LOCUS       sequence-226247-        9753 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 9753)
  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 9753)
  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..9753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     85..104
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     188..207
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        188..206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             295..317
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             337..366
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             376..1476
                     /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     1450..1473
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1531..1551
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5470..5488)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5484..5531
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5897..5902,5903..5919,5920..5925))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5952..5970)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6038..6142
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6143..6211,6212..7003)
                     /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(6361..6380)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7174..7762
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7663..7682
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7916..7933
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7948..8087
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8013..8032)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8173..8195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8189..8380)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
       61 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
      121 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
      181 gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag tgccggctcc
      241 ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa taattttgtt
      301 taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca ccatcaccat
      361 caccatggtt cttctatgaa aatcgaagaa ggtaaactgg taatctggat taacggcgat
      421 aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa
      481 gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc
      541 gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc
      601 ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg
      661 gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg
      721 ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg
      781 ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg
      841 tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc
      901 aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc
      961 ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa
     1021 gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac
     1081 atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca
     1141 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag
     1201 ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat
     1261 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat
     1321 ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg
     1381 cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt
     1441 cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa caacaacaac
     1501 aataacaata acaacaacct cgggatcgag gaaaacctgt acttccaatc caatgcaatg
     1561 ttatttcaag actttactca cctatacccc ttatcaaaaa ccgtgcgctt tgaattaaag
     1621 cccattggta agacattaga gcatatccat gccaaaaact ttttgaacca agatgagacc
     1681 atggcggaca tgtaccaaaa ggtgaaggcg atactggacg attatcatcg tgattttatc
     1741 gctgatatga tgggcgaagt taagctaact aagctggcag aattttatga tgtgtattta
     1801 aaatttagaa aaaatcccaa agacgatgga ttgcaaaaac agctaaagga cttgcaagca
     1861 gttttaagaa aagagattgt gaagcctatc ggtaatggag gaaaatacaa ggcgggttat
     1921 gaccgcttat ttggggcaaa gctctttaag gacggcaaag agcttggtga tttggcgaaa
     1981 tttgtcatcg cccaagaggg tgagtcatcg ccaaagcttg ctcaccttgc ccattttgag
     2041 aagtttagca cttactttac tggttttcat gacaaccgca aaaacatgta cagcgatgag
     2101 gataaacaca ccgccattgc ctatcgtctg attcatgaaa acttaccacg ctttattgac
     2161 aacctacaaa tactggcaac catcaaacaa aagcactcgg cgttgtatga ccagattata
     2221 aatgagctga ctgcgagtgg cttggatgtg tcgctggcaa gtcatctgga cggctatcac
     2281 aagcttttaa cccaagaggg tatcacggct tataataccc tgcttggcgg tatcagtggc
     2341 gaggcaggtt ctcgcaagat acaaggtatc aatgagctta tcaatagtca tcacaaccaa
     2401 cattgccaca aatccgaacg tatcgccaaa ttacgccccc tacacaaaca aatcctaagc
     2461 gacggcatgg gtgtgtcatt tttgccaagc aaatttgccg atgatagcga agtgtgtcaa
     2521 gcggtcaatg aattttaccg ccattatgct gatgtttttg ccaaggtgca aagcttattt
     2581 gatgggtttg atgactatca aaaggacggc atttatgttg agtataaaaa tttaaatgaa
     2641 ctatccaagc aggcatttgg tgattttgct ttgttgggtc gggtgctgga cggctattat
     2701 gtggatgtgg tgaatccaga gtttaatgag agatttgcca aagccaaaac cgacaatgcc
     2761 aaagcaaaac tgacaaaaga aaaagacaaa ttcatcaaag gcgtgcattc cttggcaagc
     2821 cttgagcagg cgatagagca ttatactgct aggcatgatg atgagtctgt gcaggcgggc
     2881 aaacttggac agtatttcaa acacggtctg gcaggcgtgg ataatccaat ccaaaaaata
     2941 cacaacaacc acagcaccat taagggtttt ttggagcgtg aacgtccagc aggcgagcga
     3001 gcattgccca aaattaagtc agacaaaagc cctgaaatca ggcaactaaa agagctgcta
     3061 gacaatgccc taaatgtggc gcattttgcc aagttattaa cgaccaaaac caccctacac
     3121 aaccaggacg gcaattttta tggtgagttt ggggcgttgt atgatgagct tgccaagatt
     3181 gccacgcttt ataacaaagt gcgtgattat ctgtcgcaaa agccatttag caccgaaaaa
     3241 tacaaattaa actttggcaa tccgacatta ttaaacgggt gggatttgaa taaagaaaaa
     3301 gataattttg gggttatctt acaaaaagac ggctgttatt atttggcatt attggataaa
     3361 gctcataaaa aagtatttga taatgctcca aatacaggta aaagcgttta tcaaaaaatg
     3421 atttataaat tattgcccgg acctaataaa atgctaccca aagtattttt tgccaagagt
     3481 aatttggatt attataaccc ttctgctgaa ctactggata aatatgcaca aggcacgcac
     3541 aaaaaaggcg ataattttaa tctaaaagac tgccatgcgt tgattgattt ttttaaggcg
     3601 ggtattaata aacatccaga atggcaacat tttggtttta aattttcacc aaccagtagc
     3661 tatcaggatt tgagtgattt ttatagagaa gtcgaaccac aaggctatca agtaaaattt
     3721 gtggatatca acgccgatta tattaacgag ctggttgaac aaggtcagtt gtatttattc
     3781 cagatttata ataaagattt ctcgcccaag gctcacggta agcccaattt gcatactttg
     3841 tattttaagg cgttatttag cgaagataat cttgtcaatc caatttataa attaaatggt
     3901 gaggcggaga tattttatcg taaggcgtct ttggatatga atgaaactac cattcatcgt
     3961 gcaggtgagg tattagaaaa taaaaatcct gataatccaa aaaagcgtca atttgtctat
     4021 gacatcatca aagataaacg ctatacccaa gataagttta tgctacatgt gcctattacc
     4081 atgaattttg gtgtgcaggg catgacgatt aaagaattta ataaaaaagt taatcaaagc
     4141 atacagcaat atgatgaggt gaatgtaata ggcatagacc gtggcgagcg acatttgcta
     4201 tatctgacag tgattaatag caaaggcgaa atcttagaac agcgtagtct caatgacatc
     4261 actaccgcat cggcaaatgg tacacaaatg accacgcctt atcataagat actggacaag
     4321 agagaaatag aacgcctaaa tgctcgtgtc ggttggggtg agattgagac cataaaagag
     4381 ctaaaatcgg gctatctaag ccatgtggta catcaaatca gccagcttat gcttaaatat
     4441 aatgccattg tggtgttaga ggatttaaat tttgggttta agcgtggtcg ctttaaggtg
     4501 gaaaaacaaa tttaccaaaa ctttgaaaac gctctaatca aaaagctaaa ccatttggta
     4561 ttaaaagata aggcagatga tgagattggc tcatataaaa atgctctgca attgaccaat
     4621 aattttactg atctaaaaag cattggcaaa caaacgggct ttttattcta tgtgcctgca
     4681 tggaatacca gcaaaataga ccctgaaacg ggctttgtgg atttgttaaa accacgttat
     4741 gaaaatatcg ctcagtcgca agcgtttttt ggtaaatttg ataagatttg ttataacgca
     4801 gataggggtt attttgaatt tcatattgat tatgccaaat ttaatgacaa agccaaaaac
     4861 tctcgccaaa tatggaagat ttgctcgcat ggcgataaac gctatgtgta tgacaaaacc
     4921 gccaatcaaa ataaaggggc gaccattggt gttaatgtca atgatgaatt aaaatcgcta
     4981 tttactcgct atcacatcaa tgacaaacag ccaaatttgg ttatggatat ttgccaaaat
     5041 aacgataaag aatttcataa atcgctgatg tatctattaa agacattgct tgcattacga
     5101 tatagtaacg ccagtagcga tgaagatttt attttatcgc ctgtggcaaa tgatgagggt
     5161 gtgtttttta attcggcatt ggcggatgat acacagccac aaaatgcgga tgccaatggg
     5221 gcatatcata tcgcattaaa gggcttatgg ctattaaatg aactaaaaaa cagtgatgat
     5281 ttgaataaag tcaaacttgc cattgacaat caaacatggt taaacttcgc tcaaaaccgc
     5341 taataaatat tggaagtgga taacggatcc gcgatcgcgg cgcgccacct ggtggccggc
     5401 cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     5461 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5521 ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc
     5581 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca
     5641 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc
     5701 gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg
     5761 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     5821 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     5881 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     5941 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6001 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6061 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6121 ataacattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6181 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6241 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     6301 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     6361 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     6421 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     6481 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     6541 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6601 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6661 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     6721 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6781 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6841 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6901 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6961 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     7021 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     7081 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     7141 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     7201 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     7261 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     7321 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     7381 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     7441 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     7501 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     7561 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7621 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7681 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7741 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7801 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7861 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     7921 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     7981 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     8041 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     8101 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     8161 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     8221 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     8281 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     8341 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     8401 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     8461 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     8521 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     8581 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     8641 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     8701 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     8761 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     8821 acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg
     8881 tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat
     8941 tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga
     9001 ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc
     9061 ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct
     9121 cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt
     9181 aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag
     9241 catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa
     9301 ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat
     9361 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     9421 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     9481 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     9541 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     9601 gaaggagctg actgggttga aggctctcaa gggcatcggt cgacgctctc ccttatgcga
     9661 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     9721 gaatggtgca tgcaaggaga tggcgcccaa cag
//
