LOCUS       sequence-360126-        4423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replicating plasmid with sgRNA cassette containing a killing spacer 
            #3 targeting the wild type polC gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4423)
  AUTHORS   Lanahan A, Zakowicz K, Tian L, Olson DG, Lynd LR
  TITLE     A Single Nucleotide Change in the polC DNA Polymerase III in 
            Clostridium thermocellum Is Sufficient To Create a Hypermutator 
            Phenotype.
  JOURNAL   Appl Environ Microbiol. 2022 Jan 11;88(1):e0153121. doi: 
            10.1128/AEM.01531-21. Epub 2021 Oct 20.
  PUBMED    35015978
REFERENCE   2  (bases 1 to 4423)
  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: "Appl 
            Environ Microbiol."; date: "2022-01-11"; volume: "88(1):e0153121. 
            doi"; pages: " 10.1128/AEM.01531-21. Epub 2021 Oct 20"
COMMENT     Sequence Label: pDGO186N-KS3
FEATURES             Location/Qualifiers
     source          1..4423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..27)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    35..51
                     /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             complement(718..1722)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     primer_bind     complement(2243..2260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2414..3002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2494..2513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3285..3301)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3309..3325
                     /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)."
     promoter        complement(join(3333..3339,3340..3357,3358..3363))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3378..3399
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
       61 acatacgaga tttcgtctcc ctttctgtct ccctatttga aattgttgcc ggggaagcgg
      121 ggagacttcg cttgtcagcc agtttgcaag gctggcctat ccccggcaaa aattttattc
      181 ttcctattgc gtttttacgc ttcaggcgaa tataaagcct taaccttatt atacagtcct
      241 attcattttc gcttaaggct ttatactata aaaccatata taacattagg aacttggagt
      301 agccagaaag tttcaattcc tcataggtac gatacaaacg tgcgtgtgaa gcgggagagc
      361 tttaccgtgc gattctgttt caattcctca taggtacgat acaaacagtt tttttccccc
      421 tttaatgata attaagatat tgcttataca ataaattata ttataaatta atctaaaaaa
      481 aaacaacatg aaataattac tatttttagt gaaaatatta cgttacaact caaattgcgc
      541 cgttttgctt atcctacgtc agttttttag cttattgtat taattttacc gggaggcaaa
      601 atataccgct gcctgaaaca tttatagcca atacagttat cttgcctgtt tcgtcggctg
      661 caaaatgcat tatttgacgg gaattgcaac gtggtcggca tattaaggat ccttgaacta
      721 ctctttaata aaataatttt tccgttccca attccacatt gcaataatag aaaatccatc
      781 ttcatcggct ttttcgtcat catctgtatg aatcaaatcg ccttcttctg tgtcatcaag
      841 gtttaatttt ttatgtattt cttttaacaa accaccatag gagattaacc ttttacggtg
      901 taaaccttcc tccaaatcag acaaacgttt caaattcttt tcttcatcat cggtcataaa
      961 atccgtatcc tttacaggat attttgcagt ttcgtcaatt gccgattgta tatccgattt
     1021 atatttattt ttcggtcgaa tcatttgaac ttttacattt ggatcatagt ctaatttcat
     1081 tgcctttttc caaaattgaa tccattgttt ttgattcacg tagttttctg tattcttaaa
     1141 ataagttggt tccacacata ccaatacatg catgtgctga ttataagaat tatctttatt
     1201 atttattgtc acttccgttg cacgcataaa accaacaaga tttttattaa tttttttata
     1261 ttgcatcatt cggcgaaatc cttgagccat atctgacaaa ctcttattta attcttcgcc
     1321 atcataaaca tttttaactg ttaatgtgag aaacaaccaa cgaactgttg gcttttgttt
     1381 aataacttca gcaacaacct tttgtgactg aatgccatgt ttcattgctc tcctccagtt
     1441 gcacattgga caaagcctgg atttacaaaa ccacactcga tacaactttc tttcgcctgt
     1501 ttcacgattt tgtttatact ctaatatttc agcacaatct tttactcttt cagccttttt
     1561 aaattcaaga atatgcagaa gttcaaagta atcaacatta gcgattttct tttctctcca
     1621 tggtctcact tttccacttt ttgtcttgtc cactaaaacc cttgattttt catctgaata
     1681 aatgctacta ttaggacaca taatattaaa agaaaccccc atctatttag ttatttgttt
     1741 ggtcacttat aactttaaca gatggggttt ttctgtgcaa ccaattttaa gggttttcaa
     1801 tactttaaaa cacatacata ccaacacttc aacgcacctt tcagcaacta aaataaaaat
     1861 gacgttattt ctatatgtat caagaataga aagaactcgt ttttcgctac gctcaaaacg
     1921 caaaaaaagc actcattcga gtgctttttc ttatcgctcc aaatcatgcg attttttcct
     1981 ctttgctttt ctttgctcac gaagttctcg atcacgctgc aaaacatctt gaagcgaaaa
     2041 agtattcttc ttttcttccg atcgctcatg ctgacgcacg aaaagccctc taggcgcata
     2101 ggaacaactc ctaaatgcat gtgaggggtt ttctcgtcca tgtgaacagt cgcatacgca
     2161 atattttgtt tcccatactg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2221 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2281 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2341 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2821 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3121 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3181 gcttaatcag tgaggcacct atctcagcga tctgtcaatt cgagctcggt acccggggat
     3241 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     3301 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3361 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3421 gctttccagt cgggaaacct gtcgtgccag cccttcaaac ttcccaaagg cgagccctag
     3481 tgacattaga aaaccgactg taaaaagtac agtcggcatt atctcatatc aaaatggtat
     3541 gcgttttgac acatccacta tatatccgtg tcgttctgtc cactcctgaa tcccattcca
     3601 gaaattctct agcgattcca gaagtttctc agagtcggaa agttgaccag acattacgaa
     3661 ctggcacaga tggtcataac ctgaaggaag atctgattgc ttaactgctt cagttaagac
     3721 cgaagcgttc gtcgtataac agatgcgatg atgcagacca atcaacatgg cacctgccat
     3781 tgctacctgt acagtcaagg atggtagaaa tgttgtcggt ccttgcacac gaatattacg
     3841 ccatttgcct gcatattcaa acagctcttc tacgataagg gcacaaatcg catcgtggaa
     3901 cgtttgggct tctaccgatt tagcagtttg atacactttc tctaagtatc cacctgaatc
     3961 ataaatcggc aaaatagaga aaaattgacc atgtgtaagc ggccaatctg attccacctg
     4021 agatgcataa tctagtagaa tctcttcgct atcaaaattc acttccacct tccactcacc
     4081 ggttgtccat tcatggctga actctgcttc ctctgttgac atgacacaca tcatctcaat
     4141 atccgaatag ggcccatcag tctgacgacc aagagagcca taaacaccaa tagccttaac
     4201 atcatcccca tatttatcca atattcgttc cttaatttca tgaacaatct tcattctttc
     4261 ttctctagtc attattattg gtccattcac catttgatat gcctcctaaa tttttatcta
     4321 aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc ttttttaaaa
     4381 gtcaatcccg tttgtaaagt taaacaaaat tatttctatt agg
//
