LOCUS       sequence-349052-        7225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SIBR Intron 2 (Int2) 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: pSIBR002
FEATURES             Location/Qualifiers
     source          1..7225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(466..552)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     567..589
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        join(596..601,602..619,620..626)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     promoter        join(4961..4966,4967..4984,4985..4991)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(5245..5264)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5256..6071
                     /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(6142..6804)
                     /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(6805..7225,1..351)
                     /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 ggcttcgccc ttcgactgcc cccactcgca taggcttggg tcgttccagg cgcgtcaagg
       61 ccaagccgct gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg
      121 gcaagcgaag cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat taaaaaaatt
      181 gatggggcaa ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg
      241 tagccggtgg gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt
      301 gtccagcagg gttgtccacg ggccgagcga agcgagccag ccggtggccg ctcgcggcca
      361 tcgtccacat atccacgggc tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga
      421 gagcaagccc gtaggggggg cgcgcccagc tgtctagggc ggcggatttg tcctactcag
      481 gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt
      541 tcgttttatt tgatgccttt aattaaagcg gataacaatt tcacacagga ggccgtttac
      601 actttatgct tccggctcgt ataatgtgtg gggaaaccac aacggtttcc ctctagaaat
      661 aattttgttt aactataaga aggagatata catatgtcct cgggttaatt gaggcctgag
      721 tataaggtga cttatacttg taatctatct aaacggggaa cctctctagt agacaatccc
      781 gtgctaaatt gataccagca tcgtcttgat gcccttggca gcataaatgc ctaacgacta
      841 tccctttggg gagtagggtc aagtgactcg aaacgataga caacttgctt taacaagttg
      901 gagatatagt ctgctctgca tggtgacatg cagctggata taattccggg gtaagattaa
      961 cgaccttatc tgaacataat gctatcaatt tatcaagaat ttgttaataa atatagttta
     1021 agtaaaactc taagatttga gttaatccca cagggtaaaa cacttgaaaa cataaaagca
     1081 agaggtttga ttttagatga tgagaaaaga gctaaagact acaaaaaggc taaacaaata
     1141 attgataaat atcatcagtt ttttatagag gagatattaa gttcggtttg tattagcgaa
     1201 gatttattac aaaactattc tgatgtttat tttaaactta aaaagagtga tgatgataat
     1261 ctacaaaaag attttaaaag tgcaaaagat acgataaaga aacaaatatc tgaatatata
     1321 aaggactcag agaaatttaa gaatttgttt aatcaaaacc ttatcgatgc taaaaaaggg
     1381 caagagtcag atttaattct atggctaaag caatctaagg ataatggtat agaactattt
     1441 aaagccaata gtgatatcac agatatagat gaggcgttag aaataatcaa atcttttaaa
     1501 ggttggacaa cttattttaa gggttttcat gaaaatagaa aaaatgttta tagtagcaat
     1561 gatattccta catctattat ttataggata gtagatgata atttgcctaa atttctagaa
     1621 aataaagcta agtatgagag tttaaaagac aaagctccag aagctataaa ctatgaacaa
     1681 attaaaaaag atttggcaga agagctaacc tttgatattg actacaaaac atctgaagtt
     1741 aatcaaagag ttttttcact tgatgaagtt tttgagatag caaactttaa taattatcta
     1801 aatcaaagtg gtattactaa atttaatact attattggtg gtaaatttgt aaatggtgaa
     1861 aatacaaaga gaaaaggtat aaatgaatat ataaatctat actcacagca aataaatgat
     1921 aaaacactca aaaaatataa aatgagtgtt ttatttaagc aaattttaag tgatacagaa
     1981 tctaaatctt ttgtaattga taagttagaa gatgatagtg atgtagttac aacgatgcaa
     2041 agtttttatg agcaaatagc agcttttaaa acagtagaag aaaaatctat taaagaaaca
     2101 ctatctttat tatttgatga tttaaaagct caaaaacttg atttgagtaa aatttatttt
     2161 aaaaatgata aatctcttac tgatctatca caacaagttt ttgatgatta tagtgttatt
     2221 ggtacagcgg tactagaata tataactcaa caaatagcac ctaaaaatct tgataaccct
     2281 agtaagaaag agcaagaatt aatagccaaa aaaactgaaa aagcaaaata cttatctcta
     2341 gaaactataa agcttgcctt agaagaattt aataagcata gagatataga taaacagtgt
     2401 aggtttgaag aaatacttgc aaactttgcg gctattccga tgatatttga tgaaatagct
     2461 caaaacaaag acaatttggc acagatatct atcaaatatc aaaatcaagg taaaaaagac
     2521 ctacttcaag ctagtgcgga agatgatgtt aaagctatca aggatctttt agatcaaact
     2581 aataatctct tacataaact aaaaatattt catattagtc agtcagaaga taaggcaaat
     2641 attttagaca aggatgagca tttttatcta gtatttgagg agtgctactt tgagctagcg
     2701 aatatagtgc ctctttataa caaaattaga aactatataa ctcaaaagcc atatagtgat
     2761 gagaaattta agctcaattt tgagaactcg actttggcta atggttggga taaaaataaa
     2821 gagcctgaca atacggcaat tttatttatc aaagatgata aatattatct gggtgtgatg
     2881 aataagaaaa ataacaaaat atttgatgat aaagctatca aagaaaataa aggcgagggt
     2941 tataaaaaaa ttgtttataa acttttacct ggcgcaaata aaatgttacc taaggttttc
     3001 ttttctgcta aatctataaa attttataat cctagtgaag atatacttag aataagaaat
     3061 cattccacac atacaaaaaa tggtagtcct caaaaaggat atgaaaaatt tgagtttaat
     3121 attgaagatt gccgaaaatt tatagatttt tataaacagt ctataagtaa gcatccggag
     3181 tggaaagatt ttggatttag attttctgat actcaaagat ataattctat agatgaattt
     3241 tatagagaag ttgaaaatca aggctacaaa ctaacttttg aaaatatatc agagagctat
     3301 attgatagcg tagttaatca gggtaaattg tacctattcc aaatctataa taaagatttt
     3361 tcagcttata gcaaagggcg accaaatcta catactttat attggaaagc gctgtttgat
     3421 gagagaaatc ttcaagatgt ggtttataag ctaaatggtg aggcagagct tttttatcgt
     3481 aaacaatcaa tacctaaaaa aatcactcac ccagctaaag aggcaatagc taataaaaac
     3541 aaagataatc ctaaaaaaga gagtgttttt gaatatgatt taatcaaaga taaacgcttt
     3601 actgaagata agtttttctt tcactgtcct attacaatca attttaaatc tagtggagct
     3661 aataagttta atgatgaaat caatttattg ctaaaagaaa aagcaaatga tgttcatata
     3721 ttaagtatag atagaggtga aagacattta gcttactata ctttggtaga tggtaaaggc
     3781 aatatcatca aacaagatac tttcaacatc attggtaatg atagaatgaa aacaaactac
     3841 catgataagc ttgctgcaat agagaaagat agggattcag ctaggaaaga ctggaaaaag
     3901 ataaataaca tcaaagagat gaaagagggc tatctatctc aggtagttca tgaaatagct
     3961 aagctagtta tagagtataa tgctattgtg gtttttgagg atttaaattt tggatttaaa
     4021 agagggcgtt tcaaggtaga gaagcaggtc tatcaaaagt tagaaaaaat gctaattgag
     4081 aaactaaact atctagtttt caaagataat gagtttgata aaactggggg agtgcttaga
     4141 gcttatcagc taacagcacc ttttgagact tttaaaaaga tgggtaaaca aacaggtatt
     4201 atctactatg taccagctgg ttttacttca aaaatttgtc ctgtaactgg ttttgtaaat
     4261 cagttatatc ctaagtatga aagtgtcagc aaatctcaag agttctttag taagtttgac
     4321 aagatttgtt ataaccttga taagggctat tttgagttta gttttgatta taaaaacttt
     4381 ggtgacaagg ctgccaaagg caagtggact atagctagct ttgggagtag attgattaac
     4441 tttagaaatt cagataaaaa tcataattgg gatactcgag aagtttatcc aactaaagag
     4501 ttggagaaat tgctaaaaga ttattctatc gaatatgggc atggcgaatg tatcaaagca
     4561 gctatttgcg gtgagagcga caaaaagttt tttgctaagc taactagtgt cctaaatact
     4621 atcttacaaa tgcgtaactc aaaaacaggt actgagttag attatctaat ttcaccagta
     4681 gcagatgtaa atggcaattt ctttgattcg cgacaggcgc caaaaaatat gcctcaagat
     4741 gctgatgcca atggtgctta tcatattggg ctaaaaggtc tgatgctact aggtaggatc
     4801 aaaaataatc aagagggcaa aaaactcaat ttggttatca aaaatgaaga gtattttgag
     4861 ttcgtgcaga ataggaataa ctaagcggcc gcataatgat gtgttatcat tgatgcgagg
     4921 tcgcctatac ctccccgctt cggcggggtt ttttcccggg tttacacttt atgcttccgg
     4981 ctcgtataat gtgtggtcta agaactttaa ataatttcta ctgttgtaga tatgtcttcc
     5041 tgagtgacga gcaacgagac tggaagacat gtctaagaac tttaaataat ttctactgtt
     5101 gtagatagat accggacaac gtgtctcccc gcttcggcgg ggttttttcc taggttgtgt
     5161 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     5221 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcag cgtgaaacga
     5281 gctgtagccg tccgcgtctg aacagcaaca tggatgcgga tctgtatggc tataaatggg
     5341 cgcgtgataa cgtgggtcag agcggcgcga ccatttatcg tctgtatggc aaaccggatg
     5401 cgccggaact gtttctgaaa catggcaaag gcagcgtggc gaacgatgtg accgatgaaa
     5461 tggtgcgtct gaactggctg accgaattta tgccgctgcc gaccattaaa cattttattc
     5521 gcaccccgga tgatgcgtgg ctgctgacca ccgcgattcc gggcaaaacc gcgtttcagg
     5581 tgctggaaga atatccggat agcggcgaaa acattgtgga tgcgctggcc gtgtttctgc
     5641 gtcgtctgca tagcattccg gtgtgcaact gcccgtttaa cagcgatcgt gtgtttcgtc
     5701 tggcccaggc gcagagccgt atgaacaacg gcctggtgga tgcgagcgat tttgatgatg
     5761 aacgtaacgg ctggccggtg gaacaggtgt ggaaagaaat gcataaactg ctgccgttta
     5821 gcccggatag cgtggtgacc cacggcgatt ttagcctgga taacctgatt ttcgatgaag
     5881 gcaaactgat tggctgcatt gatgtgggcc gtgtgggcat tgcggatcgt tatcaggatc
     5941 tggccattct gtggaactgc ctgggcgaat ttagcccgag cctgcaaaaa cgtctgtttc
     6001 agaaatatgg cattgataat ccggatatga acaaactgca atttcatctg atgctggatg
     6061 aatttttcta ataattaatt ggaccgcggt ccgagagacg catggcagta tctcagactg
     6121 tgtatcggca cgtctcacta cttaccggcg cggcagcgtt acccgtgtcg gcggctccaa
     6181 cggctcgcca tcgtccagaa aacacggctc atcgggcatc ggcaggcgct gctgcccgcg
     6241 ccgttcccat tcctccgttt cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc
     6301 gggctggctg ggcggctcct cgccggggcc ggtcggtagt tgctgctcgc ccggatacag
     6361 ggtcgggatg cggcgcaggt cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc
     6421 aaccaccacg gcggcactga acaccgacag gcgcaactgg tcgcggggct ggccccacgc
     6481 cacgcggtca ttgaccacgt aggccgacac ggtgccgggg ccgttgagct tcacgacgga
     6541 gatccagcgc tcggccacca agtccttgac tgcgtattgg accgtccgca aagaacgtcc
     6601 gatgagcttg gaaagtgttt tctggctgac caccacggcg ttctggtggc ccatctgcgc
     6661 cacgaggtga tgcagcagca ttgccgccgt gggtttcctc gcaataagcc cggcccacgc
     6721 ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc
     6781 gatttctctg gactgcgtgg ccatgcttat ctccatgcgg taggggtgcc gcacggttgc
     6841 ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta
     6901 aaagtggcag tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc
     6961 cgcaatgagc tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt
     7021 tcgccctata tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg
     7081 ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg
     7141 cgcggccttc ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc
     7201 gtgaggctcg gggggcaggc gggcg
//
