LOCUS       sequence-217285-        9653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His-MBP fused to LbCas12a with H759A mutation to 
            inactivate crRNA processing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9653)
  AUTHORS   Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, 
            Doudna JA
  TITLE     CRISPR-Cas12a target binding unleashes indiscriminate 
            single-stranded DNase activity.
  JOURNAL   Science. 2018 Apr 27;360(6387):436-439. doi: 
            10.1126/science.aar6245. Epub 2018 Feb 15.
  PUBMED    29449511
REFERENCE   2  (bases 1 to 9653)
  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.aar6245"; journalName: "Science"; date: 
            "2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "436-439"
COMMENT     Sequence Label: pMBP-LbCas12a H759A
FEATURES             Location/Qualifiers
     source          1..9653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..42
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     126..145
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        126..144
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             233..255
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             275..304
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             314..1414
                     /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     1388..1411
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1469..1489
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5308..5326)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5322..5369
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5735..5740,5741..5757,5758..5763))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5790..5808)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5876..5980
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5981..6049,6050..6841)
                     /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(6199..6218)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7012..7600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7501..7520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7754..7771
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7786..7925
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7851..7870)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8011..8033
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8027..8218)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
       61 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
      121 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg
      181 agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta
      241 actttaagaa ggagatatac atatgaaatc ttctcaccat caccatcacc atcaccatca
      301 ccatggttct tctatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa
      361 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt
      421 caccgttgag catccggata aactggaaga gaaattccca caggttgcgg caactggcga
      481 tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct
      541 gttggctgaa atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga
      601 tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct
      661 gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
      721 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
      781 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa
      841 gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
      901 ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc
      961 tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat
     1021 cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
     1081 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct
     1141 ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa
     1201 agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
     1261 acgtattgcc gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca
     1321 gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca
     1381 gactgtcgat gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa
     1441 taacaataac aacaacctcg ggatcgagga aaacctgtac ttccaatcca atgcaatgag
     1501 caaactggag aagttcacga actgttattc tctgtcgaaa accttacgtt ttaaggccat
     1561 tccggtaggt aaaacgcaag aaaatatcga caataaacgt ctcctggttg aagatgaaaa
     1621 gcgtgccgaa gattataaag gtgttaaaaa gctgcttgat cgctattacc tgtcttttat
     1681 caacgatgtt ctgcattcca tcaaattgaa aaacttgaat aattacatct cgttatttcg
     1741 caaaaaaacc cgtaccgaaa aagaaaacaa ggagctggaa aaccttgaaa ttaatctgcg
     1801 taaagaaatc gcgaaagcat ttaaaggtaa tgaaggatat aaatctctgt ttaaaaaaga
     1861 cattattgaa accatcctgc ctgagttctt ggatgataaa gatgaaattg ccctggttaa
     1921 tagctttaat ggctttacga ctgcatttac cggctttttt gataaccgtg aaaacatgtt
     1981 cagcgaagaa gcgaaatcga cttccattgc cttccgttgc attaatgaaa acctgacccg
     2041 ctacatttct aatatggaca tctttgaaaa agttgatgcc attttcgaca aacatgaagt
     2101 tcaagaaatc aaagaaaaga ttttaaactc cgattatgat gttgaagatt tctttgaggg
     2161 agaatttttc aattttgttc tcacccaaga gggtattgat gtctataacg ccattatcgg
     2221 aggatttgtt acggaatcag gcgaaaagat taaaggactg aatgaataca ttaatttata
     2281 caatcagaaa accaaacaga aattgccaaa attcaaacca ctgtataaac aggtattaag
     2341 cgaccgtgaa tccttgtcct tttatggcga aggctataca agcgatgaag aggtgctcga
     2401 agtattccgt aatactttga acaaaaactc ggaaatcttc tccagcatta aaaaactgga
     2461 aaaattgttt aaaaatttcg atgaatatag tagcgcaggc atcttcgtca aaaatggccc
     2521 tgcgatttca accatttcta aagacatttt cggtgaatgg aacgtgatcc gcgataaatg
     2581 gaacgcggag tatgacgaca ttcatctgaa aaaaaaagcc gttgttaccg aaaagtatga
     2641 agatgaccgc cgcaagagct ttaaaaaaat tggtagtttc tcactggaac agttacaaga
     2701 atatgcagac gccgatctgt cggttgtgga aaagttaaaa gagattatta tccagaaagt
     2761 cgatgaaatt tataaagtgt acggctccag cgaaaagctc tttgatgccg actttgtttt
     2821 agaaaaatcg ctgaaaaaaa acgatgccgt ggtggccatc atgaaagatc tgttggattc
     2881 ggtcaaatcg ttcgaaaact atatcaaggc gttctttggc gaagggaaag agaccaatcg
     2941 tgacgaatcg ttctatggcg attttgtgtt ggcgtatgat attcttctga aagtagatca
     3001 catctatgat gctatccgca actacgtgac ccagaaaccg tattcgaagg ataaatttaa
     3061 gctgtatttt caaaaccctc agtttatggg tggctgggat aaagataaag aaaccgacta
     3121 ccgcgcgacg atcttgcgct atggctctaa gtactattta gccatcatgg ataaaaagta
     3181 tgcaaaatgc ctccagaaaa tcgataagga tgatgtcaac gggaactacg aaaaaatcaa
     3241 ctacaaactc ttgcctggcc cgaataaaat gctgccgaaa gtcttttttt ccaaaaaatg
     3301 gatggcctat tataaccctt ccgaagatat tcagaagatt tacaagaacg gtacctttaa
     3361 aaaaggtgac atgtttaatc tgaatgactg tcacaagctt attgatttct tcaaagatag
     3421 tatttctcgc taccctaaat ggtcaaacgc gtacgatttt aatttctcag aaacagagaa
     3481 atacaaagat attgcggggt tttatcgcga agtcgaagaa caagggtata aggtgtcgtt
     3541 cgaatcggct tccaaaaagg aagtggataa acttgttgaa gaaggcaaac tctatatgtt
     3601 ccagatctac aacaaagatt tctccgacaa gagccatggc acgccgaact tacataccat
     3661 gtactttaaa ttactgttcg atgaaaataa tcatggtcag attcgcttga gcggtggcgc
     3721 cgaactgttt atgcgccgcg catcattgaa aaaggaagaa cttgttgtgg cgccagcaaa
     3781 cagtccgatt gcaaacaaaa atccggataa cccgaagaag actaccacct taagctatga
     3841 tgtatataaa gataaacgtt ttagcgaaga tcagtatgaa ctccacattc cgatcgcgat
     3901 taacaaatgt cctaagaaca tctttaaaat caataccgag gttcgtgtac tgctcaaaca
     3961 cgacgacaac ccctatgtta ttggcattga ccgcggcgaa cgcaacctgc tgtatattgt
     4021 cgttgtggat ggcaaaggta acattgttga acagtatagc cttaatgaaa tcatcaataa
     4081 tttcaacggc attcgtatta aaacggacta tcatagcctg ctggacaaaa aagagaaaga
     4141 acgcttcgaa gcccgtcaga attggacctc tatcgaaaat atcaaggaac tgaaagctgg
     4201 gtacatttct caagtggtcc acaagatttg cgaactggtg gaaaagtatg acgcggtgat
     4261 cgcactggag gatctgaatt cgggctttaa aaatagccgt gtgaaggtgg agaaacaggt
     4321 ttaccaaaag ttcgaaaaaa tgctgattga caaacttaat tacatggtgg acaaaaaatc
     4381 taatccatgt gcgaccggcg gggctctcaa aggctatcag attacgaata aattcgaaag
     4441 cttcaaatcc atgtctaccc agaacggatt tatcttttac atccccgcct ggctgacttc
     4501 caaaattgat ccgagtaccg gttttgttaa tctgctgaaa accaaatata cgtcgatcgc
     4561 cgattcaaaa aaattcatta gcagcttcga ccgtattatg tacgtcccgg aagaagacct
     4621 gttcgaattc gcccttgact ataaaaattt ctcgcgtacg gatgccgatt atatcaaaaa
     4681 atggaaactg tacagctacg gcaaccgcat ccgcattttt cgcaacccaa agaagaacaa
     4741 cgtatttgac tgggaggaag tttgcttgac gagcgcttac aaagagctct ttaacaaata
     4801 tgggatcaat tatcagcaag gggacatccg tgcgttgctg tgtgagcagt cagataaagc
     4861 gttctacagc tctttcatgg ctttaatgtc gctgatgttg cagatgcgca attcgatcac
     4921 tggccgcacc gatgtggatt tcctgattag cccggtcaaa aatagcgatg gcatttttta
     4981 cgattcacgt aactatgaag cccaggaaaa cgctattctg ccgaagaacg ctgatgcgaa
     5041 cggcgcgtac aatattgccc gtaaggtact gtgggcaatt ggtcagttca agaaagcgga
     5101 agatgaaaag ttggataagg tcaaaattgc gatctcgaat aaagaatggc tggaatatgc
     5161 gcagaccagc gtgaaacatt aataacattg gaagtggata acggatccgc gatcgcggcg
     5221 cgccacctgg tggccggccg gtaccacgcg tgcgcgctga tccggctgct aacaaagccc
     5281 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     5341 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac
     5401 aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc
     5461 aggactgggc ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat
     5521 tgcatcaacg catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg
     5581 acgatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
     5641 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
     5701 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
     5761 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5821 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5881 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5941 accctgataa atgcttcaat aacattgaaa aaggaagagt atgagtattc aacatttccg
     6001 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6061 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6121 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6181 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     6241 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6301 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6361 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6421 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6481 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     6541 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6601 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6661 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6721 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6781 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6841 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6901 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6961 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     7021 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     7081 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     7141 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     7201 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     7261 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7321 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7381 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7441 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7501 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7561 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7621 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     7681 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     7741 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     7801 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     7861 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     7921 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     7981 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     8041 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     8101 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     8161 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     8221 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     8281 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     8341 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     8401 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     8461 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     8521 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     8581 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     8641 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
     8701 tgcccgagat gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc
     8761 aagggttggt ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag
     8821 tggtgaatcc gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca
     8881 tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc
     8941 caacccgttc catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat
     9001 gatcgaagtt aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc
     9061 atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag
     9121 aagaatcata atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc
     9181 cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     9241 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     9301 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     9361 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     9421 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     9481 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     9541 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     9601 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agt
//
