LOCUS       sequence-479782-        7213 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  Expresses the SIBR2.0-818-Cas12a system and a NT crRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7213)
  AUTHORS   Della Valle S, Orsi E, Creutzburg SCA, Jansen LFM, Pentari E, Beisel
            CL, Steel H, Nikel PI, Staals RHJ, Claassens NJ, van der Oost J, 
            Huang WE, Patinios C
  TITLE     Streamlined and efficient genome editing in Cupriavidus necator H16 
            using an optimised SIBR-Cas system
  JOURNAL   bioRxiv 2024.11.24.625072; doi: 10.1101/2024.11.24.625072
REFERENCE   2  (bases 1 to 7213)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.24.625072; doi: 10.1101/2024.11.24.625072"
COMMENT     Sequence Label: pSIBR2.0-818-Cas12a-NT
FEATURES             Location/Qualifiers
     source          1..7213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..816
                     /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(887..1549)
                     /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      1550..2321
                     /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(2436..2522)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2537..2559
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        join(2566..2571,2572..2589,2590..2596)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     promoter        join(6919..6924,6925..6942,6943..6949)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(join(7203..7213,1..9))
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 atgagccata ttcagcgtga aacgagctgt agccgtccgc gtctgaacag caacatggat
       61 gcggatctgt atggctataa atgggcgcgt gataacgtgg gtcagagcgg cgcgaccatt
      121 tatcgtctgt atggcaaacc ggatgcgccg gaactgtttc tgaaacatgg caaaggcagc
      181 gtggcgaacg atgtgaccga tgaaatggtg cgtctgaact ggctgaccga atttatgccg
      241 ctgccgacca ttaaacattt tattcgcacc ccggatgatg cgtggctgct gaccaccgcg
      301 attccgggca aaaccgcgtt tcaggtgctg gaagaatatc cggatagcgg cgaaaacatt
      361 gtggatgcgc tggccgtgtt tctgcgtcgt ctgcatagca ttccggtgtg caactgcccg
      421 tttaacagcg atcgtgtgtt tcgtctggcc caggcgcaga gccgtatgaa caacggcctg
      481 gtggatgcga gcgattttga tgatgaacgt aacggctggc cggtggaaca ggtgtggaaa
      541 gaaatgcata aactgctgcc gtttagcccg gatagcgtgg tgacccacgg cgattttagc
      601 ctggataacc tgattttcga tgaaggcaaa ctgattggct gcattgatgt gggccgtgtg
      661 ggcattgcgg atcgttatca ggatctggcc attctgtgga actgcctggg cgaatttagc
      721 ccgagcctgc aaaaacgtct gtttcagaaa tatggcattg ataatccgga tatgaacaaa
      781 ctgcaatttc atctgatgct ggatgaattt ttctaataat taattggacc gcggtccgag
      841 agacgcatgg cagtatctca gactgtgtat cggcacgtct cactacttac cggcgcggca
      901 gcgttacccg tgtcggcggc tccaacggct cgccatcgtc cagaaaacac ggctcatcgg
      961 gcatcggcag gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca
     1021 ggtctggttc catgcccgga atgccgggct ggctgggcgg ctcctcgccg gggccggtcg
     1081 gtagttgctg ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca
     1141 gcgattcgtc ctggtcgtcg tgatcaacca ccacggcggc actgaacacc gacaggcgca
     1201 actggtcgcg gggctggccc cacgccacgc ggtcattgac cacgtaggcc gacacggtgc
     1261 cggggccgtt gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt
     1321 attggaccgt ccgcaaagaa cgtccgatga gcttggaaag tgttttctgg ctgaccacca
     1381 cggcgttctg gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt
     1441 tcctcgcaat aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc acccagtgac
     1501 cgggcttgtt cttggcttga atgccgattt ctctggactg cgtggccatg cttatctcca
     1561 tgcggtaggg gtgccgcacg gttgcggcac catgcgcaat cagctgcaac ttttcggcag
     1621 cgcgacaaca attatgcgtt gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta
     1681 cgcaatgagc tattgcgggg ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt
     1741 tgttgatttt taagtctttc gcatttcgcc ctatatctag ttctttggtg cccaaagaag
     1801 ggcacccctg cggggttccc ccacgccttc ggcgcggctc cccctccggc aaaaagtggc
     1861 ccctccgggg cttgttgatc gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc
     1921 ccttggaacc cccgcactcg ccgccgtgag gctcgggggg caggcgggcg ggcttcgccc
     1981 ttcgactgcc cccactcgca taggcttggg tcgttccagg cgcgtcaagg ccaagccgct
     2041 gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag
     2101 cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat taaaaaaatt gatggggcaa
     2161 ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg tagccggtgg
     2221 gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg
     2281 gttgtccacg ggccgagcga agcgagccag ccggtggccg ctcgcggcca tcgtccacat
     2341 atccacgggc tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga gagcaagccc
     2401 gtaggggggg cgcgcccagc tgtctagggc ggcggatttg tcctactcag gagagcgttc
     2461 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     2521 tgatgccttt aattaaagcg gataacaatt tcacacagga ggccgtttac actttatgct
     2581 tccggctcgt ataatgtgtg gggaaaccac aacggtttcc ctctagaaat aattttgttt
     2641 aactataaga aggagatata catatgtcaa tttatcaaga atttgttaat aaatatagtt
     2701 taagtaaaac tctaagattt gagttaatcc cacagggtaa aacacttgaa aacataaaag
     2761 caagaggttt gattttagat gatgagaaaa gagctaaaga ctacaaaaag gctaaacaaa
     2821 taattgataa atatcatcag ttttttatag aggagatatt aagttcggtt tgtattagcg
     2881 aagatttatt acaaaactat tctgatgttt attttaaact taaaaagagt gatgatgata
     2941 atctacaaaa agattttaaa agtgcaaaag atacgataaa gaaacaaata tctgaatata
     3001 taaaggactc agagaaattt aagaatttgt ttaatcaaaa ccttatcgat gctaaaaaag
     3061 ggcaagagtc agatttaatt ctatggctaa agcaatctaa ggataatggt atagaactat
     3121 ttaaagccaa tagtgatatc acagatatag atgaggcgtt agaaataatc aaatctttta
     3181 aaggttggac aacttatttt aagggttttc atgaaaatag aaaaaatgtt tatagtagca
     3241 atgatattcc tacatctatt atttatagga tagtagatga taatttgcct aaatttctag
     3301 aaaataaagc taagtatgag agtttaaaag acaaagctcc agaagctata aactatgaac
     3361 aaattaaaaa agatttggca gaagagctaa cctttgatat tgactacaaa acatctgaag
     3421 ttaatcaaag agttttttca cttgatgaag tttttgagat agcaaacttt aataattatc
     3481 taaatcaaag tggtattact aaatttaata ctattattgg tggtaaattt gtaaatggtg
     3541 aaaatacaaa gagaaaaggt ataaatgaat atataaatct atactcacag caaataaatg
     3601 ataaaacact caaaaaatat aaaatgagtg ttttatttaa gcaaatttta agtgatacag
     3661 aatctaaatc ttttgtaatt gataagttag aagatgatag tgatgtagtt acaacgatgc
     3721 aaagttttta tgagcaaata gcagctttta aaacagtaga agaaaaatct attaaagaaa
     3781 cactatcttt attatttgat gatttaaaag ctcaaaaact tgatttgagt aaaatttatt
     3841 ttaaaaatga taaatctctt actgatctat cacaacaagt ttttgatgat tatagtgtta
     3901 ttggtacagc ggtactagaa tatataactc aacaaatagc acctaaaaat cttgataacc
     3961 ctagtaagaa agagcaagaa ttaatagcca aaaaaactga aaaagcaaaa tacttatctc
     4021 tagaaactat aaagcttgcc ttagaagaat ttaataagca tagagatata gataaacagt
     4081 gtaggtttga agaaatactt gcaaactttg cggctattcc gatgatattt gatgaaatag
     4141 ctcaaaacaa agacaatttg gcacagatat ctatcaaata tcaaaatcaa ggtaaaaaag
     4201 acctacttca agctagtgcg gaagatgatg ttaaagctat caaggatctt ttagatcaaa
     4261 ctaataatct cttacataaa ctaaaaatat ttcatattag tcagtcagaa gataaggcaa
     4321 atattttaga caaggatgag catttttatc tagtatttga ggagtgctac tttgagctag
     4381 cgaatatagt gcctctttat aacaaaatta gaaactatat aactcaaaag ccatatagtg
     4441 atgagaaatt taagctcaat tttgagaact cgactttggc taatggttgg gataaaaata
     4501 aagagcctga caatacggca attttattta tcaaagatga taaatattat ctgggtgtga
     4561 tgaataagaa aaataacaaa atatttgatg ataaagctat caaagaaaat aaaggcgagg
     4621 gttataaaaa aattgtttat aaacttttac ctggcgcaaa taaaatgtta cctaaggttt
     4681 tcttttctgc taaatctata aaattttata atcctagtga agatatactt agaataagaa
     4741 atcattccac acatacaaaa aatggtagtc ctcaaaaagg atatgaaaaa tttgagttta
     4801 atattgaaga ttgccgaaaa tttatagatt tttataaaca gtctataagt aagcatccgg
     4861 agtggaaaga ttttggattt agattttctg atactcaaag atataattct atagatgaat
     4921 tttatagaga agttgaaaat caaggctaca aactaacttt tgaaaatata tcagagagct
     4981 atattgatag cgtagttaat cagggtaaat tgtacctatt ccaaatctat aataaagatt
     5041 tttcagctta tagcaaaggg cgaccaaatc tacatacttt atattggaaa gcgctgtttg
     5101 atgagagaaa tcttcaggat gtggttaatt gaggcctgag tataaggtga cttatacttg
     5161 taatctatct aaacggggaa cctctctagt agacaatccc gtgctaaatt gataccagca
     5221 tcgtcttgat gcccttggca gcataaatgc ctaacgacta tccctttggg gagtagggtc
     5281 aagtgactcg aaacgataga caacttgctt taacaagttg gagatatagt ctgctctgca
     5341 tggtgacatg cagctggata taattccggg gtaagattaa cgaccttatc tgaacataat
     5401 gctacaagct aaatggtgag gcagagcttt tttatcgtaa acaatcaata cctaaaaaaa
     5461 tcactcaccc agctaaagag gcaatagcta ataaaaacaa agataatcct aaaaaagaga
     5521 gtgtttttga atatgattta atcaaagata aacgctttac tgaagataag tttttctttc
     5581 actgtcctat tacaatcaat tttaaatcta gtggagctaa taagtttaat gatgaaatca
     5641 atttattgct aaaagaaaaa gcaaatgatg ttcatatatt aagtatagat agaggtgaaa
     5701 gacatttagc ttactatact ttggtagatg gtaaaggcaa tatcatcaaa caagatactt
     5761 tcaacatcat tggtaatgat agaatgaaaa caaactacca tgataagctt gctgcaatag
     5821 agaaagatag ggattcagct aggaaagact ggaaaaagat aaataacatc aaagagatga
     5881 aagagggcta tctatctcag gtagttcatg aaatagctaa gctagttata gagtataatg
     5941 ctattgtggt ttttgaggat ttaaattttg gatttaaaag agggcgtttc aaggtagaga
     6001 agcaggtcta tcaaaagtta gaaaaaatgc taattgagaa actaaactat ctagttttca
     6061 aagataatga gtttgataaa actgggggag tgcttagagc ttatcagcta acagcacctt
     6121 ttgagacttt taaaaagatg ggtaaacaaa caggtattat ctactatgta ccagctggtt
     6181 ttacttcaaa aatttgtcct gtaactggtt ttgtaaatca gttatatcct aagtatgaaa
     6241 gtgtcagcaa atctcaagag ttctttagta agtttgacaa gatttgttat aaccttgata
     6301 agggctattt tgagtttagt tttgattata aaaactttgg tgacaaggct gccaaaggca
     6361 agtggactat agctagcttt gggagtagat tgattaactt tagaaattca gataaaaatc
     6421 ataattggga tactcgagaa gtttatccaa ctaaagagtt ggagaaattg ctaaaagatt
     6481 attctatcga atatgggcat ggcgaatgta tcaaagcagc tatttgcggt gagagcgaca
     6541 aaaagttttt tgctaagcta actagtgtcc taaatactat cttacaaatg cgtaactcaa
     6601 aaacaggtac tgagttagat tatctaattt caccagtagc agatgtaaat ggcaatttct
     6661 ttgattcgcg acaggcgcca aaaaatatgc ctcaagatgc tgatgccaat ggtgcttatc
     6721 atattgggct aaaaggtctg atgctactag gtaggatcaa aaataatcaa gagggcaaaa
     6781 aactcaattt ggttatcaaa aatgaagagt attttgagtt cgtgcagaat aggaataact
     6841 aagcggccgc ataatgatgt gttatcattg atgcgaggtc gcctatacct ccccgcttcg
     6901 gcggggtttt ttcccgggtt tacactttat gcttccggct cgtataatgt gtggtctaag
     6961 aactttaaat aatttctact gttgtagatg agtcttcctg agtgacgagc aacgagactg
     7021 gaagacatgt ctaagaactt taaataattt ctactgttgt agatagatac cggacaacgt
     7081 gtctccccgc ttcggcgggg ttttttccta ggttgtgtct caaaatctct gatgttacat
     7141 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     7201 tacaaggggt gtt
//
