LOCUS       sequence-395378-        8908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tetracycline-Inducible Fncas12a gene expression in E. coli and 
            clostridia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8908)
  AUTHORS   Zhang Y, Kubiak AM, Bailey TS, Claessen L, Hittmeyer P, Dubois L, 
            Theys J, Lambin P
  TITLE     Development of a CRISPR-Cas12a system for efficient genome 
            engineering in clostridia.
  JOURNAL   Microbiol Spectr. 2023 Nov 10:e0245923. doi: 
            10.1128/spectrum.02459-23.
  PUBMED    37947521
REFERENCE   2  (bases 1 to 8908)
  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 
            Spectr. 2023 Nov 10:e0245923. doi: 10.1128/spectrum.02459-23."
COMMENT     Sequence Label: pPIPL12-FnCas12a
FEATURES             Location/Qualifiers
     source          1..8908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      811..1356
                     /direction=RIGHT
                     /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     1257..1276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            1604..1713
                     /note="incP origin of transfer"
     CDS             1746..2117
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     primer_bind     2305..2321
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2305..2321
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             3168..3176
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(7106..7123)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7106..7122)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tgaacaaaaa tataaaatat tctcaaaact ttttaacgag tgaaaaagta ctcaaccaaa
       61 taataaaaca attgaattta aaagaaaccg ataccgttta cgaaattgga acaggtaaag
      121 ggcatttaac gacgaaactg gctaaaataa gtaaacaggt aacgtctatt gaattagaca
      181 gtcatctatt caacttatcg tcagaaaaat taaaactgaa tactcgtgtc actttaattc
      241 accaagatat tctacagttt caattcccta acaaacagag gtataaaatt gttgggagta
      301 ttccttacca tttaagcaca caaattatta aaaaagtggt ttttgaaagc catgcgtctg
      361 acatctatct gattgttgaa gaaggattct acaagcgtac cttggatatt caccgaacac
      421 tagggttgct cttgcacact caagtctcga ttcagcaatt gcttaagctg ccagcggaat
      481 gctttcatcc taaaccaaaa gtaaacagtg tcttaataaa acttacccgc cataccacag
      541 atgttccaga taaatattgg aagctatata cgtactttgt ttcaaaatgg gtcaatcgag
      601 aatatcgtca actgtttact aaaaatcagt ttcatcaagc aatgaaacac gccaaagtaa
      661 acaatttaag taccgttact tatgagcaag tattgtctat ttttaatagt tatctattat
      721 ttaacgggag gaaataattc tatgagtcgc ttttgtaaat ttggaaagtt acacgttact
      781 aaagggaatg tgttaaacaa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc
      841 ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac
      901 caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc
      961 agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg
     1021 ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg
     1081 ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa
     1141 ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga
     1201 atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga
     1261 aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt
     1321 tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca
     1381 cttccctgtt aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca
     1441 tttccgctcg ccgcagtcga acgaccgagc gtagcgagtg ggccccctgc ttcggggtca
     1501 ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa
     1561 gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag
     1621 taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc
     1681 tcaacgggaa tcctgctctg cgaggctggc cggctaccgc cggcgtaaca gatgagggca
     1741 agcggatggc tgatgaaacc aagccaacca ggaagggcag cccacctatc aaggtgtact
     1801 gccttccaga cgaacgaaga gcgattgagg aaaaggcggc ggcggccggc atgagcctgt
     1861 cggcctacct gctggccgtc ggccagggct acaaaatcac gggcgtcgtg gactatgagc
     1921 acgtccgcga gctggcccgc atcaatggcg acctgggccg cctgggcggc ctgctgaaac
     1981 tctggctcac cgacgacccg cgcacggcgc ggttcggtga tgccacgatc ctcgccctgc
     2041 tggcgaagat cgaagagaag caggacgagc ttggcaaggt catgatgggc gtggtccgcc
     2101 cgagggcaga gccatgactt ttttagccgc taaaacggcc ggggggtgcg cgtgattgcc
     2161 aagcacgtcc ccatgcgctc catcaagaag agcgacttcg cggagctggt gaagtacatc
     2221 accgacgagc aaggcaagac cgatcgggcc ccctgcagga taatcaatcg tcccttcgtg
     2281 taaacgaagg ggcgtttttt atttcaggaa acagctatga ccgcggccgc tgtatccatt
     2341 aagacccact ttcacattta agttgttttt ctaatccgca aattattaat tcaagaccga
     2401 ataaaaatgc tggctctgca ccttggtgat caaataattc tatagcttgc cttaataatg
     2461 gtggcatact atcagtagta ggtgtttccc tttcttcttt agcaacttga tgctcttgat
     2521 cttccaatac gcaacctaaa gtaaaatgcc ccacagcgct taatgcatat aatgcattct
     2581 ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttctaaagtt tcatactgtt
     2641 tttctgtagg tcttgtacct aaatgtactt ttgctccatc cctatgacta agtaaagcac
     2701 atctaaaact tttagcgtta tttcttaaaa aatcttgcca gctttcccct tctaaagggc
     2761 aaaagtgagt atgatgtcta tctaacatct caatagctaa ggcatccaat aaagctctct
     2821 tattttttac atgccaatac aatgtaggct gctctacacc caacttctga gctaattttc
     2881 tggttgttaa accttctatt cctacctcat taagtaactc taatgctgag ttaattactt
     2941 tacttttatc taatcttgac attctaacta acctcctaaa ttttgatacg gggtaacaga
     3001 taaaccattt caatctattt cataagttcc atagtttatc cctaatttat acgttttctc
     3061 taacaactta attttaactt taaaaaataa atttgtcaaa aattataagt attgagcttc
     3121 tatcattgat aggttataat gaacatcgta taaagttgtg taatttttaa ggaggtgtgt
     3181 tacatatgtc aatttatcaa gaatttgtta ataaatatag tttaagtaaa actctaagat
     3241 ttgagttaat cccacagggt aaaacacttg aaaacataaa agcaagaggt ttgattttag
     3301 atgatgagaa aagagctaaa gactacaaaa aggctaaaca aataattgat aaatatcatc
     3361 agttttttat agaggagata ttaagttcgg tttgtattag cgaagattta ttacaaaact
     3421 attctgatgt ttattttaaa cttaaaaaga gtgatgatga taatctacaa aaagatttta
     3481 aaagtgcaaa agatacgata aagaaacaaa tatctgaata tataaaggac tcagagaaat
     3541 ttaagaattt gtttaatcaa aaccttatcg atgctaaaaa agggcaagag tcagatttaa
     3601 ttctatggct aaagcaatct aaggataatg gtatagaact atttaaagcc aatagtgata
     3661 tcacagatat agatgaggcg ttagaaataa tcaaatcttt taaaggttgg acaacttatt
     3721 ttaagggttt tcatgaaaat agaaaaaatg tttatagtag caatgatatt cctacatcta
     3781 ttatttatag gatagtagat gataatttgc ctaaatttct agaaaataaa gctaagtatg
     3841 agagtttaaa agacaaagct ccagaagcta taaactatga acaaattaaa aaagatttgg
     3901 cagaagagct aacctttgat attgactaca aaacatctga agttaatcaa agagtttttt
     3961 cacttgatga agtttttgag atagcaaact ttaataatta tctaaatcaa agtggtatta
     4021 ctaaatttaa tactattatt ggtggtaaat ttgtaaatgg tgaaaataca aagagaaaag
     4081 gtataaatga atatataaat ctatactcac agcaaataaa tgataaaaca ctcaaaaaat
     4141 ataaaatgag tgttttattt aagcaaattt taagtgatac agaatctaaa tcttttgtaa
     4201 ttgataagtt agaagatgat agtgatgtag ttacaacgat gcaaagtttt tatgagcaaa
     4261 tagcagcttt taaaacagta gaagaaaaat ctattaaaga aacactatct ttattatttg
     4321 atgatttaaa agctcaaaaa cttgatttga gtaaaattta ttttaaaaat gataaatctc
     4381 ttactgatct atcacaacaa gtttttgatg attatagtgt tattggtaca gcggtactag
     4441 aatatataac tcaacaaata gcacctaaaa atcttgataa ccctagtaag aaagagcaag
     4501 aattaatagc caaaaaaact gaaaaagcaa aatacttatc tctagaaact ataaagcttg
     4561 ccttagaaga atttaataag catagagata tagataaaca gtgtaggttt gaagaaatac
     4621 ttgcaaactt tgcggctatt ccgatgatat ttgatgaaat agctcaaaac aaagacaatt
     4681 tggcacagat atctatcaaa tatcaaaatc aaggtaaaaa agacctactt caagctagtg
     4741 cggaagatga tgttaaagct atcaaggatc ttttagatca aactaataat ctcttacata
     4801 aactaaaaat atttcatatt agtcagtcag aagataaggc aaatatttta gacaaggatg
     4861 agcattttta tctagtattt gaggagtgct actttgagct agcgaatata gtgcctcttt
     4921 ataacaaaat tagaaactat ataactcaaa agccatatag tgatgagaaa tttaagctca
     4981 attttgagaa ctcgactttg gctaatggtt gggataaaaa taaagagcct gacaatacgg
     5041 caattttatt tatcaaagat gataaatatt atctgggtgt gatgaataag aaaaataaca
     5101 aaatatttga tgataaagct atcaaagaaa ataaaggcga gggttataaa aaaattgttt
     5161 ataaactttt acctggcgca aataaaatgt tacctaaggt tttcttttct gctaaatcta
     5221 taaaatttta taatcctagt gaagatatac ttagaataag aaatcattcc acacatacaa
     5281 aaaatggtag tcctcaaaaa ggatatgaaa aatttgagtt taatattgaa gattgccgaa
     5341 aatttataga tttttataaa cagtctataa gtaagcatcc ggagtggaaa gattttggat
     5401 ttagattttc tgatactcaa agatataatt ctatagatga attttataga gaagttgaaa
     5461 atcaaggcta caaactaact tttgaaaata tatcagagag ctatattgat agcgtagtta
     5521 atcagggtaa attgtaccta ttccaaatct ataataaaga tttttcagct tatagcaaag
     5581 ggcgaccaaa tctacatact ttatattgga aagcgctgtt tgatgagaga aatcttcaag
     5641 atgtggttta taagctaaat ggtgaggcag agctttttta tcgtaaacaa tcaataccta
     5701 aaaaaatcac tcacccagct aaagaggcaa tagctaataa aaacaaagat aatcctaaaa
     5761 aagagagtgt ttttgaatat gatttaatca aagataaacg ctttactgaa gataagtttt
     5821 tctttcactg tcctattaca atcaatttta aatctagtgg agctaataag tttaatgatg
     5881 aaatcaattt attgctaaaa gaaaaagcaa atgatgttca tatattaagt atagatagag
     5941 gtgaaagaca tttagcttac tatactttgg tagatggtaa aggcaatatc atcaaacaag
     6001 atactttcaa catcattggt aatgatagaa tgaaaacaaa ctaccatgat aagcttgctg
     6061 caatagagaa agatagggat tcagctagga aagactggaa aaagataaat aacatcaaag
     6121 agatgaaaga gggctatcta tctcaggtag ttcatgaaat agctaagcta gttatagagt
     6181 ataatgctat tgtggttttt gaggatttaa attttggatt taaaagaggg cgtttcaagg
     6241 tagagaagca ggtctatcaa aagttagaaa aaatgctaat tgagaaacta aactatctag
     6301 ttttcaaaga taatgagttt gataaaactg ggggagtgct tagagcttat cagctaacag
     6361 caccttttga gacttttaaa aagatgggta aacaaacagg tattatctac tatgtaccag
     6421 ctggttttac ttcaaaaatt tgtcctgtaa ctggttttgt aaatcagtta tatcctaagt
     6481 atgaaagtgt cagcaaatct caagagttct ttagtaagtt tgacaagatt tgttataacc
     6541 ttgataaggg ctattttgag tttagttttg attataaaaa ctttggtgac aaggctgcca
     6601 aaggcaagtg gactatagct agctttggga gtagattgat taactttaga aattcagata
     6661 aaaatcataa ttgggatact cgagaagttt atccaactaa agagttggag aaattgctaa
     6721 aagattattc tatcgaatat gggcatggcg aatgtatcaa agcagctatt tgcggtgaga
     6781 gcgacaaaaa gttttttgct aagctaacta gtgtcctaaa tactatctta caaatgcgta
     6841 actcaaaaac aggtactgag ttagattatc taatttcacc agtagcagat gtaaatggca
     6901 atttctttga ttcgcgacag gcgccaaaaa atatgcctca agatgctgat gccaatggtg
     6961 cttatcatat tgggctaaaa ggtctgatgc tactaggtag gatcaaaaat aatcaagagg
     7021 gcaaaaaact caatttggtt atcaaaaatg aagagtattt tgagttcgtg cagaatagga
     7081 ataactaaag acatgcaagc ttggcactgg ccgtcgtttt acactagcat aaaaataaga
     7141 agcctgcatt tgcaggcttc ttatttttat ggcgcgccgc cattattttt ttgaacaatt
     7201 gacaattcat ttcttatttt ttattaagtg atagtcaaaa ggcataacag tgctgaatag
     7261 aaagaaattt acagaaaaga aaattataga atttagtatg attaattata ctcatttatg
     7321 aatgtttaat tgaatacaaa aaaaaatact tgttatgtat tcaattacgg gttaaaatat
     7381 agacaagttg aaaaatttaa taaaaaaata agtcctcagc tcttatatat taagctacca
     7441 acttagtata taagccaaaa cttaaatgtg ctaccaacac atcaagccgt tagagaactc
     7501 tatctatagc aatatttcaa atgtaccgac atacaagaga aacattaact atatatattc
     7561 aatttatgag attatcttaa cagatataaa tgtaaattgc aataagtaag atttagaagt
     7621 ttatagcctt tgtgtattgg aagcagtacg caaaggcttt tttatttgat aaaaattaga
     7681 agtatattta ttttttcata attaatttat gaaaatgaaa gggggtgagc aaagtgacag
     7741 aggaaagcag tatcttatca aataacaagg tattagcaat atcattattg actttagcag
     7801 taaacattat gacttttata gtgcttgtag ctaagtagta cgaaaggggg agctttaaaa
     7861 agctccttgg aatacataga attcataaat taatttatga aaagaagggc gtatatgaaa
     7921 acttgtaaaa attgcaaaga gtttattaaa gatactgaaa tatgcaaaat acattcgttg
     7981 atgattcatg ataaaacagt agcaacctat tgcagtaaat acaatgagtc aagatgttta
     8041 cataaaggga aagtccaatg tattaattgt tcaaagatga accgatatgg atggtgtgcc
     8101 ataaaaatga gatgttttac agaggaagaa cagaaaaaag aacgtacatg cattaaatat
     8161 tatgcaagga gctttaaaaa agctcatgta aagaagagta aaaagaaaaa ataatttatt
     8221 tattaattta atattgagag tgccgacaca gtatgcacta aaaaatatat ctgtggtgta
     8281 gtgagccgat acaaaaggat agtcactcgc attttcataa tacatcttat gttatgatta
     8341 tgtgtcggtg ggacttcacg acgaaaaccc acaataaaaa aagagttcgg ggtagggtta
     8401 agcatagttg aggcaactaa acaatcaagc taggatatgc agtagcagac cgtaaggtcg
     8461 ttgtttaggt gtgttgtaat acatacgcta ttaagatgta aaaatacgga taccaatgaa
     8521 gggaaaagta taatttttgg atgtagtttg tttgttcatc tatgggcaaa ctacgtccaa
     8581 agccgtttcc aaatctgcta aaaagtatat cctttctaaa atcaaagtca agtatgaaat
     8641 cataaataaa gtttaatttt gaagttatta tgatattatg tttttctatt aaaataaatt
     8701 aagtatatag aatagtttaa taatagtata tacttaatgt gataagtgtc tgacagtgtc
     8761 acagaaagga tgattgttat ggattataag cggccggccg aagcaaactt aagagtgtgt
     8821 tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt agatgctaaa
     8881 aatttgtaat taagaaggag tgattaca
//
