LOCUS       sequence-225437-        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"
     terminator      3268..3339
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3355..3382
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3410..3427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3544..4089)
                     /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(3624..3643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4203..4297)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4318..4977)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4892..4911
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4978..5080)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(5110..5736)
                     /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     5641..5660
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        5752..5807
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5788..5806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             5825..5836
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 ggataaacac accgccattg cctatcgtct gattcatgaa aacttaccac gctttattga
       61 caacctacaa atactggcaa ccatcaaaca aaagcactcg gcgttgtatg accagattat
      121 aaatgagctg actgcgagtg gcttggatgt gtcgctggca agtcatctgg acggctatca
      181 caagctttta acccaagagg gtatcacggc ttataatacc ctgcttggcg gtatcagtgg
      241 cgaggcaggt tctcgcaaga tacaaggtat caatgagctt atcaatagtc atcacaacca
      301 acattgccac aaatccgaac gtatcgccaa attacgcccc ctacacaaac aaatcctaag
      361 cgacggcatg ggtgtgtcat ttttgccaag caaatttgcc gatgatagcg aagtgtgtca
      421 agcggtcaat gaattttacc gccattatgc tgatgttttt gccaaggtgc aaagcttatt
      481 tgatgggttt gatgactatc aaaaggacgg catttatgtt gagtataaaa atttaaatga
      541 actatccaag caggcatttg gtgattttgc tttgttgggt cgggtgctgg acggctatta
      601 tgtggatgtg gtgaatccag agtttaatga gagatttgcc aaagccaaaa ccgacaatgc
      661 caaagcaaaa ctgacaaaag aaaaagacaa attcatcaaa ggcgtgcatt ccttggcaag
      721 ccttgagcag gcgatagagc attatactgc taggcatgat gatgagtctg tgcaggcggg
      781 caaacttgga cagtatttca aacacggtct ggcaggcgtg gataatccaa tccaaaaaat
      841 acacaacaac cacagcacca ttaagggttt tttggagcgt gaacgtccag caggcgagcg
      901 agcattgccc aaaattaagt cagacaaaag ccctgaaatc aggcaactaa aagagctgct
      961 agacaatgcc ctaaatgtgg cgcattttgc caagttatta acgaccaaaa ccaccctaca
     1021 caaccaggac ggcaattttt atggtgagtt tggggcgttg tatgatgagc ttgccaagat
     1081 tgccacgctt tataacaaag tgcgtgatta tctgtcgcaa aagccattta gcaccgaaaa
     1141 atacaaatta aactttggca atccgacatt attaaacggg tgggatttga ataaagaaaa
     1201 agataatttt ggggttatct tacaaaaaga cggctgttat tatttggcat tattggataa
     1261 agctcataaa aaagtatttg ataatgctcc aaatacaggt aaaagcgttt atcaaaaaat
     1321 gatttataaa ttattgcccg gacctaataa aatgctaccc aaagtatttt ttgccaagag
     1381 taatttggat tattataacc cttctgctga actactggat aaatatgcac aaggcacgca
     1441 caaaaaaggc gataatttta atctaaaaga ctgccatgcg ttgattgatt tttttaaggc
     1501 gggtattaat aaacatccag aatggcaaca ttttggtttt aaattttcac caaccagtag
     1561 ctatcaggat ttgagtgatt tttatagaga agtcgaacca caaggctatc aagtaaaatt
     1621 tgtggatatc aacgccgatt atattaacga gctggttgaa caaggtcagt tgtatttatt
     1681 ccagatttat aataaagatt tctcgcccaa ggctcacggt aagcccaatt tgcatacttt
     1741 gtattttaag gcgttattta gcgaagataa tcttgtcaat ccaatttata aattaaatgg
     1801 tgaggcggag atattttatc gtaaggcgtc tttggatatg aatgaaacta ccattcatcg
     1861 tgcaggtgag gtattagaaa ataaaaatcc tgataatcca aaaaagcgtc aatttgtcta
     1921 tgacatcatc aaagataaac gctataccca agataagttt atgctacatg tgcctattac
     1981 catgaatttt ggtgtgcagg gcatgacgat taaagaattt aataaaaaag ttaatcaaag
     2041 catacagcaa tatgatgagg tgaatgtaat aggcatagac cgtggcgagc gacatttgct
     2101 atatctgaca gtgattaata gcaaaggcga aatcttagaa cagcgtagtc tcaatgacat
     2161 cactaccgca tcggcaaatg gtacacaaat gaccacgcct tatcataaga tactggacaa
     2221 gagagaaata gaacgcctaa atgctcgtgt cggttggggt gagattgaga ccataaaaga
     2281 gctaaaatcg ggctatctaa gccatgtggt acatcaaatc agccagctta tgcttaaata
     2341 taatgccatt gtggtgttag aggatttaaa ttttgggttt aagcgtggtc gctttaaggt
     2401 ggaaaaacaa atttaccaaa actttgaaaa cgctctaatc aaaaagctaa accatttggt
     2461 attaaaagat aaggcagatg atgagattgg ctcatataaa aatgctctgc aattgaccaa
     2521 taattttact gatctaaaaa gcattggcaa acaaacgggc tttttattct atgtgcctgc
     2581 atggaatacc agcaaaatag accctgaaac gggctttgtg gatttgttaa aaccacgtta
     2641 tgaaaatatc gctcagtcgc aagcgttttt tggtaaattt gataagattt gttataacgc
     2701 agataggggt tattttgaat ttcatattga ttatgccaaa tttaatgaca aagccaaaaa
     2761 ctctcgccaa atatggaaga tttgctcgca tggcgataaa cgctatgtgt atgacaaaac
     2821 cgccaatcaa aataaagggg cgaccattgg tgttaatgtc aatgatgaat taaaatcgct
     2881 atttactcgc tatcacatca atgacaaaca gccaaatttg gttatggata tttgccaaaa
     2941 taacgataaa gaatttcata aatcgctgat gtatctatta aagacattgc ttgcattacg
     3001 atatagtaac gccagtagcg atgaagattt tattttatcg cctgtggcaa atgatgaggg
     3061 tgtgtttttt aattcggcat tggcggatga tacacagcca caaaatgcgg atgccaatgg
     3121 ggcatatcat atcgcattaa agggcttatg gctattaaat gaactaaaaa acagtgatga
     3181 tttgaataaa gtcaaacttg ccattgacaa tcaaacatgg ctaaacttcg ctcaaaaccg
     3241 ctaactcgac tgcaatagtc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     3301 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca
     3361 ccttcgggtg ggcctttctg cgtttatacc tagggatata ttccgcttcc tcgctcactg
     3421 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     3481 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     3541 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     3601 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     3661 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     3721 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     3781 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3841 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     3901 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     3961 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     4021 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     4081 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt
     4141 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgctt
     4201 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     4261 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta
     4321 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     4381 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     4441 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
     4501 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     4561 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     4621 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     4681 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     4741 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat
     4801 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     4861 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     4921 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     4981 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     5041 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat
     5101 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     5161 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     5221 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     5281 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     5341 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     5401 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     5461 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     5521 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     5581 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     5641 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     5701 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     5761 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     5821 atctaaagag gagaaaggat ctatgttatt tcaagacttt actcacctat accccttatc
     5881 aaaaaccgtg cgctttgaat taaagcccat tggtaagaca ttagagcata tccatgccaa
     5941 aaactttttg aaccaagatg agaccatggc ggacatgtac caaaaggtga aggcgatact
     6001 ggacgattat catcgtgatt ttatcgctga tatgatgggc gaagttaagc taactaagct
     6061 ggcagaattt tatgatgtgt atttaaaatt tagaaaaaat cccaaagacg atggattgca
     6121 aaaacagcta aaggacttgc aagcagtttt aagaaaagag attgtgaagc ctatcggtaa
     6181 tggaggaaaa tacaaggcgg gttatgaccg cttatttggg gcaaagctct ttaaggacgg
     6241 caaagagctt ggtgatttgg cgaaatttgt catcgcccaa gagggtgagt catcgccaaa
     6301 gcttgctcac cttgcccatt ttgagaagtt tagcacttac tttactggtt ttcatgacaa
     6361 ccgcaaaaac atgtacagcg atga
//
