LOCUS       sequence-268044-        5350 bp    DNA     circular SYN 14-APR-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 Apr 14, 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"
     polyA_signal    155..276
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(192..211)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     246..265
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1158..1206
                     /note="synthetic polyadenylation signal"
     terminator      1224..1270
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    1275..1346
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(1491..1627)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             complement(1640..2434)
                     /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             complement(1640..2434)
                     /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(1751..1770)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2361..2380
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     terminator      complement(2691..2826)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     CDS             complement(2833..3543)
                     /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"
     promoter        complement(3560..3628)
                     /label=recA (Delta-LexA) promoter
                     /note="constitutively active E. coli recA promoter lacking 
                     the LexA binding site (Brent and Ptashne, 1981)"
     rep_origin      4248..4836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4737..4756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5062..5133
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(5178..5314)
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     terminator      complement(join(5344..5350,1..40))
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cccctcaaga cccgtttaga ggccccaagg ggttatgcta cgataccgat agagatgggc
       61 caataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
      121 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
      181 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
      241 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtgagc tgaaggtacg
      301 ctgtatctca gtcagtcaag ctagcagagg cctgcggatc cagaaatcga ttgtatcagt
      361 cagtcagtgc aggaggagac aacttctaga aacctcgagg tagatatcgc ggtaccccat
      421 ggtgggtacc tcctttgaat tcgagagctc gaattcccac aacatcagca aggagaaagg
      481 ggctaccggc gaaccagcag cccctttata aaggcgcttc agtagtcaga ccagcatcag
      541 tcctgaaaag gcgggcctgc gcccgcctcc aggttgctac ttaccggatt cgtaagccat
      601 gaaagccgcc acctccctgt gtccgtctct gtaacgaatc tcgcacagcg attttcgtgt
      661 cagataagtg aatatcaaca gtgtgagaca cacgatcaac acacaccaga caagggaact
      721 tcgtggtagt ttcatggcct tcttctcctt gcgcaaagcg cggtaagagg ctatcctgat
      781 gtggactaga catagggatg cctcgtggtg gttaatgaaa attaacttac tacggggcta
      841 tcttctttct gccacacaac acggcaacaa accaccttca cgtcatgagg cagaaagcct
      901 caagcgccgg gcacatcata gcccatatac ctgcacgctg accacactca ctttccctga
      961 aaataatccg ctcattcaga ccgttcacgg gaaatccgtg tgattgttgc cgcatcacgc
     1021 tgcctcccgg agtttgttcg gccggatcct ctagagtcga cctgcaggca tgcaagcttg
     1081 gcagcggccg ctggcgggtg tgtcgagtgg atggtaggat cgacaaagat ctggctacac
     1141 tcgatcagca gttagataat aaaatcgcta tccatcgaag atggatgtgt gttggttttt
     1201 tgtgtgtgta acgcaacgat tgatagcata accccttggg gcctctaaac gggtcttgag
     1261 gggttttttg aattgagagg tagctgaagc tgctgttatg accactagag ggaagaagat
     1321 acctgtggag ctaatggtcc aagatgtcgg gagccgcgat cgctgccaaa aaggtaagct
     1381 gacgatagct gacgagattc gatgcccgac gagcaaatga gcgccacggc tgttcgcgta
     1441 agctcgaaac gacggagatt cgatagagcc agagtcgagt ttaaacgacc ttaaggataa
     1501 atttctggta aggaggacac gtatggaagt gggcaagttg gggaagccgt atccgttgct
     1561 gaatctggca tatgtgggag tataagacgc gcagcgtcgc atcaggcatt tttttctgcg
     1621 ccaatgcacc tagaatcggt cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     1681 gcgaatcggg cgcggcgata ccgtaaagca caaggaagcg gtcagcccat tcgccgccaa
     1741 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1801 gacggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     1861 aggcatcgcc gtgggtcacg acgagatcct cgccgtcggg catagacgcc ttgagcctgg
     1921 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     1981 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     2041 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     2101 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     2161 gccagtccct tcccgcttca gtgacaacgt cgagcacagc cgcgcaagga acgcccgtcg
     2221 tcgccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt
     2281 cggtcttgac aaaaagaacc ggacgcccct gcgctgacag ccggaacacg gcggcatcag
     2341 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagccgccg
     2401 gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa gcctcctgct gatcctgtct
     2461 cttgatagat gtttgcaaaa gctaggactc gattatacgt tgaccgatca gactatcaat
     2521 atctaggttt aaacagatcg gtacgctgcc gtgaccaggc ttgagtacaa tatctgacga
     2581 ttggacttcc tgggtcccaa gctggattcc gactggctga ctgccaagca acgatatgga
     2641 tcgtcgagac tcgcactgag aggatcgttc tcggtacacc aggcgcgccc ttaaggataa
     2701 atttctggta aggaggacac gtatggaagt gggcaagttg gggaagccgt atccgttgct
     2761 gaatctggca tatgtgggag tataagacgc gcagcgtcgc atcaggcatt tttttctgcg
     2821 ccaatggcta gcctactggt atgtatcaag atccaccgcc ttcactactt cgaccaggca
     2881 catatggtcc cttctctcat ctctgtcttt tgagagtttt gtgtggtagg taatatggtg
     2941 ataccggggg atatggacgg ctgctgatcc cgcaaggggg cgattcatct ggctgcattt
     3001 ggtgacgagc ttttcagtca caccctcaat atcgtatgcc tggttgaact ccactctgat
     3061 cccattattc accgtgtccg ggagaatata cagaatcgag ggcggacatt gaaatgccac
     3121 gtttttccgc aagatatgcc cgtccttctt aaagttctcc ccagtaagag ttaccctgtt
     3181 gtagatggac ccgcgctcgt acgttaccat cgcgcgtgtc ttatacactc cgtccccctc
     3241 aaaggagatg gttcgttctt gtgtgtaacc ttctggcatg gccgacttga aaaagtcctt
     3301 aatgtgcgat ggatatttcg cgaagcactg gacaccatac gagagcgtgc ttacaagcgt
     3361 cgcccagggg acaggcaaat cgcccgtggt acaaatgtat ttggccttga tcgttccggt
     3421 cgttgcgtca cccgttcctt ctcccttgat gatgaatttc atcccttcga catctccttc
     3481 caactctgtg atgtacggga tctccttctc gaacagtttc gctccctctg tcagagctgt
     3541 catgttggct acctcctttg ccgggtaata ccggatagtc aatatgttct gttgaagcaa
     3601 ttatagcagc tacgtggacc ttatcaagcg atcgagcaca cggcgtttct acatagctag
     3661 atcgaagcct atcgctagat cgagaccaat tgctagatgg atcggttcac aactcgatag
     3721 ctggctagag caagcggtca caacagcttg gcgcgccccg atttcgatat tcctcggatc
     3781 tcggatagat cgggattgag gatgtaggtt gaacttctct cgggtgatcc cgatctcgga
     3841 tattcgggat tggagtaata cctggcgata ggcgctgcca agcaatagcc aaggcgcaag
     3901 gccggccaag agacttgcga tagttcgttc gagaggtagc ctgcaagatg gatcggacga
     3961 aaggtttgaa gattttccga cgagggcctt ggagagttcg gaaatttaaa tgcaactatg
     4021 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4081 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4141 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4201 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4261 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4321 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4381 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4441 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4501 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4561 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4621 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4681 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4741 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4801 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4861 cggttcctgg ccttttgctg gccttttgct cacatgattt aaatgaaact cgtaatcgga
     4921 gaacgggtgc aatagcaagg accagatcga acttgactga cgcaattctg agcgtagtcg
     4981 aaacccagac aagtcgatct cgccaagtag ctgacttgac tggttaatta agtttcgata
     5041 gcccaaggta accaatgaga ttagggaggg aagaaagttt gatgaactac ttctgacccc
     5101 tagtggtgtc cagaaaagac cattaaagga atgtcttgat cgaaatcgag agatcgcctg
     5161 caggatagct gacgacctta aggataaatt tctggtaagg aggacacgta tggaagtggg
     5221 caagttgggg aagccgtatc cgttgctgaa tctggcatat gtgggagtat aagacgcgca
     5281 gcgtcgcatc aggcattttt ttctgcgcca atgccggtag atagcaatcc ggcccgaggg
     5341 gcacaaaaaa
//
