LOCUS       sequence-349050-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SIBR Intron 3 (Int3) interrupts the CDS of FnCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7225)
  AUTHORS   Patinios C, Creutzburg SCA, Arifah AQ, Adiego-Perez B, Gyimah EA, 
            Ingham CJ, Kengen SWM, van der Oost J, Staals RHJ
  TITLE     Streamlined CRISPR genome engineering in wild-type bacteria using 
            SIBR-Cas.
  JOURNAL   Nucleic Acids Res. 2021 Oct 6. pii: 6382381. doi: 
            10.1093/nar/gkab893.
  PUBMED    34614191
REFERENCE   2  (bases 1 to 7225)
  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: "Nucleic 
            Acids Res. 2021 Oct 6. pii: 6382381. doi: 10.1093/nar/gkab893."
COMMENT     Sequence Label: pSIBR003
FEATURES             Location/Qualifiers
     source          1..7225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(459..545)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     560..582
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        join(589..594,595..612,613..619)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     promoter        join(4954..4959,4960..4977,4978..4984)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(5238..5257)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5249..6064
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6135..6797)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      join(6798..7225,1..344)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca aggccaagcc
       61 gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa ccggcaagcg
      121 aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa attgatgggg
      181 caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct gggtagccgg
      241 tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag cttgtccagc
      301 agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg ccatcgtcca
      361 catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc ggagagcaag
      421 cccgtagggg gggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg
      481 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
      541 atttgatgcc tttaattaaa gcggataaca atttcacaca ggaggccgtt tacactttat
      601 gcttccggct cgtataatgt gtggggaaac cacaacggtt tccctctaga aataattttg
      661 tttaactata agaaggagat atacatatgt ccttgggtta attgaggcct gagtataagg
      721 tgacttatac ttgtaatcta tctaaacggg gaacctctct agtagacaat cccgtgctaa
      781 attgatacca gcatcgtctt gatgcccttg gcagcataaa tgcctaacga ctatcccttt
      841 ggggagtagg gtcaagtgac tcgaaacgat agacaacttg ctttaacaag ttggagatat
      901 agtctgctct gcatggtgac atgcagctgg atataattcc ggggtaagat taacgacctt
      961 atctgaacat aatgctatca atttatcaag aatttgttaa taaatatagt ttaagtaaaa
     1021 ctctaagatt tgagttaatc ccacagggta aaacacttga aaacataaaa gcaagaggtt
     1081 tgattttaga tgatgagaaa agagctaaag actacaaaaa ggctaaacaa ataattgata
     1141 aatatcatca gttttttata gaggagatat taagttcggt ttgtattagc gaagatttat
     1201 tacaaaacta ttctgatgtt tattttaaac ttaaaaagag tgatgatgat aatctacaaa
     1261 aagattttaa aagtgcaaaa gatacgataa agaaacaaat atctgaatat ataaaggact
     1321 cagagaaatt taagaatttg tttaatcaaa accttatcga tgctaaaaaa gggcaagagt
     1381 cagatttaat tctatggcta aagcaatcta aggataatgg tatagaacta tttaaagcca
     1441 atagtgatat cacagatata gatgaggcgt tagaaataat caaatctttt aaaggttgga
     1501 caacttattt taagggtttt catgaaaata gaaaaaatgt ttatagtagc aatgatattc
     1561 ctacatctat tatttatagg atagtagatg ataatttgcc taaatttcta gaaaataaag
     1621 ctaagtatga gagtttaaaa gacaaagctc cagaagctat aaactatgaa caaattaaaa
     1681 aagatttggc agaagagcta acctttgata ttgactacaa aacatctgaa gttaatcaaa
     1741 gagttttttc acttgatgaa gtttttgaga tagcaaactt taataattat ctaaatcaaa
     1801 gtggtattac taaatttaat actattattg gtggtaaatt tgtaaatggt gaaaatacaa
     1861 agagaaaagg tataaatgaa tatataaatc tatactcaca gcaaataaat gataaaacac
     1921 tcaaaaaata taaaatgagt gttttattta agcaaatttt aagtgataca gaatctaaat
     1981 cttttgtaat tgataagtta gaagatgata gtgatgtagt tacaacgatg caaagttttt
     2041 atgagcaaat agcagctttt aaaacagtag aagaaaaatc tattaaagaa acactatctt
     2101 tattatttga tgatttaaaa gctcaaaaac ttgatttgag taaaatttat tttaaaaatg
     2161 ataaatctct tactgatcta tcacaacaag tttttgatga ttatagtgtt attggtacag
     2221 cggtactaga atatataact caacaaatag cacctaaaaa tcttgataac cctagtaaga
     2281 aagagcaaga attaatagcc aaaaaaactg aaaaagcaaa atacttatct ctagaaacta
     2341 taaagcttgc cttagaagaa tttaataagc atagagatat agataaacag tgtaggtttg
     2401 aagaaatact tgcaaacttt gcggctattc cgatgatatt tgatgaaata gctcaaaaca
     2461 aagacaattt ggcacagata tctatcaaat atcaaaatca aggtaaaaaa gacctacttc
     2521 aagctagtgc ggaagatgat gttaaagcta tcaaggatct tttagatcaa actaataatc
     2581 tcttacataa actaaaaata tttcatatta gtcagtcaga agataaggca aatattttag
     2641 acaaggatga gcatttttat ctagtatttg aggagtgcta ctttgagcta gcgaatatag
     2701 tgcctcttta taacaaaatt agaaactata taactcaaaa gccatatagt gatgagaaat
     2761 ttaagctcaa ttttgagaac tcgactttgg ctaatggttg ggataaaaat aaagagcctg
     2821 acaatacggc aattttattt atcaaagatg ataaatatta tctgggtgtg atgaataaga
     2881 aaaataacaa aatatttgat gataaagcta tcaaagaaaa taaaggcgag ggttataaaa
     2941 aaattgttta taaactttta cctggcgcaa ataaaatgtt acctaaggtt ttcttttctg
     3001 ctaaatctat aaaattttat aatcctagtg aagatatact tagaataaga aatcattcca
     3061 cacatacaaa aaatggtagt cctcaaaaag gatatgaaaa atttgagttt aatattgaag
     3121 attgccgaaa atttatagat ttttataaac agtctataag taagcatccg gagtggaaag
     3181 attttggatt tagattttct gatactcaaa gatataattc tatagatgaa ttttatagag
     3241 aagttgaaaa tcaaggctac aaactaactt ttgaaaatat atcagagagc tatattgata
     3301 gcgtagttaa tcagggtaaa ttgtacctat tccaaatcta taataaagat ttttcagctt
     3361 atagcaaagg gcgaccaaat ctacatactt tatattggaa agcgctgttt gatgagagaa
     3421 atcttcaaga tgtggtttat aagctaaatg gtgaggcaga gcttttttat cgtaaacaat
     3481 caatacctaa aaaaatcact cacccagcta aagaggcaat agctaataaa aacaaagata
     3541 atcctaaaaa agagagtgtt tttgaatatg atttaatcaa agataaacgc tttactgaag
     3601 ataagttttt ctttcactgt cctattacaa tcaattttaa atctagtgga gctaataagt
     3661 ttaatgatga aatcaattta ttgctaaaag aaaaagcaaa tgatgttcat atattaagta
     3721 tagatagagg tgaaagacat ttagcttact atactttggt agatggtaaa ggcaatatca
     3781 tcaaacaaga tactttcaac atcattggta atgatagaat gaaaacaaac taccatgata
     3841 agcttgctgc aatagagaaa gatagggatt cagctaggaa agactggaaa aagataaata
     3901 acatcaaaga gatgaaagag ggctatctat ctcaggtagt tcatgaaata gctaagctag
     3961 ttatagagta taatgctatt gtggtttttg aggatttaaa ttttggattt aaaagagggc
     4021 gtttcaaggt agagaagcag gtctatcaaa agttagaaaa aatgctaatt gagaaactaa
     4081 actatctagt tttcaaagat aatgagtttg ataaaactgg gggagtgctt agagcttatc
     4141 agctaacagc accttttgag acttttaaaa agatgggtaa acaaacaggt attatctact
     4201 atgtaccagc tggttttact tcaaaaattt gtcctgtaac tggttttgta aatcagttat
     4261 atcctaagta tgaaagtgtc agcaaatctc aagagttctt tagtaagttt gacaagattt
     4321 gttataacct tgataagggc tattttgagt ttagttttga ttataaaaac tttggtgaca
     4381 aggctgccaa aggcaagtgg actatagcta gctttgggag tagattgatt aactttagaa
     4441 attcagataa aaatcataat tgggatactc gagaagttta tccaactaaa gagttggaga
     4501 aattgctaaa agattattct atcgaatatg ggcatggcga atgtatcaaa gcagctattt
     4561 gcggtgagag cgacaaaaag ttttttgcta agctaactag tgtcctaaat actatcttac
     4621 aaatgcgtaa ctcaaaaaca ggtactgagt tagattatct aatttcacca gtagcagatg
     4681 taaatggcaa tttctttgat tcgcgacagg cgccaaaaaa tatgcctcaa gatgctgatg
     4741 ccaatggtgc ttatcatatt gggctaaaag gtctgatgct actaggtagg atcaaaaata
     4801 atcaagaggg caaaaaactc aatttggtta tcaaaaatga agagtatttt gagttcgtgc
     4861 agaataggaa taactaagcg gccgcataat gatgtgttat cattgatgcg aggtcgccta
     4921 tacctccccg cttcggcggg gttttttccc gggtttacac tttatgcttc cggctcgtat
     4981 aatgtgtggt ctaagaactt taaataattt ctactgttgt agatatgtct tcctgagtga
     5041 cgagcaacga gactggaaga catgtctaag aactttaaat aatttctact gttgtagata
     5101 gataccggac aacgtgtctc cccgcttcgg cggggttttt tcctaggttg tgtctcaaaa
     5161 tctctgatgt tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
     5221 ttacataaac agtaatacaa ggggtgttat gagccatatt cagcgtgaaa cgagctgtag
     5281 ccgtccgcgt ctgaacagca acatggatgc ggatctgtat ggctataaat gggcgcgtga
     5341 taacgtgggt cagagcggcg cgaccattta tcgtctgtat ggcaaaccgg atgcgccgga
     5401 actgtttctg aaacatggca aaggcagcgt ggcgaacgat gtgaccgatg aaatggtgcg
     5461 tctgaactgg ctgaccgaat ttatgccgct gccgaccatt aaacatttta ttcgcacccc
     5521 ggatgatgcg tggctgctga ccaccgcgat tccgggcaaa accgcgtttc aggtgctgga
     5581 agaatatccg gatagcggcg aaaacattgt ggatgcgctg gccgtgtttc tgcgtcgtct
     5641 gcatagcatt ccggtgtgca actgcccgtt taacagcgat cgtgtgtttc gtctggccca
     5701 ggcgcagagc cgtatgaaca acggcctggt ggatgcgagc gattttgatg atgaacgtaa
     5761 cggctggccg gtggaacagg tgtggaaaga aatgcataaa ctgctgccgt ttagcccgga
     5821 tagcgtggtg acccacggcg attttagcct ggataacctg attttcgatg aaggcaaact
     5881 gattggctgc attgatgtgg gccgtgtggg cattgcggat cgttatcagg atctggccat
     5941 tctgtggaac tgcctgggcg aatttagccc gagcctgcaa aaacgtctgt ttcagaaata
     6001 tggcattgat aatccggata tgaacaaact gcaatttcat ctgatgctgg atgaattttt
     6061 ctaataatta attggaccgc ggtccgagag acgcatggca gtatctcaga ctgtgtatcg
     6121 gcacgtctca ctacttaccg gcgcggcagc gttacccgtg tcggcggctc caacggctcg
     6181 ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc
     6241 cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg
     6301 ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg
     6361 atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc
     6421 acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg
     6481 tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag
     6541 cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc
     6601 ttggaaagtg ttttctggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg
     6661 tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc
     6721 gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct
     6781 ctggactgcg tggccatgct tatctccatg cggtaggggt gccgcacggt tgcggcacca
     6841 tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg
     6901 cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg
     6961 agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct
     7021 atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg
     7081 cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc
     7141 ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc
     7201 tcggggggca ggcgggcggg cttcg
//
