatttctg
     5401 gaaaaaaggc gtatttacca gtggatgctt tctcctttct atgacatgaa acacagtgca
     5461 acaggaccga tcaatgacta acaggaattt cagacaaatt ataaatctgc ttgatttgcg
     5521 ctggcaacgt cgtgttccgg ttattcatca gacggagacc gctgaatgtg gactggcctg
     5581 cctagcaatg atatgcggtc attttggtaa gaatattgac ctgatatatc ttcgccggaa
     5641 gtttaatctc tctgcccgtg gagcaaccct tgcaggaatc aatggaatag cggagcaact
     5701 ggggatggcc acccgggctc tttcactgga gttggatgaa cttcgagtcc tcaaaacgcc
     5761 gtgtattctc cactgggatt tcagtcactt cgtcgttctg gtcagcgtaa agcgtaaccg
     5821 ttatgtactg catgatccgg ccaggggcat aagatatatc agccgggagg aaatgagccg
     5881 atattttaca ggcgttgcac ttgaggtctg gcccggaagt gaattccagt cggaaaccct
     5941 gcagacccgc ataagtcttc gttcactgat taacagtatt tacggtatta aaagaacgct
     6001 ggcgaaaatt ttctgtctgt cagttgtaat tgaagcaatc aatctgctaa tgccggtggg
     6061 gacacagctg gttatggatc atgctattcc tgcgggggac agagggctac tgacgctaat
     6121 ttctgctgct cttatgtttt ttatattact caaagctgca acgagtacgc tgcgcgcatg
     6181 gtcttcactg gttatgagca cgctcatcaa tgtacagtgg cagtcggggc tgttcgatca
     6241 tcttctcaga ctaccgctgg cgttttttga acgccgaaaa ttaggtgata tccagtcacg
     6301 ttttgactcc cttgacacat tgagggccac atttaccacc agtgtgatcg ggttcataat
     6361 ggacagcatt atggttgtcg gtgtttgtgt gatgatgctg ttatacggag gatatctcac
     6421 ctggatagtt ctctgcttta ccacaattta catttttatt cgactggtga catacggcaa
     6481 ttaccgacag atatcagaag aatgtcttgt cagggaggcc cgtgccgcct cctattttat
     6541 ggaaacatta tatggtattg ccacggtaaa aatccagggg atggtcggaa ttcggggggc
     6601 acactggctt aatatgaaaa tagatgcgat aaattcgggt attaagctaa ccaggatgga
     6661 tttgctcttc ggaggaataa atacctttgt taccgcctgt gatcagattg taattttatg
     6721 gctgggagca ggccttgtga tcgataatca gatgacaata ggaatgtttg tagcgtttag
     6781 ttcttttcgt gggcagtttt cggaaagagt tgcctctctg accagttttc ttcttcagct
     6841 aagaataatg agtctgcaca atgagcgcat tgcagatatt gcattacatg aaaaggagga
     6901 aaagaaacct gaaattgaaa tcgttgctga tatggggcca atatccctgg aaaccaatgg
     6961 tttaagctat cgttatgaca gtcagtcagc accgatattc agtgctctga gtttatctgt
     7021 agctccgggg gaaagtgtgg ctataactgg tgcttccggt gcgggaaaaa ccacattaat
     7081 gaaagtacta tgtggactat ttgaacctga tagcgggagg gtactgataa atggtataga
     7141 tatacgccaa attggaataa ataattatca ccggatgata gcctgtgtta tgcaggatga
     7201 ccggctattt tcaggctcaa ttcgtgaaaa tatctgtggt tttgcagagg aaatggatga
     7261 agagtggatg gtagaatgtg ccagagcaag tcatattcat gatgttataa tgaatatgcc
     7321 aatgggatat gaaacattaa taggtgaact tggggaaggt ctttctggcg gtcaaaaaca
     7381 gcgtatattt attgcacgag ccttataccg gaaaccagga atattattta tggatgaggc
     7441 aaccagtgct cttgattcag agagtgaaca tttcgtgaat gttgccataa aaaacatgaa
     7501 tatcaccagg gtaattattg cacacagaga aacaacgttg agaactgttg atagagttat
     7561 ttctatttaa accatagagg aattacaagc gtatgaggaa tatttcttcc tgttataatt
     7621 cctcgttatg ctcagatatc tgttggaggt ggaatggaag atagacaatc caccaagaag
     7681 aaatatcatt ctgtgtggat tgtccaataa ctgttctttc ttatattaaa taatactatt
     7741 tataaacaaa catcactaag attatttgga ctccaattac acaatcttcc cgcagcatag
     7801 ttccatgctt ctgaaggtat cccttcgggt ttttgcttaa ttgttccccc taaaccggat
     7861 ggagacattg caggattagg tttgtgagtg gatgcatagt catatattgc acctccagcc
     7921 acacccccag cagctgctcc aattcctcct gcaacaaatt gcccggatag tgttcctata
     7981 gccatcgcaa tatcacgccc tgaagcacca ccagaaacag aatctaattc atttagagtc
     8041 agagttctca tatgatctcc tttttatctt atcggatatt gaataataat tatcaccaac
     8101 aaagtaacat attgcagaca ttaatgcaga gaagcaaaat gtatgcatgg ataaaaagtc
     8161 ctttcctcta aaaacacaat catatatagc taatgcaata tatattgcgg tggcatttat
     8221 tataaatgca aataacaact ctaattttgt tctttttcta tccattactt tttatcccat
     8281 tactttctat cccattacca cacaaacact aacgataatg attatcgtta acatagtcaa
     8341 gagtgaaggg taggaggccc tcaaccccct ataaggggtc cgcttggaaa acggatttcc
     8401 ccacgtcaag agaattgatt tgaacgagtg gcttcgctac aaacagctcc cgtttgtgtg
     8461 tgaaaaaccc ctctcaacag atctcaactc tgcacgatat ctacaggaga ttgctccaaa
     8521 aggcagtatc gtgtctttta actaccgata taaccccgtc atcgacatga taatgcgtct
     8581 gagaaatgag aagaaattag gtgaactgca tttcttctca gcagaattta ataaaaattc
     8641 tgcactgacc cgtctagaaa cctcgaggta gatatcgcgg taccccatgg tgggtaccgc
     8701 gatatctacc tcgaggtttc tagaagttgt ctcctcctgc actgactgac tgatacaatc
     8761 gatttctgga tccgcaggcc tctgctagct tgactgactg agatacagcg taccttcagc
     8821 tcacagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     8881 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     8941 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     9001 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtattggc ccatctctat
     9061 cggtatcgta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc
     9121 ctcgggccgg attgctatct accggcattg gcgcagaaaa aaatgcctga tgcgacgctg
     9181 cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc
     9241 cacttccata cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag ctatcctgca
     9301 ggcgatctct cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg
     9361 ggtcagaagt agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta
     9421 tcgaaactta attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg
     9481 actacgctca gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc
     9541 cgattacgag tttcatttaa atcatgtgag caaaaggcca gcaaaag
//
