LOCUS       sequence-218337-        9756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MbCas12 (from M. bovoculi 33362) in bacteria via T7 RNAP, 
            for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9756)
  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 9756)
  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: pKEW336-MBP-TEV-MbCas12a-33362
FEATURES             Location/Qualifiers
     source          1..9756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(147..152,153..169,170..175))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(202..220)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        288..392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(393..461,462..1253)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(611..630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1424..2012
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1913..1932
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2166..2183
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2198..2340
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2266..2285)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2426..2448
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2442..2633)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4091..4110
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4194..4213
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4194..4212
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4301..4323
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4343..4372
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4382..5482
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     5456..5479
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5537..5557
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(9476..9494)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      9490..9537
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
       61 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
      121 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
      181 agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
      241 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      301 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      361 aaatgcttca ataacattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      421 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      481 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      541 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      601 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      661 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      721 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      781 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      841 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      901 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
      961 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1021 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1081 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1141 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1201 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1261 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1321 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1381 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1441 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1501 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1561 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1621 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1681 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1741 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1801 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1861 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1921 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1981 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2041 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2101 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2161 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2221 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     2281 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     2341 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     2401 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2461 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     2521 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     2581 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     2641 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     2701 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     2761 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     2821 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     2881 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     2941 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     3001 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     3061 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc aacgctgccc
     3121 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg
     3181 ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg
     3241 aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac
     3301 cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc
     3361 cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccagtgatcg
     3421 aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta
     3481 cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa
     3541 tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg
     3601 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     3661 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     3721 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     3781 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     3841 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgacgct
     3901 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     3961 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     4021 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     4081 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     4141 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     4201 actcactata gggagaccac aacggtttcc ctctagtgcc ggctccggag agctctttaa
     4261 ttaagcggcc gccctgcagg actcgagttc tagaaataat tttgtttaac tttaagaagg
     4321 agatatacat atgaaatctt ctcaccatca ccatcaccat caccatcacc atggttcttc
     4381 tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     4441 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     4501 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     4561 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     4621 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     4681 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     4741 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     4801 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     4861 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     4921 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     4981 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     5041 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     5101 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     5161 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     5221 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     5281 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     5341 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     5401 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     5461 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     5521 caacctcggg atcgaggaaa acctgtactt ccaatccaat gcaatgttat ttcaagactt
     5581 tactcaccta taccccttat caaaaaccgt gcgctttgaa ttaaagccca ttggtaagac
     5641 attagagcat atccatgcca aaaacttttt gaaccaagat gagaccatgg cggacatgta
     5701 ccaaaaggtg aaggcgatac tggacgatta tcatcgtgat tttatcgctg atatgatggg
     5761 cgaagttaag ctaactaagc tggcagaatt ttatgatgtg tatttaaaat ttagaaaaaa
     5821 tcccaaagac gatggattgc aaaaacagct aaaggacttg caagcagttt taagaaaaga
     5881 gattgtgaag cctatcggta atggaggaaa atacaaggcg ggttatgacc gcttatttgg
     5941 ggcaaagctc tttaaggacg gcaaagagct tggtgatttg gcgaaatttg tcatcgccca
     6001 agagggtgag tcatcgccaa agcttgctca ccttgcccat tttgagaagt ttagcactta
     6061 ctttactggt tttcatgaca accgcaaaaa catgtacagc gatgaggata aacacaccgc
     6121 cattgcctat cgtctgattc atgaaaactt accacgcttt attgacaacc tacaaatact
     6181 ggcaaccatc aaacaaaagc actcggcgtt gtatgaccag attataaatg agctgactgc
     6241 gagtggcttg gatgtgtcgc tggcaagtca tctggacggc tatcacaagc ttttaaccca
     6301 agagggtatc acggcttata ataccctgct tggcggtatc agtggcgagg caggttctcg
     6361 caagatacaa ggtatcaatg agcttatcaa tagtcatcac aaccaacatt gccacaaatc
     6421 cgaacgtatc gccaaattac gccccctaca caaacaaatc ctaagcgacg gcatgggtgt
     6481 gtcatttttg ccaagcaaat ttgccgatga tagcgaagtg tgtcaagcgg tcaatgaatt
     6541 ttaccgccat tatgctgatg tttttgccaa ggtgcaaagc ttatttgatg ggtttgatga
     6601 ctatcaaaag gacggcattt atgttgagta taaaaattta aatgaactat ccaagcaggc
     6661 atttggtgat tttgctttgt tgggtcgggt gctggacggc tattatgtgg atgtggtgaa
     6721 tccagagttt aatgagagat ttgccaaagc caaaaccgac aatgccaaag caaaactgac
     6781 aaaagaaaaa gacaaattca tcaaaggcgt gcattccttg gcaagccttg agcaggcgat
     6841 agagcattat actgctaggc atgatgatga gtctgtgcag gcgggcaaac ttggacagta
     6901 tttcaaacac ggtctggcag gcgtggataa tccaatccaa aaaatacaca acaaccacag
     6961 caccattaag ggttttttgg agcgtgaacg tccagcaggc gagcgagcat tgcccaaaat
     7021 taagtcagac aaaagccctg aaatcaggca actaaaagag ctgctagaca atgccctaaa
     7081 tgtggcgcat tttgccaagt tattaacgac caaaaccacc ctacacaacc aggacggcaa
     7141 tttttatggt gagtttgggg cgttgtatga tgagcttgcc aagattgcca cgctttataa
     7201 caaagtgcgt gattatctgt cgcaaaagcc atttagcacc gaaaaataca aattaaactt
     7261 tggcaatccg acattattaa acgggtggga tttgaataaa gaaaaagata attttggggt
     7321 tatcttacaa aaagacggct gttattattt ggcattattg gataaagctc ataaaaaagt
     7381 atttgataat gctccaaata caggtaaaag cgtttatcaa aaaatgattt ataaattatt
     7441 gcccggacct aataaaatgc tacccaaagt attttttgcc aagagtaatt tggattatta
     7501 taacccttct gctgaactac tggataaata tgcacaaggc acgcacaaaa aaggcgataa
     7561 ttttaatcta aaagactgcc atgcgttgat tgattttttt aaggcgggta ttaataaaca
     7621 tccagaatgg caacattttg gttttaaatt ttcaccaacc agtagctatc aggatttgag
     7681 tgatttttat agagaagtcg aaccacaagg ctatcaagta aaatttgtgg atatcaacgc
     7741 cgattatatt aacgagctgg ttgaacaagg tcagttgtat ttattccaga tttataataa
     7801 agatttctcg cccaaggctc acggtaagcc caatttgcat actttgtatt ttaaggcgtt
     7861 atttagcgaa gataatcttg tcaatccaat ttataaatta aatggtgagg cggagatatt
     7921 ttatcgtaag gcgtctttgg atatgaatga aactaccatt catcgtgcag gtgaggtatt
     7981 agaaaataaa aatcctgata atccaaaaaa gcgtcaattt gtctatgaca tcatcaaaga
     8041 taaacgctat acccaagata agtttatgct acatgtgcct attaccatga attttggtgt
     8101 gcagggcatg acgattaaag aatttaataa aaaagttaat caaagcatac agcaatatga
     8161 tgaggtgaat gtaataggca tagaccgtgg cgagcgacat ttgctatatc tgacagtgat
     8221 taatagcaaa ggcgaaatct tagaacagcg tagtctcaat gacatcacta ccgcatcggc
     8281 aaatggtaca caaatgacca cgccttatca taagatactg gacaagagag aaatagaacg
     8341 cctaaatgct cgtgtcggtt ggggtgagat tgagaccata aaagagctaa aatcgggcta
     8401 tctaagccat gtggtacatc aaatcagcca gcttatgctt aaatataatg ccattgtggt
     8461 gttagaggat ttaaattttg ggtttaagcg tggtcgcttt aaggtggaaa aacaaattta
     8521 ccaaaacttt gaaaacgctc taatcaaaaa gctaaaccat ttggtattaa aagataaggc
     8581 agatgatgag attggctcat ataaaaatgc tctgcaattg accaataatt ttactgatct
     8641 aaaaagcatt ggcaaacaaa cgggcttttt attctatgtg cctgcatgga ataccagcaa
     8701 aatagaccct gaaacgggct ttgtggattt gttaaaacca cgttatgaaa atatcgctca
     8761 gtcgcaagcg ttttttggta aatttgataa gatttgttat aacgcagata ggggttattt
     8821 tgaatttcat attgattatg ccaaatttaa tgacaaagcc aaaaactctc gccaaatatg
     8881 gaagatttgc tcgcatggcg ataaacgcta tgtgtatgac aaaaccgcca atcaaaataa
     8941 aggggcgacc attggtgtta atgtcaatga tgaattaaaa tcgctattta ctcgctatca
     9001 catcaatgac aaacagccaa atttggttat ggatatttgc caaaataacg ataaagaatt
     9061 tcataaatcg ctgatgtatc tattaaagac attgcttgca ttacgatata gtaacgccag
     9121 tagcgatgaa gattttattt tatcgcctgt ggcaaatgat gagggtgtgt tttttaattc
     9181 ggcattggcg gatgatacac agccacaaaa tgcggatgcc aatggggcat atcatatcgc
     9241 attaaagggc ttatggctat taaatgaact aaaaaacagt gatgatttga ataaagtcaa
     9301 acttgccatt gacaatcaaa catggttaaa cttcgctcaa aaccgctaat aaatattgga
     9361 agtggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt accacgcgtg
     9421 cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     9481 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     9541 aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga tcgcgtagtc
     9601 gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc ggtcggacag
     9661 tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc
     9721 atagtgactg gcgatgctgt cggaatggac gatatc
//
