LOCUS       sequence-399868-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression for FnCas12a in E. coli and clostridia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  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 6352)
  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: pPfetFn-Target_v2
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1617..2162
                     /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     2063..2082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            2410..2519
                     /note="incP origin of transfer"
     CDS             2552..2923
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     RBS             3122..3130
                     /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)"
     CDS             join(3143..3226,3227..4561)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="NIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISHI
                     TRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYHI
                     VFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEWS
                     GSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDGK
                     TYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKAY
                     YGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDEI
                     ERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLKM
                     DLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSVV
                     KDSILEQGQLTVNK"
     primer_bind     4614..4630
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4614..4630
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4769..4786)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4769..4785)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 tactttatat atatatgatt aaaaaaataa aaaacaacag cctattaggt tgttgttttt
       61 tattttcttt attaattttt ttaattttta gtttttagtt cttttttaaa ataagtttca
      121 gcctcttttt caatattttt taaagaagga gtatttgcat gaattgcctt ttttctaaca
      181 gacttaggaa atattttaac agtatcttct tgcgccggtg attttggaac ttcataactt
      241 actaatttat aattattatt ttctttttta attgtaacag ttgcaaaaga agctgaacct
      301 gttccttcaa ctagtttatc atcttcaata taatattctt gacctatata gtataaatat
      361 atttttatta tatttttact tttttctgaa tctattattt tataatcata aaaagtttta
      421 ccaccaaaag aaggttgtac tccttctggt ccaacatatt tttttactat attatctaaa
      481 taatttttgg gaactggtgt tgtaatttga ttaatcgaac aaccagttat acttaaagga
      541 attataacta taaaaatata taggattatc tttttaaatt tcattattgg cctccttttt
      601 attaaattta tgttaccata aaaaggacat aacgggaata tgtagaatat ttttaatgta
      661 gacaaaattt tacataaata taaagaaagg aagtgtttgt ttaaatttta tagcaaacta
      721 tcaaaaatta gggggataaa aatttatgaa aaaaaggttt tcgatgttat ttttatgttt
      781 aactttaata gtttgtggtt tatttacaaa ttcggccggc cagtgggcaa gttgaaaaat
      841 tcacaaaaat gtggtataat atctttgttc attagagcga taaacttgaa tttgagaggg
      901 gacttagatg gtatttgaaa aaattgataa aaatagttgg aacagaaaag agtattttga
      961 ccactacttt gcaagtgtac cttgtaccta cagcatgacc gttaaagtgg atatcacaca
     1021 aataaaggaa aagggaatga aactatatcc tgcaatgctt tattatattg caatgattgt
     1081 aaaccgccat tcagagttta ggacggcaat caatcaagat ggtgaattgg ggatatatga
     1141 tgagatgata ccaagctata caatatttca caatgatact gaaacatttt ccagcctttg
     1201 gactgagtgt aagtctgact ttaaatcatt tttagcagat tatgaaagtg atacgcaacg
     1261 gtatggaaac aatcatagaa tggaaggaaa gccaaatgct ccggaaaaca tttttaatgt
     1321 atctatgata ccgtggtcaa ccttcgatgg ctttaatctg aatttgcaga aaggatatga
     1381 ttatttgatt cctattttta ctatggggaa atattataaa gaagataaca aaattatact
     1441 tcctttggca attcaagttc atcacgcagt atgtgacgga tttcacattt gccgttttgt
     1501 aaacgaattg caggaattga taaatagtta acttcaggtt tgtctgtaac taaaaacaag
     1561 tatttaagca aaaacatcgt agaaatacgg tgttttttgt taccctaagt ttcttcttga
     1621 gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc
     1681 ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga
     1741 gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact
     1801 aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg
     1861 gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc
     1921 gtgcatacag tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat
     1981 gagacaaacg cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag
     2041 agcgcacgag ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2101 gccaccactg atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga
     2161 aaaacggctt tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag
     2221 gaaatctccg ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag
     2281 cgagtgggcc ccctgcttcg gggtcattat agcgattttt tcggtatatc catccttttt
     2341 cgcacgatat acaggatttt gccaaagggt tcgtgtagac tttccttggt gtatccaacg
     2401 gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac
     2461 tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag gctggccggc
     2521 taccgccggc gtaacagatg agggcaagcg gatggctgat gaaaccaagc caaccaggaa
     2581 gggcagccca cctatcaagg tgtactgcct tccagacgaa cgaagagcga ttgaggaaaa
     2641 ggcggcggcg gccggcatga gcctgtcggc ctacctgctg gccgtcggcc agggctacaa
     2701 aatcacgggc gtcgtggact atgagcacgt ccgcgagctg gcccgcatca atggcgacct
     2761 gggccgcctg ggcggcctgc tgaaactctg gctcaccgac gacccgcgca cggcgcggtt
     2821 cggtgatgcc acgatcctcg ccctgctggc gaagatcgaa gagaagcagg acgagcttgg
     2881 caaggtcatg atgggcgtgg tccgcccgag ggcagagcca tgactttttt agccgctaaa
     2941 acggccgggg ggtgcgcgtg attgccaagc acgtccccat gcgctccatc aagaagagcg
     3001 acttcgcgga gctggtgaag tacatcaccg acgagcaagg caagaccgat cgtttttaac
     3061 aaaatatatt gataaaaata ataatagtgg gtataattaa cgtataaagt tgtgtaattt
     3121 ttaaggaggt gtgttacatt tgaacatcaa aaagtttgca aaacaagcaa cagtattaac
     3181 ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca
     3241 aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat
     3301 ccctgaacag caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa
     3361 aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga
     3421 cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa
     3481 aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga
     3541 cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc
     3601 tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa
     3661 aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac
     3721 tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta
     3781 taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga
     3841 aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa
     3901 aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga
     3961 agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag
     4021 tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg
     4081 tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc
     4141 taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta
     4201 cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat
     4261 ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac
     4321 tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt
     4381 cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg
     4441 attctacgca gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa
     4501 gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata
     4561 actgcaggat aatcaatcgt cccttcgtgt aaacgaaggg gcgtttttta tttcaggaaa
     4621 cagctatgac cgcggccgct gtatccaaaa ttactttaaa atctatcatt gatagggtaa
     4681 aatataaatc aatttctact gttgtagatt gagacgattc gtctccaatt tctactgttg
     4741 tagatttttt tagacatgca agcttggcac tggccgtcgt tttacactag cataaaaata
     4801 agaagcctgc atttgcaggc ttcttatttt tatggcgcga cgtcgttctg aatccttagc
     4861 taatgggtcg acgtatataa aatgtgctaa tgaaaaagaa aatgcgttaa aagagcctaa
     4921 aatgagttca aatggttttg aaattgattg gtagtttaat ttaatatatt ttttctattg
     4981 gctatctcga tacctataga atcttctgtt cacttttgtt tttgaaatat aaaaaggggc
     5041 tttttagccc ctttttttta aaactccgga ggagtttctt cattcttgat actatacgta
     5101 actatttcga tttgacttca ttgtcaatta agctagtaaa atcaatggtt aaaaaacaaa
     5161 aaacttgcat ttttctacct agtaatttat aattttaagt gtcgagttta aaagtataat
     5221 ttaccaggaa aggagcaagt tttttaataa ggaaaaattt ttccttttaa aattctattt
     5281 cgttatatga ctaattataa tcaaaaaaat gaaaataaac aagaggtaaa aactgcttta
     5341 gagaaatgta ctgataaaaa aagaaaaaat cctagattta cgtcatacat agcaccttta
     5401 actactaaga aaaatattga aaggacttcc acttgtggag attatttgtt tatgttgagt
     5461 gatgcagact tagaacattt taaattacat aaaggtaatt tttgcggtaa tagattttgt
     5521 ccaatgtgta gttggcgact tgcttgtaag gatagtttag aaatatctat tcttatggag
     5581 catttaagaa aagaagaaaa taaagagttt atatttttaa ctcttacaac tccaaatgta
     5641 aaaagttatg atcttaatta ttctattaaa caatataata aatcttttaa aaaattaatg
     5701 gagcgtaagg aagttaagga tataactaaa ggttatataa gaaaattaga agtaacttac
     5761 caaaaggaaa aatacataac aaaggattta tggaaaataa aaaaagatta ttatcaaaaa
     5821 aaaggacttg aaattggtga tttagaacct aattttgata cttataatcc tcattttcat
     5881 gtagttattg cagttaataa aagttatttt acagataaaa attattatat aaatcgagaa
     5941 agatggttgg aattatggaa gtttgctact aaggatgatt ctataactca agttgatgtt
     6001 agaaaagcaa aaattaatga ttataaagag gtttacgaac ttgcgaaata ttcagctaaa
     6061 gacactgatt atttaatatc gaggccagta tttgaaattt tttataaagc attaaaaggc
     6121 aagcaggtat tagtttttag tggatttttt aaagatgcac acaaattgta caagcaagga
     6181 aaacttgatg tttataaaaa gaaagatgaa attaaatatg tctatatagt ttattataat
     6241 tggtgcaaaa aacaatatga aaaaactaga ataagggaac ttacggaaga tgaaaaagaa
     6301 gaattaaatc aagatttaat agatggaata gaaatagatt aaagtgtaac ta
//
