LOCUS       sequence-224372-        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"
     terminator      3260..3331
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3347..3374
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3402..3419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3536..4081)
                     /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(3616..3635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4195..4289)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4310..4969)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4884..4903
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4970..5072)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(5102..5728)
                     /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     5633..5652
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5744..5799
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5780..5798
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             5817..5828
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 gcaaaaacat gtacagcgat gaggataaac acaccgccat tgcctatcgt ctgatacatg
       61 aaaacctgcc acgttttatt gacaacctac aaatactgac aaccatcaaa caaaagcact
      121 cggcgttgta tgaccagatt ataaatgagc tgactgcgag tggcttggat gtgtcgctgg
      181 caagtcatct ggacggctat cacaagcttt taacccaaga gggtatcacg gcttataata
      241 ggataatagg tgaagtgaat ggttatacga ataagcacaa tcaaatttgc cacaaatccg
      301 aacgtatcgc caaattacgc cccctacaca aacaaatcct aagcgacggc atgggtgtgt
      361 catttttgcc aagcaaattt gctgatgata gcgagatgtg tcaagcggtc aatgaatttt
      421 accgccatta tgccgatgtt tttgccaagg tgcaaagctt gtttgatggg tttgatgacc
      481 atcaaaagga cggcatttat gttgagcata aaaatttaaa tgaactatcc aagcaggcat
      541 ttggtgattt tgccttgttg ggtcgggtgc tggacggcta ttatgtggat gtggtgaatc
      601 cagagtttaa tgaaagattt gccaaagcca aaaccgacaa tgccaaagca aaactgacta
      661 aagaaaaaga caaattcatc aaaggcgtac actccttggc aagccttgag caggcgataa
      721 agcatcatac tgctaggcat gatgatgagt ctgtgcaggc gggcaaactt ggacagtatt
      781 tcaaacacgg tctggcaggc gtggataatc caatccaaaa aatacacaac aaccacagca
      841 ccattaaggg ttttttggag cgtgaacgtc cagcaggcga gcgagcattg cccaaaatta
      901 agtcaggcaa aaaccctgaa atgacgcagt taagacagct aaaagagctg ctagacaatg
      961 ccctaaacgt ggcgcatttt gccaagttat taatgaccaa aaccacgcta gacaaccaag
     1021 acggcaattt ttatggtgag tttggggtgt tgtatgatga gcttgccaag attcccacgc
     1081 tttataacaa agtgcgcgat tatctgtcgc aaaagccatt tagcaccgaa aaatataaat
     1141 taaactttgg caatccgaca ttattaaacg ggtgggattt gaataaagaa aaagataatt
     1201 ttggggttat cttacaaaaa gacggctgtt attatttggc gttattagat aaagctcata
     1261 aaaaagtatt tgataatgct ccaaatacag gcaaaaatgt ttatcaaaaa atgatttata
     1321 aattattgcc cggacctaat aaaatgctac ccagagtatt ttttgccaag agtaatttgg
     1381 attattataa cccttctgct gaactactgg ataaatatgc acaaggcacg cacaaaaaag
     1441 gcgataattt taatctaaaa gactgccatg cgttgattga tttttttaag gcgggtatta
     1501 ataaacatcc agaatggcaa aattttggtt ttaaattttc gccaaccagt agctatcgag
     1561 atttgagtga tttttataga gaagtcgaac cacaaggtta tcaagtaaaa tttgtggata
     1621 tcaacgccga ttatattgac gagttggttg aacaaggtca gttgtattta ttccagattt
     1681 ataataaaga tttctcgccc aaggctcacg gtaagcctaa tttacatact ttatatttta
     1741 gggcgttatt tagtgaagat aatcttgcca atccgattta taagttaaat ggcgaggcac
     1801 agatatttta tcgcaaggcg tctttgggta tgaatgaaac caccattcat cgtgcaggcg
     1861 agatattaga aaataaaaat cccgataatc ccaaagaacg tgtatttacc tatgacatca
     1921 tcaaagatag acgctatacc caagataagt ttatgctaca tgtgcctatt accatgaatt
     1981 ttggcgtgca gggcatgacg attaaagaat ttaataaaaa agtgaatcaa agcatacggc
     2041 aatatgatga tgtcaatgtc ataggcatag accgtggcga gcgacatttg ctatatctga
     2101 ccgtgattaa tagcaaaggt gaaatcttag aacagcgtag tctcaatgac atcactaccg
     2161 catcagcaaa tggcacacaa atgaccacgc cttatcataa gatactggac aaaagagaaa
     2221 tagaacgcct aaatgctcgt gtcggttggg gtgagattga gaccataaaa gagctaaaat
     2281 cgggctatct aagccatgtg gtacatcaag tcagccagct tatgcttaaa tataatgcca
     2341 ttgtggtatt agaggattta aattttgggt ttaagcgtgg tcgctttaag gtggaaaaac
     2401 aaatttacca aaactttgaa aacgccttaa tcaaaaagct aaaccatttg gaattaaaag
     2461 ataaggcaga tgatgagatt ggctcataca aaaatgccct gcaattgacc aataatttta
     2521 ctgatctaaa aaacattggc aaacaaacag gctttttatt ctatgtgcct gcatggaata
     2581 ccagtaaaat agaccctgaa acgggctttg tggatttgtt aaaaccacgt tatgaaaata
     2641 tcgctcagtc gcaagcgttt tttggtaaat ttgataagat ttgttataac gcagataagg
     2701 attattttga atttcatatt gattatgcca aatttactga caaagccaaa aactctcgcc
     2761 aaacatggac gatttgctcg catggcgata aacgttatgt gtatgacaaa accgccaatc
     2821 aaaataaagg agcgaccaag ggtattaatg ttaatgatga attaaaatcg ctatttgctc
     2881 gctatcacat caatgaaaaa cagccaaatt tggtcatgga tatttgccaa aataacgaca
     2941 aagaatttca taaatcgctg atgtatctat taaagacatt gcttgcatta cgatatagta
     3001 acgccagtag cgatgaagat tttattttat cgcctgtggc aaatgatgag ggtgtgtttt
     3061 ttaattcggc attggcggat gatacacagc cacaaaatgc ggatgccaat ggggcatatc
     3121 atatcgcatt aaagggctta tggctattaa atgagctaaa aaacagtgat gatttgaata
     3181 aagtcaaact tgccattgat aatcaaacat ggctaaactt cgctcaaaac cgctaactcg
     3241 actgcaatag tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     3301 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     3361 tgggcctttc tgcgtttata cctagggata tattccgctt cctcgctcac tgactcgcta
     3421 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     3481 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
     3541 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
     3601 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
     3661 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
     3721 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
     3781 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3841 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
     3901 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
     3961 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
     4021 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     4081 agaagatcat cttattaatc agataaaata tttctagatt tcagtgcaat ttatctcttc
     4141 aaatgtagca cctgaagtca gccccatacg atataagttg ttactagtgc ttggattctc
     4201 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     4261 ggtcattact ggatctatca acaggagtcc aagcgagctc gatatcaaat tacgccccgc
     4321 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     4381 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     4441 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     4501 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     4561 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     4621 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     4681 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     4741 ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     4801 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     4861 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     4921 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     4981 tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat
     5041 ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccag atatcgacgt
     5101 cttaagaccc actttcacat ttaagttgtt tttctaatcc gcatatgatc aattcaaggc
     5161 cgaataagaa ggctggctct gcaccttggt gatcaaataa ttcgatagct tgtcgtaata
     5221 atggcggcat actatcagta gtaggtgttt ccctttcttc tttagcgact tgatgctctt
     5281 gatcttccaa tacgcaacct aaagtaaaat gccccacagc gctgagtgca tataatgcat
     5341 tctctagtga aaaaccttgt tggcataaaa aggctaattg attttcgaga gtttcatact
     5401 gtttttctgt aggccgtgta cctaaatgta cttttgctcc atcgcgatga cttagtaaag
     5461 cacatctaaa acttttagcg ttattacgta aaaaatcttg ccagctttcc ccttctaaag
     5521 ggcaaaagtg agtatggtgc ctatctaaca tctcaatggc taaggcgtcg agcaaagccc
     5581 gcttattttt tacatgccaa tacaatgtag gctgctctac acctagcttc tgggcgagtt
     5641 tacgggttgt taaaccttcg attccgacct cattaagcag ctctaatgcg ctgttaatca
     5701 ctttactttt atctaatcta gacatcatta attcctaatt tttgttgaca ctctatcgtt
     5761 gatagagtta ttttaccact ccctatcagt gatagagaaa agaattcaaa agatctaaag
     5821 aggagaaagg atctatgttg tttcaagact ttacccattt atatccctta tcaaaaaccg
     5881 tgcgttttga attaaagccc attggcagga cattagagca tatccatgcc aaaaactttt
     5941 tgagccaaga tgagaccatg gcggacatgt accaaaaggt gaaggtgata ctcgacgatt
     6001 atcatcgtga ttttatcgct gatatgatgg gcgaagttaa gctaactaag ctggcagaat
     6061 tttatgatgt gtatttaaaa tttagaaaaa atcccaaaga cgatgaattg caaaaacagc
     6121 taaaggactt gcaagcagtt ttaagaaaag agagtgtgaa gcccatcggt aatggcggaa
     6181 aatacaaggc gggtcatgac cgcttgtttg gggcaaagct ctttaaggac ggcaaagagc
     6241 ttggtgattt ggcaaaattt gtcatcgccc aagagggtaa gtcatcgcca aagcttgctc
     6301 accttgccca ttttgagaag tttagcactt actttactgg ttttcatgat aacc
//
