LOCUS       sequence-218336-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MbCas12a in bacteria from M. bovoculi strain 58069, under 
            TetR control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Watters KE, Fellmann C, Bai HB, Ren SM, Doudna JA
  TITLE     Systematic discovery of natural CRISPR-Cas12a inhibitors.
  JOURNAL   Science. 2018 Oct 12;362(6411):236-239. doi: 
            10.1126/science.aau5138. Epub 2018 Sep 6.
  PUBMED    30190307
REFERENCE   2  (bases 1 to 6354)
  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"; doi: 
            "10.1126/science.aau5138"; journalName: "Science"; date: 
            "2018-10-12- 12"; volume: "362"; issue: "6411"; pages: "236-239"
COMMENT     Sequence Label: pKEW106-MbCas12a-58069
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..633)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      4519..4590
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4606..4633
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4661..4678)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4795..5340)
                     /direction=LEFT
                     /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     complement(4875..4894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5454..5548)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5569..6228)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6143..6162
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6229..6331)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taaagaggag aaaggatcta tgttgtttca agactttacc catttatatc ccttatcaaa
      781 aaccgtgcgt tttgaattaa agcccattgg caggacatta gagcatatcc atgccaaaaa
      841 ctttttgagc caagatgaga ccatggcgga catgtaccaa aaggtgaagg tgatactcga
      901 cgattatcat cgtgatttta tcgctgatat gatgggcgaa gttaagctaa ctaagctggc
      961 agaattttat gatgtgtatt taaaatttag aaaaaatccc aaagacgatg aattgcaaaa
     1021 acagctaaag gacttgcaag cagttttaag aaaagagagt gtgaagccca tcggtaatgg
     1081 cggaaaatac aaggcgggtc atgaccgctt gtttggggca aagctcttta aggacggcaa
     1141 agagcttggt gatttggcaa aatttgtcat cgcccaagag ggtaagtcat cgccaaagct
     1201 tgctcacctt gcccattttg agaagtttag cacttacttt actggttttc atgataaccg
     1261 caaaaacatg tacagcgatg aggataaaca caccgccatt gcctatcgtc tgatacatga
     1321 aaacctgcca cgttttattg acaacctaca aatactgaca accatcaaac aaaagcactc
     1381 ggcgttgtat gaccagatta taaatgagct gactgcgagt ggcttggatg tgtcgctggc
     1441 aagtcatctg gacggctatc acaagctttt aacccaagag ggtatcacgg cttataatag
     1501 gataataggt gaagtgaatg gttatacgaa taagcacaat caaatttgcc acaaatccga
     1561 acgtatcgcc aaattacgcc ccctacacaa acaaatccta agcgacggca tgggtgtgtc
     1621 atttttgcca agcaaatttg ctgatgatag cgagatgtgt caagcggtca atgaatttta
     1681 ccgccattat gccgatgttt ttgccaaggt gcaaagcttg tttgatgggt ttgatgacca
     1741 tcaaaaggac ggcatttatg ttgagcataa aaatttaaat gaactatcca agcaggcatt
     1801 tggtgatttt gccttgttgg gtcgggtgct ggacggctat tatgtggatg tggtgaatcc
     1861 agagtttaat gaaagatttg ccaaagccaa aaccgacaat gccaaagcaa aactgactaa
     1921 agaaaaagac aaattcatca aaggcgtaca ctccttggca agccttgagc aggcgataaa
     1981 gcatcatact gctaggcatg atgatgagtc tgtgcaggcg ggcaaacttg gacagtattt
     2041 caaacacggt ctggcaggcg tggataatcc aatccaaaaa atacacaaca accacagcac
     2101 cattaagggt tttttggagc gtgaacgtcc agcaggcgag cgagcattgc ccaaaattaa
     2161 gtcaggcaaa aaccctgaaa tgacgcagtt aagacagcta aaagagctgc tagacaatgc
     2221 cctaaacgtg gcgcattttg ccaagttatt aatgaccaaa accacgctag acaaccaaga
     2281 cggcaatttt tatggtgagt ttggggtgtt gtatgatgag cttgccaaga ttcccacgct
     2341 ttataacaaa gtgcgcgatt atctgtcgca aaagccattt agcaccgaaa aatataaatt
     2401 aaactttggc aatccgacat tattaaacgg gtgggatttg aataaagaaa aagataattt
     2461 tggggttatc ttacaaaaag acggctgtta ttatttggcg ttattagata aagctcataa
     2521 aaaagtattt gataatgctc caaatacagg caaaaatgtt tatcaaaaaa tgatttataa
     2581 attattgccc ggacctaata aaatgctacc cagagtattt tttgccaaga gtaatttgga
     2641 ttattataac ccttctgctg aactactgga taaatatgca caaggcacgc acaaaaaagg
     2701 cgataatttt aatctaaaag actgccatgc gttgattgat ttttttaagg cgggtattaa
     2761 taaacatcca gaatggcaaa attttggttt taaattttcg ccaaccagta gctatcgaga
     2821 tttgagtgat ttttatagag aagtcgaacc acaaggttat caagtaaaat ttgtggatat
     2881 caacgccgat tatattgacg agttggttga acaaggtcag ttgtatttat tccagattta
     2941 taataaagat ttctcgccca aggctcacgg taagcctaat ttacatactt tatattttag
     3001 ggcgttattt agtgaagata atcttgccaa tccgatttat aagttaaatg gcgaggcaca
     3061 gatattttat cgcaaggcgt ctttgggtat gaatgaaacc accattcatc gtgcaggcga
     3121 gatattagaa aataaaaatc ccgataatcc caaagaacgt gtatttacct atgacatcat
     3181 caaagataga cgctataccc aagataagtt tatgctacat gtgcctatta ccatgaattt
     3241 tggcgtgcag ggcatgacga ttaaagaatt taataaaaaa gtgaatcaaa gcatacggca
     3301 atatgatgat gtcaatgtca taggcataga ccgtggcgag cgacatttgc tatatctgac
     3361 cgtgattaat agcaaaggtg aaatcttaga acagcgtagt ctcaatgaca tcactaccgc
     3421 atcagcaaat ggcacacaaa tgaccacgcc ttatcataag atactggaca aaagagaaat
     3481 agaacgccta aatgctcgtg tcggttgggg tgagattgag accataaaag agctaaaatc
     3541 gggctatcta agccatgtgg tacatcaagt cagccagctt atgcttaaat ataatgccat
     3601 tgtggtatta gaggatttaa attttgggtt taagcgtggt cgctttaagg tggaaaaaca
     3661 aatttaccaa aactttgaaa acgccttaat caaaaagcta aaccatttgg aattaaaaga
     3721 taaggcagat gatgagattg gctcatacaa aaatgccctg caattgacca ataattttac
     3781 tgatctaaaa aacattggca aacaaacagg ctttttattc tatgtgcctg catggaatac
     3841 cagtaaaata gaccctgaaa cgggctttgt ggatttgtta aaaccacgtt atgaaaatat
     3901 cgctcagtcg caagcgtttt ttggtaaatt tgataagatt tgttataacg cagataagga
     3961 ttattttgaa tttcatattg attatgccaa atttactgac aaagccaaaa actctcgcca
     4021 aacatggacg atttgctcgc atggcgataa acgttatgtg tatgacaaaa ccgccaatca
     4081 aaataaagga gcgaccaagg gtattaatgt taatgatgaa ttaaaatcgc tatttgctcg
     4141 ctatcacatc aatgaaaaac agccaaattt ggtcatggat atttgccaaa ataacgacaa
     4201 agaatttcat aaatcgctga tgtatctatt aaagacattg cttgcattac gatatagtaa
     4261 cgccagtagc gatgaagatt ttattttatc gcctgtggca aatgatgagg gtgtgttttt
     4321 taattcggca ttggcggatg atacacagcc acaaaatgcg gatgccaatg gggcatatca
     4381 tatcgcatta aagggcttat ggctattaaa tgagctaaaa aacagtgatg atttgaataa
     4441 agtcaaactt gccattgata atcaaacatg gctaaacttc gctcaaaacc gctaactcga
     4501 ctgcaatagt ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     4561 tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt
     4621 gggcctttct gcgtttatac ctagggatat attccgcttc ctcgctcact gactcgctac
     4681 gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg
     4741 aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca
     4801 taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa
     4861 cccgacagga ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc
     4921 tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc
     4981 cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac
     5041 cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5101 aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc
     5161 ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct
     5221 ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc
     5281 tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa acgatctcaa
     5341 gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt tatctcttca
     5401 aatgtagcac ctgaagtcag ccccatacga tataagttgt tactagtgct tggattctca
     5461 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
     5521 gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt acgccccgcc
     5581 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     5641 acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     5701 atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     5761 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
     5821 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     5881 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     5941 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     6001 tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     6061 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     6121 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     6181 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt
     6241 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
     6301 gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaga tatc
//
