LOCUS       sequence-351669-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SIBR Intron 4 (Int4) 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: pSIBR004
FEATURES             Location/Qualifiers
     source          1..7225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(2275..2280,2281..2298,2299..2305)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(2559..2578)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2570..3385
                     /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(3456..4118)
                     /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      4119..4890
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(5005..5091)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     5106..5128
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        join(5135..5140,5141..5158,5159..5165)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
ORIGIN
        1 actttgagct agcgaatata gtgcctcttt ataacaaaat tagaaactat ataactcaaa
       61 agccatatag tgatgagaaa tttaagctca attttgagaa ctcgactttg gctaatggtt
      121 gggataaaaa taaagagcct gacaatacgg caattttatt tatcaaagat gataaatatt
      181 atctgggtgt gatgaataag aaaaataaca aaatatttga tgataaagct atcaaagaaa
      241 ataaaggcga gggttataaa aaaattgttt ataaactttt acctggcgca aataaaatgt
      301 tacctaaggt tttcttttct gctaaatcta taaaatttta taatcctagt gaagatatac
      361 ttagaataag aaatcattcc acacatacaa aaaatggtag tcctcaaaaa ggatatgaaa
      421 aatttgagtt taatattgaa gattgccgaa aatttataga tttttataaa cagtctataa
      481 gtaagcatcc ggagtggaaa gattttggat ttagattttc tgatactcaa agatataatt
      541 ctatagatga attttataga gaagttgaaa atcaaggcta caaactaact tttgaaaata
      601 tatcagagag ctatattgat agcgtagtta atcagggtaa attgtaccta ttccaaatct
      661 ataataaaga tttttcagct tatagcaaag ggcgaccaaa tctacatact ttatattgga
      721 aagcgctgtt tgatgagaga aatcttcaag atgtggttta taagctaaat ggtgaggcag
      781 agctttttta tcgtaaacaa tcaataccta aaaaaatcac tcacccagct aaagaggcaa
      841 tagctaataa aaacaaagat aatcctaaaa aagagagtgt ttttgaatat gatttaatca
      901 aagataaacg ctttactgaa gataagtttt tctttcactg tcctattaca atcaatttta
      961 aatctagtgg agctaataag tttaatgatg aaatcaattt attgctaaaa gaaaaagcaa
     1021 atgatgttca tatattaagt atagatagag gtgaaagaca tttagcttac tatactttgg
     1081 tagatggtaa aggcaatatc atcaaacaag atactttcaa catcattggt aatgatagaa
     1141 tgaaaacaaa ctaccatgat aagcttgctg caatagagaa agatagggat tcagctagga
     1201 aagactggaa aaagataaat aacatcaaag agatgaaaga gggctatcta tctcaggtag
     1261 ttcatgaaat agctaagcta gttatagagt ataatgctat tgtggttttt gaggatttaa
     1321 attttggatt taaaagaggg cgtttcaagg tagagaagca ggtctatcaa aagttagaaa
     1381 aaatgctaat tgagaaacta aactatctag ttttcaaaga taatgagttt gataaaactg
     1441 ggggagtgct tagagcttat cagctaacag caccttttga gacttttaaa aagatgggta
     1501 aacaaacagg tattatctac tatgtaccag ctggttttac ttcaaaaatt tgtcctgtaa
     1561 ctggttttgt aaatcagtta tatcctaagt atgaaagtgt cagcaaatct caagagttct
     1621 ttagtaagtt tgacaagatt tgttataacc ttgataaggg ctattttgag tttagttttg
     1681 attataaaaa ctttggtgac aaggctgcca aaggcaagtg gactatagct agctttggga
     1741 gtagattgat taactttaga aattcagata aaaatcataa ttgggatact cgagaagttt
     1801 atccaactaa agagttggag aaattgctaa aagattattc tatcgaatat gggcatggcg
     1861 aatgtatcaa agcagctatt tgcggtgaga gcgacaaaaa gttttttgct aagctaacta
     1921 gtgtcctaaa tactatctta caaatgcgta actcaaaaac aggtactgag ttagattatc
     1981 taatttcacc agtagcagat gtaaatggca atttctttga ttcgcgacag gcgccaaaaa
     2041 atatgcctca agatgctgat gccaatggtg cttatcatat tgggctaaaa ggtctgatgc
     2101 tactaggtag gatcaaaaat aatcaagagg gcaaaaaact caatttggtt atcaaaaatg
     2161 aagagtattt tgagttcgtg cagaatagga ataactaagc ggccgcataa tgatgtgtta
     2221 tcattgatgc gaggtcgcct atacctcccc gcttcggcgg ggttttttcc cgggtttaca
     2281 ctttatgctt ccggctcgta taatgtgtgg tctaagaact ttaaataatt tctactgttg
     2341 tagatatgtc ttcctgagtg acgagcaacg agactggaag acatgtctaa gaactttaaa
     2401 taatttctac tgttgtagat agataccgga caacgtgtct ccccgcttcg gcggggtttt
     2461 ttcctaggtt gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     2521 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     2581 tcagcgtgaa acgagctgta gccgtccgcg tctgaacagc aacatggatg cggatctgta
     2641 tggctataaa tgggcgcgtg ataacgtggg tcagagcggc gcgaccattt atcgtctgta
     2701 tggcaaaccg gatgcgccgg aactgtttct gaaacatggc aaaggcagcg tggcgaacga
     2761 tgtgaccgat gaaatggtgc gtctgaactg gctgaccgaa tttatgccgc tgccgaccat
     2821 taaacatttt attcgcaccc cggatgatgc gtggctgctg accaccgcga ttccgggcaa
     2881 aaccgcgttt caggtgctgg aagaatatcc ggatagcggc gaaaacattg tggatgcgct
     2941 ggccgtgttt ctgcgtcgtc tgcatagcat tccggtgtgc aactgcccgt ttaacagcga
     3001 tcgtgtgttt cgtctggccc aggcgcagag ccgtatgaac aacggcctgg tggatgcgag
     3061 cgattttgat gatgaacgta acggctggcc ggtggaacag gtgtggaaag aaatgcataa
     3121 actgctgccg tttagcccgg atagcgtggt gacccacggc gattttagcc tggataacct
     3181 gattttcgat gaaggcaaac tgattggctg cattgatgtg ggccgtgtgg gcattgcgga
     3241 tcgttatcag gatctggcca ttctgtggaa ctgcctgggc gaatttagcc cgagcctgca
     3301 aaaacgtctg tttcagaaat atggcattga taatccggat atgaacaaac tgcaatttca
     3361 tctgatgctg gatgaatttt tctaataatt aattggaccg cggtccgaga gacgcatggc
     3421 agtatctcag actgtgtatc ggcacgtctc actacttacc ggcgcggcag cgttacccgt
     3481 gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg catcggcagg
     3541 cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag gtctggttcc
     3601 atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc
     3661 tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag cgattcgtcc
     3721 tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg
     3781 ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc ggggccgttg
     3841 agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta ttggaccgtc
     3901 cgcaaagaac gtccgatgag cttggaaagt gttttctggc tgaccaccac ggcgttctgg
     3961 tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt cctcgcaata
     4021 agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc gggcttgttc
     4081 ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat gcggtagggg
     4141 tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc gcgacaacaa
     4201 ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac gcaatgagct
     4261 attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt gttgattttt
     4321 aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg gcacccctgc
     4381 ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc cctccggggc
     4441 ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc
     4501 ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgccct tcgactgccc
     4561 ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct
     4621 gcgcgagcct tgacccgcct tccacttggt gtccaaccgg caagcgaagc gcgcaggccg
     4681 caggccggag gcttttcccc agagaaaatt aaaaaaattg atggggcaag gccgcaggcc
     4741 gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg caatccctgt
     4801 ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg tccagcaggg ttgtccacgg
     4861 gccgagcgaa gcgagccagc cggtggccgc tcgcggccat cgtccacata tccacgggct
     4921 ggcaagggag cgcagcgacc gcgcagggcg aagcccggag agcaagcccg tagggggggc
     4981 gcgcccagct gtctagggcg gcggatttgt cctactcagg agagcgttca ccgacaaaca
     5041 acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgccttta
     5101 attaaagcgg ataacaattt cacacaggag gccgtttaca ctttatgctt ccggctcgta
     5161 taatgtgtgg ggaaaccaca acggtttccc tctagaaata attttgttta actataagaa
     5221 ggagatatac atatgtcctc tggttaattg aggcctgagt ataaggtgac ttatacttgt
     5281 aatctatcta aacggggaac ctctctagta gacaatcccg tgctaaattg ataccagcat
     5341 cgtcttgatg cccttggcag cataaatgcc taacgactat ccctttgggg agtagggtca
     5401 agtgactcga aacgatagac aacttgcttt aacaagttgg agatatagtc tgctctgcat
     5461 ggtgacatgc agctggatat aattccgggg taagattaac gaccttatct gaacataatg
     5521 ctatcaattt atcaagaatt tgttaataaa tatagtttaa gtaaaactct aagatttgag
     5581 ttaatcccac agggtaaaac acttgaaaac ataaaagcaa gaggtttgat tttagatgat
     5641 gagaaaagag ctaaagacta caaaaaggct aaacaaataa ttgataaata tcatcagttt
     5701 tttatagagg agatattaag ttcggtttgt attagcgaag atttattaca aaactattct
     5761 gatgtttatt ttaaacttaa aaagagtgat gatgataatc tacaaaaaga ttttaaaagt
     5821 gcaaaagata cgataaagaa acaaatatct gaatatataa aggactcaga gaaatttaag
     5881 aatttgttta atcaaaacct tatcgatgct aaaaaagggc aagagtcaga tttaattcta
     5941 tggctaaagc aatctaagga taatggtata gaactattta aagccaatag tgatatcaca
     6001 gatatagatg aggcgttaga aataatcaaa tcttttaaag gttggacaac ttattttaag
     6061 ggttttcatg aaaatagaaa aaatgtttat agtagcaatg atattcctac atctattatt
     6121 tataggatag tagatgataa tttgcctaaa tttctagaaa ataaagctaa gtatgagagt
     6181 ttaaaagaca aagctccaga agctataaac tatgaacaaa ttaaaaaaga tttggcagaa
     6241 gagctaacct ttgatattga ctacaaaaca tctgaagtta atcaaagagt tttttcactt
     6301 gatgaagttt ttgagatagc aaactttaat aattatctaa atcaaagtgg tattactaaa
     6361 tttaatacta ttattggtgg taaatttgta aatggtgaaa atacaaagag aaaaggtata
     6421 aatgaatata taaatctata ctcacagcaa ataaatgata aaacactcaa aaaatataaa
     6481 atgagtgttt tatttaagca aattttaagt gatacagaat ctaaatcttt tgtaattgat
     6541 aagttagaag atgatagtga tgtagttaca acgatgcaaa gtttttatga gcaaatagca
     6601 gcttttaaaa cagtagaaga aaaatctatt aaagaaacac tatctttatt atttgatgat
     6661 ttaaaagctc aaaaacttga tttgagtaaa atttatttta aaaatgataa atctcttact
     6721 gatctatcac aacaagtttt tgatgattat agtgttattg gtacagcggt actagaatat
     6781 ataactcaac aaatagcacc taaaaatctt gataacccta gtaagaaaga gcaagaatta
     6841 atagccaaaa aaactgaaaa agcaaaatac ttatctctag aaactataaa gcttgcctta
     6901 gaagaattta ataagcatag agatatagat aaacagtgta ggtttgaaga aatacttgca
     6961 aactttgcgg ctattccgat gatatttgat gaaatagctc aaaacaaaga caatttggca
     7021 cagatatcta tcaaatatca aaatcaaggt aaaaaagacc tacttcaagc tagtgcggaa
     7081 gatgatgtta aagctatcaa ggatctttta gatcaaacta ataatctctt acataaacta
     7141 aaaatatttc atattagtca gtcagaagat aaggcaaata ttttagacaa ggatgagcat
     7201 ttttatctag tatttgagga gtgct
//
