LOCUS       sequence-218335-        6384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MbCas12a in bacteria from M. bovoculi strain 33362, under 
            TetR control.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6384)
  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 6384)
  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: pKEW107-MbCas12a-33362
FEATURES             Location/Qualifiers
     source          1..6384
                     /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      4549..4620
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4636..4663
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4691..4708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4825..5370)
                     /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(4905..4924)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5484..5578)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5599..6258)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6173..6192
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6259..6361)
                     /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 tgttatttca agactttact cacctatacc ccttatcaaa
      781 aaccgtgcgc tttgaattaa agcccattgg taagacatta gagcatatcc atgccaaaaa
      841 ctttttgaac caagatgaga ccatggcgga catgtaccaa aaggtgaagg cgatactgga
      901 cgattatcat cgtgatttta tcgctgatat gatgggcgaa gttaagctaa ctaagctggc
      961 agaattttat gatgtgtatt taaaatttag aaaaaatccc aaagacgatg gattgcaaaa
     1021 acagctaaag gacttgcaag cagttttaag aaaagagatt gtgaagccta tcggtaatgg
     1081 aggaaaatac aaggcgggtt atgaccgctt atttggggca aagctcttta aggacggcaa
     1141 agagcttggt gatttggcga aatttgtcat cgcccaagag ggtgagtcat cgccaaagct
     1201 tgctcacctt gcccattttg agaagtttag cacttacttt actggttttc atgacaaccg
     1261 caaaaacatg tacagcgatg aggataaaca caccgccatt gcctatcgtc tgattcatga
     1321 aaacttacca cgctttattg acaacctaca aatactggca accatcaaac aaaagcactc
     1381 ggcgttgtat gaccagatta taaatgagct gactgcgagt ggcttggatg tgtcgctggc
     1441 aagtcatctg gacggctatc acaagctttt aacccaagag ggtatcacgg cttataatac
     1501 cctgcttggc ggtatcagtg gcgaggcagg ttctcgcaag atacaaggta tcaatgagct
     1561 tatcaatagt catcacaacc aacattgcca caaatccgaa cgtatcgcca aattacgccc
     1621 cctacacaaa caaatcctaa gcgacggcat gggtgtgtca tttttgccaa gcaaatttgc
     1681 cgatgatagc gaagtgtgtc aagcggtcaa tgaattttac cgccattatg ctgatgtttt
     1741 tgccaaggtg caaagcttat ttgatgggtt tgatgactat caaaaggacg gcatttatgt
     1801 tgagtataaa aatttaaatg aactatccaa gcaggcattt ggtgattttg ctttgttggg
     1861 tcgggtgctg gacggctatt atgtggatgt ggtgaatcca gagtttaatg agagatttgc
     1921 caaagccaaa accgacaatg ccaaagcaaa actgacaaaa gaaaaagaca aattcatcaa
     1981 aggcgtgcat tccttggcaa gccttgagca ggcgatagag cattatactg ctaggcatga
     2041 tgatgagtct gtgcaggcgg gcaaacttgg acagtatttc aaacacggtc tggcaggcgt
     2101 ggataatcca atccaaaaaa tacacaacaa ccacagcacc attaagggtt ttttggagcg
     2161 tgaacgtcca gcaggcgagc gagcattgcc caaaattaag tcagacaaaa gccctgaaat
     2221 caggcaacta aaagagctgc tagacaatgc cctaaatgtg gcgcattttg ccaagttatt
     2281 aacgaccaaa accaccctac acaaccagga cggcaatttt tatggtgagt ttggggcgtt
     2341 gtatgatgag cttgccaaga ttgccacgct ttataacaaa gtgcgtgatt atctgtcgca
     2401 aaagccattt agcaccgaaa aatacaaatt aaactttggc aatccgacat tattaaacgg
     2461 gtgggatttg aataaagaaa aagataattt tggggttatc ttacaaaaag acggctgtta
     2521 ttatttggca ttattggata aagctcataa aaaagtattt gataatgctc caaatacagg
     2581 taaaagcgtt tatcaaaaaa tgatttataa attattgccc ggacctaata aaatgctacc
     2641 caaagtattt tttgccaaga gtaatttgga ttattataac ccttctgctg aactactgga
     2701 taaatatgca caaggcacgc acaaaaaagg cgataatttt aatctaaaag actgccatgc
     2761 gttgattgat ttttttaagg cgggtattaa taaacatcca gaatggcaac attttggttt
     2821 taaattttca ccaaccagta gctatcagga tttgagtgat ttttatagag aagtcgaacc
     2881 acaaggctat caagtaaaat ttgtggatat caacgccgat tatattaacg agctggttga
     2941 acaaggtcag ttgtatttat tccagattta taataaagat ttctcgccca aggctcacgg
     3001 taagcccaat ttgcatactt tgtattttaa ggcgttattt agcgaagata atcttgtcaa
     3061 tccaatttat aaattaaatg gtgaggcgga gatattttat cgtaaggcgt ctttggatat
     3121 gaatgaaact accattcatc gtgcaggtga ggtattagaa aataaaaatc ctgataatcc
     3181 aaaaaagcgt caatttgtct atgacatcat caaagataaa cgctataccc aagataagtt
     3241 tatgctacat gtgcctatta ccatgaattt tggtgtgcag ggcatgacga ttaaagaatt
     3301 taataaaaaa gttaatcaaa gcatacagca atatgatgag gtgaatgtaa taggcataga
     3361 ccgtggcgag cgacatttgc tatatctgac agtgattaat agcaaaggcg aaatcttaga
     3421 acagcgtagt ctcaatgaca tcactaccgc atcggcaaat ggtacacaaa tgaccacgcc
     3481 ttatcataag atactggaca agagagaaat agaacgccta aatgctcgtg tcggttgggg
     3541 tgagattgag accataaaag agctaaaatc gggctatcta agccatgtgg tacatcaaat
     3601 cagccagctt atgcttaaat ataatgccat tgtggtgtta gaggatttaa attttgggtt
     3661 taagcgtggt cgctttaagg tggaaaaaca aatttaccaa aactttgaaa acgctctaat
     3721 caaaaagcta aaccatttgg tattaaaaga taaggcagat gatgagattg gctcatataa
     3781 aaatgctctg caattgacca ataattttac tgatctaaaa agcattggca aacaaacggg
     3841 ctttttattc tatgtgcctg catggaatac cagcaaaata gaccctgaaa cgggctttgt
     3901 ggatttgtta aaaccacgtt atgaaaatat cgctcagtcg caagcgtttt ttggtaaatt
     3961 tgataagatt tgttataacg cagatagggg ttattttgaa tttcatattg attatgccaa
     4021 atttaatgac aaagccaaaa actctcgcca aatatggaag atttgctcgc atggcgataa
     4081 acgctatgtg tatgacaaaa ccgccaatca aaataaaggg gcgaccattg gtgttaatgt
     4141 caatgatgaa ttaaaatcgc tatttactcg ctatcacatc aatgacaaac agccaaattt
     4201 ggttatggat atttgccaaa ataacgataa agaatttcat aaatcgctga tgtatctatt
     4261 aaagacattg cttgcattac gatatagtaa cgccagtagc gatgaagatt ttattttatc
     4321 gcctgtggca aatgatgagg gtgtgttttt taattcggca ttggcggatg atacacagcc
     4381 acaaaatgcg gatgccaatg gggcatatca tatcgcatta aagggcttat ggctattaaa
     4441 tgaactaaaa aacagtgatg atttgaataa agtcaaactt gccattgaca atcaaacatg
     4501 gttaaacttc gctcaaaacc gctaactcga ctgcaatagt ccaggcatca aataaaacga
     4561 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     4621 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatac ctagggatat
     4681 attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa
     4741 tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag
     4801 tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga
     4861 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4921 tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat
     4981 tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag
     5041 ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat
     5101 ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag
     5161 ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact
     5221 gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg
     5281 gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa
     5341 gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat
     5401 ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     5461 tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc
     5521 gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca
     5581 agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     5641 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     5701 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     5761 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     5821 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     5881 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     5941 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     6001 atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat
     6061 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     6121 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     6181 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     6241 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     6301 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     6361 aacgtctcat tttcgccaga tatc
//
