LOCUS       sequence-256181-        5350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ""Burdensome"" GFP expressing plasmid carrying hok/sok 
            toxin-antitoxin for plasmid stabilisation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5350)
  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 5350)
  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-HS
FEATURES             Location/Qualifiers
     source          1..5350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    18..89
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(94..140)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    158..206
                     /note="synthetic polyadenylation signal"
     polyA_signal    1088..1209
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1099..1118)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1153..1172
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1324..1370
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1400..1536
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1581..1652
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(1878..2466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1958..1977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3086..3154
                     /label=recA (Delta-LexA) promoter
                     /note="constitutively active E. coli recA promoter lacking 
                     the LexA binding site (Brent and Ptashne, 1981)"
     CDS             3171..3881
                     /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      3888..4023
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             4280..5074
                     /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             4280..5074
                     /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(4334..4353)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4944..4963
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5087..5223
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
ORIGIN
        1 gcgatcgcgg ctcccgacat cttggaccat tagctccaca ggtatcttct tccctctagt
       61 ggtcataaca gcagcttcag ctacctctca attcaaaaaa cccctcaaga cccgtttaga
      121 ggccccaagg ggttatgcta tcaatcgttg cgttacacac acaaaaaacc aacacacatc
      181 catcttcgat ggatagcgat tttattatct aactgctgat cgagtgtagc cagatctttg
      241 tcgatcctac catccactcg acacacccgc cagcggccgc tgccaagctt gcatgcctgc
      301 aggtcgactc tagaggatcc ggccgaacaa actccgggag gcagcgtgat gcggcaacaa
      361 tcacacggat ttcccgtgaa cggtctgaat gagcggatta ttttcaggga aagtgagtgt
      421 ggtcagcgtg caggtatatg ggctatgatg tgcccggcgc ttgaggcttt ctgcctcatg
      481 acgtgaaggt ggtttgttgc cgtgttgtgt ggcagaaaga agatagcccc gtagtaagtt
      541 aattttcatt aaccaccacg aggcatccct atgtctagtc cacatcagga tagcctctta
      601 ccgcgctttg cgcaaggaga agaaggccat gaaactacca cgaagttccc ttgtctggtg
      661 tgtgttgatc gtgtgtctca cactgttgat attcacttat ctgacacgaa aatcgctgtg
      721 cgagattcgt tacagagacg gacacaggga ggtggcggct ttcatggctt acgaatccgg
      781 taagtagcaa cctggaggcg ggcgcaggcc cgccttttca ggactgatgc tggtctgact
      841 actgaagcgc ctttataaag gggctgctgg ttcgccggta gcccctttct ccttgctgat
      901 gttgtgggaa ttcgagctct cgaattcaaa ggaggtaccc accatggggt accgcgatat
      961 ctacctcgag gtttctagaa gttgtctcct cctgcactga ctgactgata caatcgattt
     1021 ctggatccgc aggcctctgc tagcttgact gactgagata cagcgtacct tcagctcaca
     1081 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     1141 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     1201 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     1261 gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta
     1321 tcgtagcata accccttggg gcctctaaac gggtcttgag gggttttttg tgcccctcgg
     1381 gccggattgc tatctaccgg cattggcgca gaaaaaaatg cctgatgcga cgctgcgcgt
     1441 cttatactcc cacatatgcc agattcagca acggatacgg cttccccaac ttgcccactt
     1501 ccatacgtgt cctccttacc agaaatttat ccttaaggtc gtcagctatc ctgcaggcga
     1561 tctctcgatt tcgatcaaga cattccttta atggtctttt ctggacacca ctaggggtca
     1621 gaagtagttc atcaaacttt cttccctccc taatctcatt ggttaccttg ggctatcgaa
     1681 acttaattaa ccagtcaagt cagctacttg gcgagatcga cttgtctggg tttcgactac
     1741 gctcagaatt gcgtcagtca agttcgatct ggtccttgct attgcacccg ttctccgatt
     1801 acgagtttca tttaaatcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1861 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1921 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1981 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2041 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2101 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2161 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2221 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2281 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2341 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2401 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2461 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2521 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2581 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2641 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2701 gcatttaaat ttccgaactc tccaaggccc tcgtcggaaa atcttcaaac ctttcgtccg
     2761 atccatcttg caggctacct ctcgaacgaa ctatcgcaag tctcttggcc ggccttgcgc
     2821 cttggctatt gcttggcagc gcctatcgcc aggtattact ccaatcccga atatccgaga
     2881 tcgggatcac ccgagagaag ttcaacctac atcctcaatc ccgatctatc cgagatccga
     2941 ggaatatcga aatcggggcg cgccaagctg ttgtgaccgc ttgctctagc cagctatcga
     3001 gttgtgaacc gatccatcta gcaattggtc tcgatctagc gataggcttc gatctagcta
     3061 tgtagaaacg ccgtgtgctc gatcgcttga taaggtccac gtagctgcta taattgcttc
     3121 aacagaacat attgactatc cggtattacc cggcaaagga ggtagccaac atgacagctc
     3181 tgacagaggg agcgaaactg ttcgagaagg agatcccgta catcacagag ttggaaggag
     3241 atgtcgaagg gatgaaattc atcatcaagg gagaaggaac gggtgacgca acgaccggaa
     3301 cgatcaaggc caaatacatt tgtaccacgg gcgatttgcc tgtcccctgg gcgacgcttg
     3361 taagcacgct ctcgtatggt gtccagtgct tcgcgaaata tccatcgcac attaaggact
     3421 ttttcaagtc ggccatgcca gaaggttaca cacaagaacg aaccatctcc tttgaggggg
     3481 acggagtgta taagacacgc gcgatggtaa cgtacgagcg cgggtccatc tacaacaggg
     3541 taactcttac tggggagaac tttaagaagg acgggcatat cttgcggaaa aacgtggcat
     3601 ttcaatgtcc gccctcgatt ctgtatattc tcccggacac ggtgaataat gggatcagag
     3661 tggagttcaa ccaggcatac gatattgagg gtgtgactga aaagctcgtc accaaatgca
     3721 gccagatgaa tcgccccctt gcgggatcag cagccgtcca tatcccccgg tatcaccata
     3781 ttacctacca cacaaaactc tcaaaagaca gagatgagag aagggaccat atgtgcctgg
     3841 tcgaagtagt gaaggcggtg gatcttgata cataccagta ggctagccat tggcgcagaa
     3901 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg
     3961 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct
     4021 taagggcgcg cctggtgtac cgagaacgat cctctcagtg cgagtctcga cgatccatat
     4081 cgttgcttgg cagtcagcca gtcggaatcc agcttgggac ccaggaagtc caatcgtcag
     4141 atattgtact caagcctggt cacggcagcg taccgatctg tttaaaccta gatattgata
     4201 gtctgatcgg tcaacgtata atcgagtcct agcttttgca aacatctatc aagagacagg
     4261 atcagcagga ggctttcgca tgattgaaca agatggattg cacgcaggtt ctccggcggc
     4321 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4381 cgccgtgttc cggctgtcag cgcaggggcg tccggttctt tttgtcaaga ccgacctgtc
     4441 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg cgacgacggg
     4501 cgttccttgc gcggctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4561 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4621 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4681 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4741 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4801 caaggcgtct atgcccgacg gcgaggatct cgtcgtgacc cacggcgatg cctgcttgcc
     4861 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc gtctgggtgt
     4921 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4981 cgaatgggct gaccgcttcc ttgtgcttta cggtatcgcc gcgcccgatt cgcagcgcat
     5041 cgccttctat cgccttcttg acgagttctt ctgaccgatt ctaggtgcat tggcgcagaa
     5101 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg
     5161 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct
     5221 taaggtcgtt taaactcgac tctggctcta tcgaatctcc gtcgtttcga gcttacgcga
     5281 acagccgtgg cgctcatttg ctcgtcgggc atcgaatctc gtcagctatc gtcagcttac
     5341 ctttttggca
//
