LOCUS       sequence-372027-        8357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting plasmid for 2-plasmid C. difficile mutagenesis system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8357)
  AUTHORS   Brehm JN, Sorg JA
  TITLE     Plasmid Sequence and Availability for an Improved Clostridioides 
            difficile CRISPR-Cas9 Mutagenesis System.
  JOURNAL   Microbiol Resour Announc. 2022 Dec 15;11(12):e0083322. doi: 
            10.1128/mra.00833-22. Epub 2022 Nov 7.
  PUBMED    36342279
REFERENCE   2  (bases 1 to 8357)
  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: "Microbiol 
            Resour Announc."; date: "2022-12-15"; volume: "11(12):e0083322. 
            doi"; pages: " 10.1128/mra.00833-22. Epub 2022 Nov 7"
COMMENT     Sequence Label: pJB07
FEATURES             Location/Qualifiers
     source          1..8357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2116..2133)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2116..2132)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2125..2147)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        2447..2522
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      6026..6614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6515..6534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6768..6785
                     /label=L4440
                     /note="L4440 vector, forward primer"
     oriT            7710..7819
                     /note="incP origin of transfer"
     CDS             7852..8223
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
ORIGIN
        1 cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt
       61 atcaggaaac agctatgacc gcggccgcga cgtgattttt aatgggtaat ttaagtgtga
      121 aatttggaag tgtagaattt aaaaatccag taataatggc gtctggaact tttggatttg
      181 gaaaagaata taatgagatt tatgatatac aaaaacttgg tggtataagt agcaagggtc
      241 ttactttaaa taaaaaacct ggtaacaatg gtatgagagt tcatgagaca ccatccggta
      301 tgatgaatag tgttgggctt gaaaaccctg gagttcaagg atttatagac tatgaattac
      361 ctttttttag taaactagac ttagtcagaa tagctaatgt tggaggtggg acattagaag
      421 actacttatt aggggttcaa atgttaaatg acaagcctat agacattatt gaattaaata
      481 tatcttgccc aaatgtcaaa gctggtggaa tggcttttgg tataaaaaat gaagtggcaa
      541 gagaagttgt tcgtgaggta agaaacataa cgaaactgcc tttagtaata aaattatcac
      601 ccaatgcaga agatattgta ggaatggcaa aagtttgtga agaagaaggt gctgatggag
      661 tttcattagt taatactttt aaagcaatgg caattgatat aaaaaataga agaccagtat
      721 ttgaaaatgt atatgctgga ttatctggac cagctataaa acctatagct ttgagaatgg
      781 tacatgaagt gtgtaaaaat gtaaatatac ctgtaatggg aatgggcgga ataactaaag
      841 ccacagatgc tatagagttt ataatggcag gagctacttg tatccaagtt ggaactgcaa
      901 attttatgaa tcctagaata ggtattgaaa taatagatgg gattaatgaa tttatggata
      961 gagaaggaat aaaaagttta gacgaaataa gaggaattat ataaggtaga ataaatagta
     1021 ttttaaagat aaaaaatata gattaaataa acaataattt tataacatta acatggagga
     1081 aaaataatta aaaacttaat tatttatagt gttacttaaa aaatgtaaat tttttagagt
     1141 aaaaatttgt aatataacat ataacttaaa aataaaaagc agttagcctt ataaatgaag
     1201 gttagctgtt ttttttgtta aaaatatatt tacaaaataa gagatagtag cagtacaata
     1261 taagtataag gtaaaagtat tatatattag ggaggggtaa gtatgaaaaa tcaattaaat
     1321 gaaaatcata aaaaggttgt attttttgga gtaggtgctg taggagctac ttttgcagaa
     1381 caatttttta actctaaata tgattttaaa attctttgtg ataatgaaag aaagaaaaga
     1441 tatttagaag aaggatttat aataaatgga aaaaggtatg attttaatta tgtaactaaa
     1501 gatgagtata aacaagaggc tgattttata attataggtc taaaatataa taatttaaaa
     1561 gaaaatataa aagaattaga tggattagtt ggaaaaaata cagttataat gtctctgcta
     1621 aatggagttg atagcgaaga gataatagga gaaagatttg gaattggaaa aatggtatat
     1681 tcatatgtta ccaatataga tgcaaagaaa atcaataata atattataca cactactaat
     1741 gggataattg tatttggtaa caaagataat agtgaagata gaaaaactaa tataataact
     1801 gaagtctttg atgatgtaaa tatagaatat attttatcaa aagatattca acgagatatg
     1861 tggtggaagt acatggttaa tattggtgta aatcaaactt cagctatact tggtgcacct
     1921 tatggagttt ttcagagttc tgagcattta agagaattag caaaatctgc aatgagggaa
     1981 gttgttgcta tagcacaagc aaaagacata tctcttacag aagatgatgt tgaaaattca
     2041 ttacatagaa tactagaaca ttcaaaagaa ggaagaacat caatgcttct cgagcctgca
     2101 gacatgcaac ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2161 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2221 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctagct
     2281 gacaaagata attaaatatt ttattattag ttcataagtt agtttaatat actaacaaaa
     2341 ataaagcaag taaaatatac ctaaaatata aaaaaattag gataggaaaa cgatagttat
     2401 gaagtggcat tcaaggaggg ggtaccgaaa agtgatgcat tgttgggttt tagagctaga
     2461 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     2521 gctttttttc tatggagaaa tctagatcag catgatgtct gactagacgc gtgctagcat
     2581 aaaaataaga agcctgcatt tgcaggcttc ttatttttat ggcgcgccgt tctgaatcct
     2641 tagctaatgg ttcaacaggt aactatgacg aagatagcac cctggataag tctgtaatgg
     2701 attctaaggc atttaatgaa gacgtgtata taaaatgtgc taatgaaaaa gaaaatgcgt
     2761 taaaagagcc taaaatgagt tcaaatggtt ttgaaattga ttggtagttt aatttaatat
     2821 attttttcta ttggctatct cgatacctat agaatcttct gttcactttt gtttttgaaa
     2881 tataaaaagg ggctttttag cccctttttt ttaaaactcc ggaggagttt cttcattctt
     2941 gatactatac gtaactattt cgatttgact tcattgtcaa ttaagctagt aaaatcaatg
     3001 gttaaaaaac aaaaaacttg catttttcta cctagtaatt tataatttta agtgtcgagt
     3061 ttaaaagtat aatttaccag gaaaggagca agttttttaa taaggaaaaa tttttccttt
     3121 taaaattcta tttcgttata tgactaatta taatcaaaaa aatgaaaata aacaagaggt
     3181 aaaaactgct ttagagaaat gtactgataa aaaaagaaaa aatcctagat ttacgtcata
     3241 catagcacct ttaactacta agaaaaatat tgaaaggact tccacttgtg gagattattt
     3301 gtttatgttg agtgatgcag acttagaaca ttttaaatta cataaaggta atttttgcgg
     3361 taatagattt tgtccaatgt gtagttggcg acttgcttgt aaggatagtt tagaaatatc
     3421 tattcttatg gagcatttaa gaaaagaaga aaataaagag tttatatttt taactcttac
     3481 aactccaaat gtaaaaagtt atgatcttaa ttattctatt aaacaatata ataaatcttt
     3541 taaaaaatta atggagcgta aggaagttaa ggatataact aaaggttata taagaaaatt
     3601 agaagtaact taccaaaagg aaaaatacat aacaaaggat ttatggaaaa taaaaaaaga
     3661 ttattatcaa aaaaaaggac ttgaaattgg tgatttagaa cctaattttg atacttataa
     3721 tcctcatttt catgtagtta ttgcagttaa taaaagttat tttacagata aaaattatta
     3781 tataaatcga gaaagatggt tggaattatg gaagtttgct actaaggatg attctataac
     3841 tcaagttgat gttagaaaag caaaaattaa tgattataaa gaggtttacg aacttgcgaa
     3901 atattcagct aaagacactg attatttaat atcgaggcca gtatttgaaa ttttttataa
     3961 agcattaaaa ggcaagcagg tattagtttt tagtggattt tttaaagatg cacacaaatt
     4021 gtacaagcaa ggaaaacttg atgtttataa aaagaaagat gaaattaaat atgtctatat
     4081 agtttattat aattggtgca aaaaacaata tgaaaaaact agaataaggg aacttacgga
     4141 agatgaaaaa gaagaattaa atcaagattt aatagatgga atagaaatag attaaagtgt
     4201 aactatactt tatatatata tgattaaaaa aataaaaaac aacagcctat taggttgttg
     4261 ttttttattt tctttattaa tttttttaat ttttagtttt tagttctttt ttaaaataag
     4321 tttcagcctc tttttcaata ttttttaaag aaggagtatt tgcatgaatt gccttttttc
     4381 taacagactt aggaaatatt ttaacagtat cttcttgcgc cggtgatttt ggaacttcat
     4441 aacttactaa tttataatta ttattttctt ttttaattgt aacagttgca aaagaagctg
     4501 aacctgttcc ttcaactagt ttatcatctt caatataata ttcttgacct atatagtata
     4561 aatatatttt tattatattt ttactttttt ctgaatctat tattttataa tcataaaaag
     4621 ttttaccacc aaaagaaggt tgtactcctt ctggtccaac atattttttt actatattat
     4681 ctaaataatt tttgggaact ggtgttgtaa tttgattaat cgaacaacca gttatactta
     4741 aaggaattat aactataaaa atatatagga ttatcttttt aaatttcatt attggcctcc
     4801 tttttattaa atttatgtta ccataaaaag gacataacgg gaatatgtag aatattttta
     4861 atgtagacaa aattttacat aaatataaag aaaggaagtg tttgtttaaa ttttatagca
     4921 aactatcaaa aattaggggg ataaaaattt atgaaaaaaa ggttttcgat gttattttta
     4981 tgtttaactt taatagtttg tggtttattt acaaattcgg ccggccgaag caaacttaag
     5041 agtgtgttga tagtgcagta tcttaaaatt ttgtataata ggaattgaag ttaaattaga
     5101 tgctaaaaat ttgtaattaa gaaggagtga ttacatgaac aaaaatataa aatattctca
     5161 aaacttttta acgagtgaaa aagtactcaa ccaaataata aaacaattga atttaaaaga
     5221 aaccgatacc gtttacgaaa ttggaacagg taaagggcat ttaacgacga aactggctaa
     5281 aataagtaaa caggtaacgt ctattgaatt agacagtcat ctattcaact tatcgtcaga
     5341 aaaattaaaa ctgaatactc gtgtcacttt aattcaccaa gatattctac agtttcaatt
     5401 ccctaacaaa cagaggtata aaattgttgg gagtattcct taccatttaa gcacacaaat
     5461 tattaaaaaa gtggtttttg aaagccatgc gtctgacatc tatctgattg ttgaagaagg
     5521 attctacaag cgtaccttgg atattcaccg aacactaggg ttgctcttgc acactcaagt
     5581 ctcgattcag caattgctta agctgccagc ggaatgcttt catcctaaac caaaagtaaa
     5641 cagtgtctta ataaaactta cccgccatac cacagatgtt ccagataaat attggaagct
     5701 atatacgtac tttgtttcaa aatgggtcaa tcgagaatat cgtcaactgt ttactaaaaa
     5761 tcagtttcat caagcaatga aacacgccaa agtaaacaat ttaagtaccg ttacttatga
     5821 gcaagtattg tctattttta atagttatct attatttaac gggaggaaat aattctatga
     5881 gtcgcttttg taaatttgga aagttacacg ttactaaagg gaatgtgttt aaactccttt
     5941 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6001 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6061 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6721 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6781 gcgaggaagc ggaagagcgc ccaatacgca gggccctaac atcttctatt tttcccaaat
     6841 ccttacatct cccctaattt ttagacttaa gggcgaattc caaaggacga atatgtcgcc
     6901 taaagcataa tgctctgaat aagaatagaa accatagtcc tcatgaagaa aataggacag
     6961 tttcagttgt aagatgtttt cgaccacctg ctgtacgtct gttgtcggaa agcgaatcct
     7021 tacataatca aacagcattt caaggggagc gtcgggattg aagcgttcca gagcttccca
     7081 aagggactgc tgtaaatcct ctgatggctt gacttttcct gtttcaatat cgcttagata
     7141 ctgccttgta ataccagtcg caacagctaa acggttttga gatagtccat aagccaagcg
     7201 tttttctttt aaatgctgta accaagtttg ttcattcagt aaaaatccct ccaatcaaaa
     7261 aggcgtatgt caacttttaa agcccatttg acatacgctg aaattttgta aatcccttgt
     7321 aaccaaagga ttttctaatg tttttttgac tgtttcctgt cgatttgtac ccccctgtta
     7381 gatacggggg gttaagtgct ggcgtggcta ttgccacacc agccagcaag atcagtccac
     7441 acctgcgact tccgcttcgc acgtcgcctg cgtggactgt ctgctgttgg ataacttttt
     7501 aatttcctcc aagaaatcat atccttttgg tacaagggga gtataaaaga attctgcaga
     7561 ttacctaata atttatctac attcccttta gcctgcttcg gggtcattat agcgattttt
     7621 tcggtatatc catccttttt cgcacgatat acaggatttt gccaaagggt tcgtgtagac
     7681 tttccttggt gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg
     7741 agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct
     7801 gctctgcgag gctggccggc taccgccggc gtaacagatg agggcaagcg gatggctgat
     7861 gaaaccaagc caaccaggaa gggcagccca cctatcaagg tgtactgcct tccagacgaa
     7921 cgaagagcga ttgaggaaaa ggcggcggcg gccggcatga gcctgtcggc ctacctgctg
     7981 gccgtcggcc agggctacaa aatcacgggc gtcgtggact atgagcacgt ccgcgagctg
     8041 gcccgcatca atggcgacct gggccgcctg ggcggcctgc tgaaactctg gctcaccgac
     8101 gacccgcgca cggcgcggtt cggtgatgcc acgatcctcg ccctgctggc gaagatcgaa
     8161 gagaagcagg acgagcttgg caaggtcatg atgggcgtgg tccgcccgag ggcagagcca
     8221 tgactttttt agccgctaaa acggccgggg ggtgcgcgtg attgccaagc acgtccccat
     8281 gcgctccatc aagaagagcg acttcgcgga gctggtgaag tacatcaccg acgagcaagg
     8341 caagaccgat cgggccc
//
