LOCUS       sequence-384161-        4423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replicating plasmid with sgRNA cassette containing a killing spacer 
            #2 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-KS2
FEATURES             Location/Qualifiers
     source          1..4423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             924..1928
                     /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"
     protein_bind    2595..2611
                     /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)."
     primer_bind     2619..2635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3670..3691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3706..3711,3712..3729,3730..3736)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3744..3760
                     /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)."
     primer_bind     3768..3784
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      join(4067..4423,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagta tgggaaacaa
      481 aatattgcgt atgcgactgt tcacatggac gagaaaaccc ctcacatgca tttaggagtt
      541 gttcctatgc gcctagaggg cttttcgtgc gtcagcatga gcgatcggaa gaaaagaaga
      601 atactttttc gcttcaagat gttttgcagc gtgatcgaga acttcgtgag caaagaaaag
      661 caaagaggaa aaaatcgcat gatttggagc gataagaaaa agcactcgaa tgagtgcttt
      721 ttttgcgttt tgagcgtagc gaaaaacgag ttctttctat tcttgataca tatagaaata
      781 acgtcatttt tattttagtt gctgaaaggt gcgttgaagt gttggtatgt atgtgtttta
      841 aagtattgaa aacccttaaa attggttgca cagaaaaacc ccatctgtta aagttataag
      901 tgaccaaaca aataactaaa tagatggggg tttcttttaa tattatgtgt cctaatagta
      961 gcatttattc agatgaaaaa tcaagggttt tagtggacaa gacaaaaagt ggaaaagtga
     1021 gaccatggag agaaaagaaa atcgctaatg ttgattactt tgaacttctg catattcttg
     1081 aatttaaaaa ggctgaaaga gtaaaagatt gtgctgaaat attagagtat aaacaaaatc
     1141 gtgaaacagg cgaaagaaag ttgtatcgag tgtggttttg taaatccagg ctttgtccaa
     1201 tgtgcaactg gaggagagca atgaaacatg gcattcagtc acaaaaggtt gttgctgaag
     1261 ttattaaaca aaagccaaca gttcgttggt tgtttctcac attaacagtt aaaaatgttt
     1321 atgatggcga agaattaaat aagagtttgt cagatatggc tcaaggattt cgccgaatga
     1381 tgcaatataa aaaaattaat aaaaatcttg ttggttttat gcgtgcaacg gaagtgacaa
     1441 taaataataa agataattct tataatcagc acatgcatgt attggtatgt gtggaaccaa
     1501 cttattttaa gaatacagaa aactacgtga atcaaaaaca atggattcaa ttttggaaaa
     1561 aggcaatgaa attagactat gatccaaatg taaaagttca aatgattcga ccgaaaaata
     1621 aatataaatc ggatatacaa tcggcaattg acgaaactgc aaaatatcct gtaaaggata
     1681 cggattttat gaccgatgat gaagaaaaga atttgaaacg tttgtctgat ttggaggaag
     1741 gtttacaccg taaaaggtta atctcctatg gtggtttgtt aaaagaaata cataaaaaat
     1801 taaaccttga tgacacagaa gaaggcgatt tgattcatac agatgatgac gaaaaagccg
     1861 atgaagatgg attttctatt attgcaatgt ggaattggga acggaaaaat tattttatta
     1921 aagagtagtt caaggatcct taatatgccg accacgttgc aattcccgtc aaataatgca
     1981 ttttgcagcc gacgaaacag gcaagataac tgtattggct ataaatgttt caggcagcgg
     2041 tatattttgc ctcccggtaa aattaataca ataagctaaa aaactgacgt aggataagca
     2101 aaacggcgca atttgagttg taacgtaata ttttcactaa aaatagtaat tatttcatgt
     2161 tgtttttttt tagattaatt tataatataa tttattgtat aagcaatatc ttaattatca
     2221 ttaaaggggg aaaaaaactg tttgtatcgt acctatgagg aattgaaacg ggaagtgcgt
     2281 gtgaagcggg agagctttac cgtgcggttt gtatcgtacc tatgaggaat tgaaactttc
     2341 tggctactcc aagttcctaa tgttatatat ggttttatag tataaagcct taagcgaaaa
     2401 tgaataggac tgtataataa ggttaaggct ttatattcgc ctgaagcgta aaaacgcaat
     2461 aggaagaata aaatttttgc cggggatagg ccagccttgc aaactggctg acaagcgaag
     2521 tctccccgct tccccggcaa caatttcaaa tagggagaca gaaagggaga cgaaatctcg
     2581 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2641 ttacgcctaa tagaaataat tttgtttaac tttacaaacg ggattgactt ttaaaaaagg
     2701 attgattcta atgaagaaag cagacaagta agcctcctaa attcacttta gataaaaatt
     2761 taggaggcat atcaaatggt gaatggacca ataataatga ctagagaaga aagaatgaag
     2821 attgttcatg aaattaagga acgaatattg gataaatatg gggatgatgt taaggctatt
     2881 ggtgtttatg gctctcttgg tcgtcagact gatgggccct attcggatat tgagatgatg
     2941 tgtgtcatgt caacagagga agcagagttc agccatgaat ggacaaccgg tgagtggaag
     3001 gtggaagtga attttgatag cgaagagatt ctactagatt atgcatctca ggtggaatca
     3061 gattggccgc ttacacatgg tcaatttttc tctattttgc cgatttatga ttcaggtgga
     3121 tacttagaga aagtgtatca aactgctaaa tcggtagaag cccaaacgtt ccacgatgcg
     3181 atttgtgccc ttatcgtaga agagctgttt gaatatgcag gcaaatggcg taatattcgt
     3241 gtgcaaggac cgacaacatt tctaccatcc ttgactgtac aggtagcaat ggcaggtgcc
     3301 atgttgattg gtctgcatca tcgcatctgt tatacgacga acgcttcggt cttaactgaa
     3361 gcagttaagc aatcagatct tccttcaggt tatgaccatc tgtgccagtt cgtaatgtct
     3421 ggtcaacttt ccgactctga gaaacttctg gaatcgctag agaatttctg gaatgggatt
     3481 caggagtgga cagaacgaca cggatatata gtggatgtgt caaaacgcat accattttga
     3541 tatgagataa tgccgactgt actttttaca gtcggttttc taatgtcact agggctcgcc
     3601 tttgggaagt ttgaagggct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     3661 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     3721 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     3781 tgaccatgat tacgccaagc ttgcatgcct gcaggtcgac tctagaggat ccccgggtac
     3841 cgagctcgaa ttgacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3901 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3961 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4081 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4141 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4201 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4261 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4321 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4381 gctgaacggg gggttcgtgc acacagccca gcttggagcg aac
//
