LOCUS       sequence-214281-        9656 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 9656)
  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 9656)
  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..9656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     420..439
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     523..542
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        523..541
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             630..652
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             672..701
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             711..1811
                     /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     1785..1808
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1866..1886
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5705..5723)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5719..5766
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6132..6137,6138..6154,6155..6160))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6187..6205)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6273..6377
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6378..6446,6447..7238)
                     /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(6596..6615)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7409..7997
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7898..7917
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8151..8168
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8183..8325
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8251..8270)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8411..8433
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8427..8618)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
       61 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
      121 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
      181 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacgctc tcccttatgc
      241 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
      301 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
      361 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
      421 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
      481 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
      541 ggagaccaca acggtttccc tctagtgccg gctccggaga gctctttaat taagcggccg
      601 ccctgcagga ctcgagttct agaaataatt ttgtttaact ttaagaagga gatatacata
      661 tgaaatcttc tcaccatcac catcaccatc accatcacca tggttcttct atgaaaatcg
      721 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
      781 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
      841 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
      901 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
      961 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
     1021 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
     1081 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
     1141 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
     1201 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
     1261 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
     1321 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
     1381 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
     1441 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
     1501 gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact
     1561 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     1621 cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc actatggaaa
     1681 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     1741 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     1801 acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac aacctcggga
     1861 tcgaggaaaa cctgtacttc caatccaatg caatgagcaa actggagaag ttcacgaact
     1921 gttattctct gtcgaaaacc ttacgtttta aggccattcc ggtaggtaaa acgcaagaaa
     1981 atatcgacaa taaacgtctc ctggttgaag atgaaaagcg tgccgaagat tataaaggtg
     2041 ttaaaaagct gcttgatcgc tattacctgt cttttatcaa cgatgttctg cattccatca
     2101 aattgaaaaa cttgaataat tacatctcgt tatttcgcaa aaaaacccgt accgaaaaag
     2161 aaaacaagga gctggaaaac cttgaaatta atctgcgtaa agaaatcgcg aaagcattta
     2221 aaggtaatga aggatataaa tctctgttta aaaaagacat tattgaaacc atcctgcctg
     2281 agttcttgga tgataaagat gaaattgccc tggttaatag ctttaatggc tttacgactg
     2341 catttaccgg cttttttgat aaccgtgaaa acatgttcag cgaagaagcg aaatcgactt
     2401 ccattgcctt ccgttgcatt aatgaaaacc tgacccgcta catttctaat atggacatct
     2461 ttgaaaaagt tgatgccatt ttcgacaaac atgaagttca agaaatcaaa gaaaagattt
     2521 taaactccga ttatgatgtt gaagatttct ttgagggaga atttttcaat tttgttctca
     2581 cccaagaggg tattgatgtc tataacgcca ttatcggagg atttgttacg gaatcaggcg
     2641 aaaagattaa aggactgaat gaatacatta atttatacaa tcagaaaacc aaacagaaat
     2701 tgccaaaatt caaaccactg tataaacagg tattaagcga ccgtgaatcc ttgtcctttt
     2761 atggcgaagg ctatacaagc gatgaagagg tgctcgaagt attccgtaat actttgaaca
     2821 aaaactcgga aatcttctcc agcattaaaa aactggaaaa attgtttaaa aatttcgatg
     2881 aatatagtag cgcaggcatc ttcgtcaaaa atggccctgc gatttcaacc atttctaaag
     2941 acattttcgg tgaatggaac gtgatccgcg ataaatggaa cgcggagtat gacgacattc
     3001 atctgaaaaa aaaagccgtt gttaccgaaa agtatgaaga tgaccgccgc aagagcttta
     3061 aaaaaattgg tagtttctca ctggaacagt tacaagaata tgcagacgcc gatctgtcgg
     3121 ttgtggaaaa gttaaaagag attattatcc agaaagtcga tgaaatttat aaagtgtacg
     3181 gctccagcga aaagctcttt gatgccgact ttgttttaga aaaatcgctg aaaaaaaacg
     3241 atgccgtggt ggccatcatg aaagatctgt tggattcggt caaatcgttc gaaaactata
     3301 tcaaggcgtt ctttggcgaa gggaaagaga ccaatcgtga cgaatcgttc tatggcgatt
     3361 ttgtgttggc gtatgatatt cttctgaaag tagatcacat ctatgatgct atccgcaact
     3421 acgtgaccca gaaaccgtat tcgaaggata aatttaagct gtattttcaa aaccctcagt
     3481 ttatgggtgg ctgggataaa gataaagaaa ccgactaccg cgcgacgatc ttgcgctatg
     3541 gctctaagta ctatttagcc atcatggata aaaagtatgc aaaatgcctc cagaaaatcg
     3601 ataaggatga tgtcaacggg aactacgaaa aaatcaacta caaactcttg cctggcccga
     3661 ataaaatgct gccgaaagtc tttttttcca aaaaatggat ggcctattat aacccttccg
     3721 aagatattca gaagatttac aagaacggta cctttaaaaa aggtgacatg tttaatctga
     3781 atgactgtca caagcttatt gatttcttca aagatagtat ttctcgctac cctaaatggt
     3841 caaacgcgta cgattttaat ttctcagaaa cagagaaata caaagatatt gcggggtttt
     3901 atcgcgaagt cgaagaacaa gggtataagg tgtcgttcga atcggcttcc aaaaaggaag
     3961 tggataaact tgttgaagaa ggcaaactct atatgttcca gatctacaac aaagatttct
     4021 ccgacaagag ccatggcacg ccgaacttac ataccatgta ctttaaatta ctgttcgatg
     4081 aaaataatca tggtcagatt cgcttgagcg gtggcgccga actgtttatg cgccgcgcat
     4141 cattgaaaaa ggaagaactt gttgtggcgc cagcaaacag tccgattgca aacaaaaatc
     4201 cggataaccc gaagaagact accaccttaa gctatgatgt atataaagat aaacgtttta
     4261 gcgaagatca gtatgaactc cacattccga tcgcgattaa caaatgtcct aagaacatct
     4321 ttaaaatcaa taccgaggtt cgtgtactgc tcaaacacga cgacaacccc tatgttattg
     4381 gcattgaccg cggcgaacgc aacctgctgt atattgtcgt tgtggatggc aaaggtaaca
     4441 ttgttgaaca gtatagcctt aatgaaatca tcaataattt caacggcatt cgtattaaaa
     4501 cggactatca tagcctgctg gacaaaaaag agaaagaacg cttcgaagcc cgtcagaatt
     4561 ggacctctat cgaaaatatc aaggaactga aagctgggta catttctcaa gtggtccaca
     4621 agatttgcga actggtggaa aagtatgacg cggtgatcgc actggaggat ctgaattcgg
     4681 gctttaaaaa tagccgtgtg aaggtggaga aacaggttta ccaaaagttc gaaaaaatgc
     4741 tgattgacaa acttaattac atggtggaca aaaaatctaa tccatgtgcg accggcgggg
     4801 ctctcaaagg ctatcagatt acgaataaat tcgaaagctt caaatccatg tctacccaga
     4861 acggatttat cttttacatc cccgcctggc tgacttccaa aattgatccg agtaccggtt
     4921 ttgttaatct gctgaaaacc aaatatacgt cgatcgccga ttcaaaaaaa ttcattagca
     4981 gcttcgaccg tattatgtac gtcccggaag aagacctgtt cgaattcgcc cttgactata
     5041 aaaatttctc gcgtacggat gccgattata tcaaaaaatg gaaactgtac agctacggca
     5101 accgcatccg catttttcgc aacccaaaga agaacaacgt atttgactgg gaggaagttt
     5161 gcttgacgag cgcttacaaa gagctcttta acaaatatgg gatcaattat cagcaagggg
     5221 acatccgtgc gttgctgtgt gagcagtcag ataaagcgtt ctacagctct ttcatggctt
     5281 taatgtcgct gatgttgcag atgcgcaatt cgatcactgg ccgcaccgat gtggatttcc
     5341 tgattagccc ggtcaaaaat agcgatggca ttttttacga ttcacgtaac tatgaagccc
     5401 aggaaaacgc tattctgccg aagaacgctg atgcgaacgg cgcgtacaat attgcccgta
     5461 aggtactgtg ggcaattggt cagttcaaga aagcggaaga tgaaaagttg gataaggtca
     5521 aaattgcgat ctcgaataaa gaatggctgg aatatgcgca gaccagcgtg aaacattaat
     5581 aacattggaa gtggataacg gatccgcgat cgcggcgcgc cacctggtgg ccggccggta
     5641 ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     5701 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     5761 tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat
     5821 cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg
     5881 gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag
     5941 cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg
     6001 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     6061 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     6121 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     6181 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     6241 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6301 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac
     6361 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6421 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6481 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6541 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6601 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     6661 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6721 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6781 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6841 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6901 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6961 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7021 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     7081 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     7141 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     7201 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7261 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7321 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     7381 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7441 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7501 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     7561 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7621 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7681 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7741 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7801 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7861 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7921 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7981 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     8041 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     8101 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     8161 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     8221 tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     8281 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     8341 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     8401 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     8461 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     8521 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     8581 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     8641 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     8701 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     8761 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     8821 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     8881 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     8941 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     9001 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     9061 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     9121 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     9181 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     9241 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     9301 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     9361 ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa
     9421 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     9481 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     9541 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     9601 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaag
//
