LOCUS       sequence-466415-        7815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA cloning backbone for expression of sgRNA in C. difficile. 
            Contains TA system to enable maintenence without antibiotic 
            selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7815)
  AUTHORS   Chua M, Erickson D, Collins J
  TITLE     Plasmid sequences and availability of a two-plasmid system for 
            CRISPRi knockdown of Clostridioides difficile genes without 
            antibiotic selection.
  JOURNAL   Microbiol Resour Announc. 2025 Apr 28:e0011625. doi: 
            10.1128/mra.00116-25.
  PUBMED    40293262
REFERENCE   2  (bases 1 to 7815)
  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. 2025 Apr 28:e0011625. doi: 10.1128/mra.00116-25."
COMMENT     Sequence Label: pJC.15A.sgRNA.TA
FEATURES             Location/Qualifiers
     source          1..7815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    67..85
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     misc_feature    968..986
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(1362..1733)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1766..1875
                     /note="incP origin of transfer"
     primer_bind     complement(3066..3083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3195..3740)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(3275..3294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcaaaaaac cccgcttcgg cggggttttt tcgcaattcg gtcgacagat cttcgtcggc
       61 agcgtcagat gtgtataaga gacagccatt ctacagttta ttcttgacat tgcactgtcc
      121 ccctggtata ataactatat gagacgagga ggaaaattaa tgagcaaagg agaagaagat
      181 aacatggcaa tcatcaaaga atttatgcgt ttcaaagttc acatggaagg ttctgtaaac
      241 ggacacgaat ttgaaattga aggtgaaggt gaaggccgtc cttatgaagg aacacaaacg
      301 gcaaagctga aagtaacaaa aggcggaccg cttccgtttg catgggatat cctttctccg
      361 caattcatgt acggttcaaa agcatacgtg aagcatccgg ctgatattcc tgattatttg
      421 aagctgtcat tccctgaagg cttcaaatgg gagcgtgtga tgaactttga agatggcggt
      481 gttgttactg ttactcaaga ttcaagcctt caagacggtg aatttattta caaagtgaag
      541 ctgcgcggaa caaacttccc atctgacgga cctgtcatgc aaaagaaaac aatgggctgg
      601 gaagcaagct ctgaacgcat gtatccagag gacggtgctt taaaaggaga aatcaaacag
      661 cgtttgaagc tgaaagacgg cggacactat gacgctgaag tgaaaacaac ttacaaagcg
      721 aaaaagccgg ttcagcttcc aggtgcttac aacgtaaaca tcaaacttga tattacaagc
      781 cacaatgaag attatacgat tgttgaacaa tatgaacgcg ctgaaggccg tcattcaact
      841 ggcggaatgg atgagcttta caaataacgt ctcggtttaa gagctatgct ggaaacagca
      901 tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      961 tttttttctg tctcttatac acatctccga gcccacgcaa caactctcct ggcgcaccgg
     1021 atcctataag ttttaataaa actttaaata gaaaaaggct tctctcatga gaagtctttt
     1081 ttatttaaaa taaatataaa ataaaataga ggctataaat agcctctatt ttatgtgaga
     1141 aatccctaaa taaaaagatg ccagtgtgct ggaattcgcc cttaggctgc gctagggacc
     1201 tctttagctc cttggaagct gtcagtagta ggggatcggt cttgccttgc tcgtcggtga
     1261 tgtacttcac cagctccgcg aagtcgctct tcttgatgga gcgcatgggg acgtgcttgg
     1321 caatcacgcg caccccccgg ccgttttagc ggctaaaaaa gtcatggctc tgccctcggg
     1381 cggaccacgc ccatcatgac cttgccaagc tcgtcctgct tctcttcgat cttcgccagc
     1441 agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt gagccagagt
     1501 ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc gggccagctc gcggacgtgc
     1561 tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag gtaggccgac
     1621 aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc tggaaggcag
     1681 tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc catccgcttg
     1741 ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt cccgttgagc
     1801 accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg tgggcctact
     1861 tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac acgaaccctt
     1921 tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg
     1981 accccgaagc aggaagggcg aattctgcag atctgaccgg tctctgaaaa tataaaaacc
     2041 acagattgat actaaaacct tggttgtgtt gcttttcggg gcttaaatca aggaaaaatc
     2101 cttgttttaa gcctttcaaa aagaaacaca aggtctttgt actaacctgt ggttatgtat
     2161 aaaattgtag attttagggt aacaaaaaac accgtatttc tacgatgttt ttgcttaaat
     2221 acttgttttt agttacagac aaacctgaag ttaactattt atcaattcct gcaattcgtt
     2281 tacaaacctg caaatgtgaa atccgtcaca tactgcgtga tgaacttgaa ttgccaaagg
     2341 aagtataatt ttgttatctt ctttataata cttacccata gtaaaaatag gaatcaaata
     2401 atcatatcct ttctgcaaat tcaaattaaa tccatcgaaa gttgaccaag gtatcataga
     2461 tacattaaaa atgttttcag gagcatttgg ctttccttcc attctatgat tgtttccata
     2521 cctttgagta tcactttcat aatctgctaa aaatgactta aagtcagact tacactcagt
     2581 ccaaagactt gaaaatgttt cagtatcatt gtgaaatata gtatatgatg gtatcatctc
     2641 atcatatatt cccaattcac catcttgatt tattgctgtc ctaaactctg aatgtctgtt
     2701 tacaatcatt gcaatataat aaagcattgc aggatatagt ttcattccct tttcctttat
     2761 ttgtgttata tcaactttaa ctgtcattga gtatgtgcaa ggaacacttg caaagtagtg
     2821 gtcaaaatac tcttttctgt tccaactatt tttatcaatt ttttcaaata ccatctaagt
     2881 tccctctcaa attcaagttt atcgctctaa tgaacaaaga tattatacca catttttgtg
     2941 aatttttcaa cttgcccact tcgactgcac tcccgactta ataacttctt gaacacttgc
     3001 cgaaaaagaa aaactgccgg gtacgtaccc ggggatcgat ccccggccga gcgctcttcc
     3061 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3121 cactcaaagg cggtaatacg gttcggccta ggagatactt aacagggaag tgagagggcc
     3181 gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga aatctgacgc
     3241 tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggc
     3301 ggctcccccg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta
     3361 tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa
     3421 gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta
     3481 tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa
     3541 ttgatttaga ggagttagtc ttgaagccat gcgccggtta aggctaaact gaaaggacaa
     3601 gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga
     3661 gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc
     3721 gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat ttctaggatc
     3781 taaaacacta ggccccgtca ggatggcctt ctgcttaatt tgctatttta gaatctagca
     3841 tttccaatgc ttactaatac tatttttgat ttttaacttg ttttaaaaaa gcacctacta
     3901 attaggattc tagctctata ttctagttat acattctcta tattctagct ctatattcta
     3961 gttatacatt ctctatattc tagctctata ttctagttat acattctcta tattctagct
     4021 ctatattcta gttatacatt ctctatattc tagctctata ttctagttat acattctcta
     4081 tattctagct ctatattcta gttatacatt ctctatattc tagctctata ttctagttat
     4141 acattctcta aaaaacatat atatatatac tacatatata tgtacaatta ttttaccaaa
     4201 agaaattttt aaaacggctt aatttcaata ctttgatgcc attttttttt ttgcaattct
     4261 cgtacaatta ttttaccaaa agaaattttt attaattagt ttttttgcat aaaaatagga
     4321 ctagaaaatc aattctaatc ctatcaaaaa tctacttgtt taatctatat gtatagtaca
     4381 taaatcctct atcatttatt ttagtttccc atacatctaa aattttagta tctaataaag
     4441 attctatcat atttgaaatt ctttgctgtg ttactcctaa atcttttgct aattcctctt
     4501 gattaattct taatatattt ccttttattt tatcatcttt gaccaataaa tcttgcttat
     4561 atctcatatg agtatataat cttaattctt cagttgaaat tattccccaa attactgcca
     4621 tggtaacaaa gtaactaata gtatattgtt tatctatatt acatctaact tcttttacat
     4681 tgtatgtatc ttttattcct agctttgtaa caccttttgt ttttgtaata taatcattat
     4741 gttgaagttc ttttaatgtt tctcttaatg tcttttcact cataacaatg tttttaactt
     4801 tcttcttacg tttataggtt atcagctcca ttatatcgtc tatgtttaat tctctatctt
     4861 tattgttaag taacacatta tagataaaca attgattacc agtcattatt ttaacccctt
     4921 tcctattctt atatttcaaa tcctttgagt gcttatgtgg catattgaac aaagtatctt
     4981 catcactata tataaaatct gattctattt tattccaaca atcactatta aactcacaac
     5041 ctgtacaacc aaaattaacc gttttaagct tctcatacat atacttgact tgatattcta
     5101 gctcactatt tgaaagacta ttttgtgata tatagttcca ttctctagca acggatatta
     5161 cttgacttaa acttttattc ctcaatctta aagtcacaac tattttttgc aaatctaggt
     5221 ttttatgtcc atctttacta ccatttttta aaatatctac tatacaattt ggtagcttac
     5281 tatctaatat atattttgta ttagtattac ctactgtttt tacatctttg caatattttt
     5341 tagctaactt ttctatgtca tatctatata tatttttgtc ttggtgtatt atttttactt
     5401 gtttagtagt atttttaata ttatatgtat aaggtactct taatacttga gtagataaat
     5461 ttgcttgttt atctgcaccc aatttattta ttaatgtatt tgtaacttct gatactaact
     5521 taatgttatt agttttatta attagcacat aaacatggaa tccatttcca ctatcaacta
     5581 ggatatgata atgtaatcct atcttagtaa ataaattaat tatatcttta taattaaaac
     5641 cttgtcctaa ttcttttttg tcaaaatcaa atcctaatgc atatctattt attaagtttt
     5701 cattggttcc tgcttttcca tcagttgtac ataggttaaa aaatgtattt tcataataaa
     5761 tattttttgc gaaggtaaat tctactatat catctatatt tttaaaaaaa gttgttttac
     5821 tatattcttt aaattcttct ccgagttcta attgtttact attactttta ttattttgaa
     5881 atactctaac atattcatta tcaataagat tatttccttg tcctatacca tctctaaata
     5941 ctgccattag aaattttttc aacttatatt ttgaatctaa ttgttccata tatttattat
     6001 ctccttatct agttttttta agagataaca ctacattaaa tatatactta ttgactaatc
     6061 aacttttaca actattgaat aattttagct tagtatgtat aataatattg ttaagatgct
     6121 aaaattgata gcaatattat tatataacta cagatactta atcagttaga ccgatattgg
     6181 agttggtagc tccataaact caattggtag ttgagttgtc attgtctagt agtattatct
     6241 cattttgttg ttatcttaaa atattatttt aaaaatactc tgcttggtcg gcttagggta
     6301 ttttttttgt ttaattccaa tattggttca aactcctgtg taatactttt gacacttcca
     6361 tctgtattga attgacagtt gatatggtat ctcattattg gcttgttctt atgtttttct
     6421 atgtatttat tttttacttt caaataattt gtatttccat ttttagttat gtttaacaca
     6481 ctatccttat tactcaataa aattattttt tgttttaaca tatgatttat agaatctaaa
     6541 aattgatatt gacttataat agtactttct acctctctag ctttttctag tgtttcaaat
     6601 tcttcaaact ctgctatatt gctatagtat agcttatcat taataatatt atctgacaac
     6661 tttaattcca ttttataaaa caattccaag ttatgtataa aatttttaat gttacaacat
     6721 atagacttat tatttgttct tatatggtat ctccatttac tacttctagc tctcatgtta
     6781 tatgcacaac tactcccaac ataaacatat attgcttgtt ctaactcata tttttgtaaa
     6841 atttcaacat catatccagt caaatcgtac tctctcttat atacaaatgg tttttctaat
     6901 tttttgttca ataaatctcc tcctatttct tacttaattt agctaaaaat ctatttatat
     6961 ttcacctcct ttttgacttt aagcctacga ataccatatt tatgattata acatcttcta
     7021 tttttcccaa atccttacat ctcccctaat ttttagactt aagggcgaat tctgcatcaa
     7081 gctagcataa aaataagaag cctgcatttg caggcttctt atttttatgg taccttaaga
     7141 cccactttca cagccgcctc aatacttata aagatagagg atgaagagga agcaatgaaa
     7201 gaagctgaaa gagagagaaa gaggggacga agaagatagc aaaataaaaa aataaatata
     7261 gaataggtaa aatatgtaat aattatatgt tataatattt ttagcaagaa gatgtaatct
     7321 acaatttata gagtggagtt catactggga taaaacctac ttcctaatga aaggaggtgg
     7381 gaagtatgaa taacttttta cttaatgtaa tagctggcgt tattgctagt ttaatatttt
     7441 gcttaatttg taaagtattt ctaaaagtaa aaagccactc aactcgtggc aagagtaaaa
     7501 gtggctggga atttgatttt aaaatcaagt tccataagtt caaatagatt catttaatta
     7561 tgaacttcac tctaccgcaa aatagattgt agttcttctt gcttttatta taccacaaat
     7621 tagaaaaaat attgtttata taaaataaaa aataaaaatt tttattaaaa aattgaaaac
     7681 ttgattataa agcaattaat ttataaaata tatataaata agtaggtatt tatttacttg
     7741 aatttcattg tttatataaa aaaaatgaat aatcattgat tgctcgagcg ctaaaactat
     7801 taatcttatc atatc
//
