LOCUS       sequence-214018-        9872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 10xHis-MBP fused to FnCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9872)
  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 9872)
  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-FnCas12a
FEATURES             Location/Qualifiers
     source          1..9872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     849..868
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1059..1081
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1101..1130
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1140..2240
                     /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     2214..2237
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2295..2315
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6350..6368)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6364..6411
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6777..6782,6783..6799,6800..6805))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6832..6850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6918..7022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7023..7091,7092..7883)
                     /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(7241..7260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8054..8642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8543..8562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8796..8813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8828..8970
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8896..8915)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9056..9078
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(9072..9263)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg
       61 cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg
      121 ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc agtgatcgaa
      181 gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc
      241 tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc
      301 ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg
      361 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
      421 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
      481 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
      541 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
      601 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct
      661 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
      721 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
      781 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
      841 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
      901 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
      961 tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag ctctttaatt
     1021 aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt taagaaggag
     1081 atatacatat gaaatcttct caccatcacc atcaccatca ccatcaccat ggttcttcta
     1141 tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
     1201 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
     1261 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1321 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1381 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1441 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1501 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1561 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     1621 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     1681 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     1741 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
     1801 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     1861 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     1921 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     1981 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     2041 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     2101 ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     2161 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     2221 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     2281 acctcgggat cgaggaaaac ctgtacttcc aatccaatgc aatgtcaatt tatcaagaat
     2341 ttgttaataa atatagttta agtaaaactc taagatttga gttaatccca cagggtaaaa
     2401 cacttgaaaa cataaaagca agaggtttga ttttagatga tgagaaaaga gctaaagact
     2461 acaaaaaggc taaacaaata attgataaat atcatcagtt ttttatagag gagatattaa
     2521 gttcggtttg tattagcgaa gatttattac aaaactattc tgatgtttat tttaaactta
     2581 aaaagagtga tgatgataat ctacaaaaag attttaaaag tgcaaaagat acgataaaga
     2641 aacaaatatc tgaatatata aaggactcag agaaatttaa gaatttgttt aatcaaaacc
     2701 ttatcgatgc taaaaaaggg caagagtcag atttaattct atggctaaag caatctaagg
     2761 ataatggtat agaactattt aaagccaata gtgatatcac agatatagat gaggcgttag
     2821 aaataatcaa atcttttaaa ggttggacaa cttattttaa gggttttcat gaaaatagaa
     2881 aaaatgttta tagtagcaat gatattccta catctattat ttataggata gtagatgata
     2941 atttgcctaa atttctagaa aataaagcta agtatgagag tttaaaagac aaagctccag
     3001 aagctataaa ctatgaacaa attaaaaaag atttggcaga agagctaacc tttgatattg
     3061 actacaaaac atctgaagtt aatcaaagag ttttttcact tgatgaagtt tttgagatag
     3121 caaactttaa taattatcta aatcaaagtg gtattactaa atttaatact attattggtg
     3181 gtaaatttgt aaatggtgaa aatacaaaga gaaaaggtat aaatgaatat ataaatctat
     3241 actcacagca aataaatgat aaaacactca aaaaatataa aatgagtgtt ttatttaagc
     3301 aaattttaag tgatacagaa tctaaatctt ttgtaattga taagttagaa gatgatagtg
     3361 atgtagttac aacgatgcaa agtttttatg agcaaatagc agcttttaaa acagtagaag
     3421 aaaaatctat taaagaaaca ctatctttat tatttgatga tttaaaagct caaaaacttg
     3481 atttgagtaa aatttatttt aaaaatgata aatctcttac tgatctatca caacaagttt
     3541 ttgatgatta tagtgttatt ggtacagcgg tactagaata tataactcaa caaatagcac
     3601 ctaaaaatct tgataaccct agtaagaaag agcaagaatt aatagccaaa aaaactgaaa
     3661 aagcaaaata cttatctcta gaaactataa agcttgcctt agaagaattt aataagcata
     3721 gagatataga taaacagtgt aggtttgaag aaatacttgc aaactttgcg gctattccga
     3781 tgatatttga tgaaatagct caaaacaaag acaatttggc acagatatct atcaaatatc
     3841 aaaatcaagg taaaaaagac ctacttcaag ctagtgcgga agatgatgtt aaagctatca
     3901 aggatctttt agatcaaact aataatctct tacataaact aaaaatattt catattagtc
     3961 agtcagaaga taaggcaaat attttagaca aggatgagca tttttatcta gtatttgagg
     4021 agtgctactt tgagctagcg aatatagtgc ctctttataa caaaattaga aactatataa
     4081 ctcaaaagcc atatagtgat gagaaattta agctcaattt tgagaactcg actttggcta
     4141 atggttggga taaaaataaa gagcctgaca atacggcaat tttatttatc aaagatgata
     4201 aatattatct gggtgtgatg aataagaaaa ataacaaaat atttgatgat aaagctatca
     4261 aagaaaataa aggcgagggt tataaaaaaa ttgtttataa acttttacct ggcgcaaata
     4321 aaatgttacc taaggttttc ttttctgcta aatctataaa attttataat cctagtgaag
     4381 atatacttag aataagaaat cattccacac atacaaaaaa tggtagtcct caaaaaggat
     4441 atgaaaaatt tgagtttaat attgaagatt gccgaaaatt tatagatttt tataaacagt
     4501 ctataagtaa gcatccggag tggaaagatt ttggatttag attttctgat actcaaagat
     4561 ataattctat agatgaattt tatagagaag ttgaaaatca aggctacaaa ctaacttttg
     4621 aaaatatatc agagagctat attgatagcg tagttaatca gggtaaattg tacctattcc
     4681 aaatctataa taaagatttt tcagcttata gcaaagggcg accaaatcta catactttat
     4741 attggaaagc gctgtttgat gagagaaatc ttcaagatgt ggtttataag ctaaatggtg
     4801 aggcagagct tttttatcgt aaacaatcaa tacctaaaaa aatcactcac ccagctaaag
     4861 aggcaatagc taataaaaac aaagataatc ctaaaaaaga gagtgttttt gaatatgatt
     4921 taatcaaaga taaacgcttt actgaagata agtttttctt tcactgtcct attacaatca
     4981 attttaaatc tagtggagct aataagttta atgatgaaat caatttattg ctaaaagaaa
     5041 aagcaaatga tgttcatata ttaagtatag atagaggtga aagacattta gcttactata
     5101 ctttggtaga tggtaaaggc aatatcatca aacaagatac tttcaacatc attggtaatg
     5161 atagaatgaa aacaaactac catgataagc ttgctgcaat agagaaagat agggattcag
     5221 ctaggaaaga ctggaaaaag ataaataaca tcaaagagat gaaagagggc tatctatctc
     5281 aggtagttca tgaaatagct aagctagtta tagagtataa tgctattgtg gtttttgagg
     5341 atttaaattt tggatttaaa agagggcgtt tcaaggtaga gaagcaggtc tatcaaaagt
     5401 tagaaaaaat gctaattgag aaactaaact atctagtttt caaagataat gagtttgata
     5461 aaactggggg agtgcttaga gcttatcagc taacagcacc ttttgagact tttaaaaaga
     5521 tgggtaaaca aacaggtatt atctactatg taccagctgg ttttacttca aaaatttgtc
     5581 ctgtaactgg ttttgtaaat cagttatatc ctaagtatga aagtgtcagc aaatctcaag
     5641 agttctttag taagtttgac aagatttgtt ataaccttga taagggctat tttgagttta
     5701 gttttgatta taaaaacttt ggtgacaagg ctgccaaagg caagtggact atagctagct
     5761 ttgggagtag attgattaac tttagaaatt cagataaaaa tcataattgg gatactcgag
     5821 aagtttatcc aactaaagag ttggagaaat tgctaaaaga ttattctatc gaatatgggc
     5881 atggcgaatg tatcaaagca gctatttgcg gtgagagcga caaaaagttt tttgctaagc
     5941 taactagtgt cctaaatact atcttacaaa tgcgtaactc aaaaacaggt actgagttag
     6001 attatctaat ttcaccagta gcagatgtaa atggcaattt ctttgattcg cgacaggcgc
     6061 caaaaaatat gcctcaagat gctgatgcca atggtgctta tcatattggg ctaaaaggtc
     6121 tgatgctact aggtaggatc aaaaataatc aagagggcaa aaaactcaat ttggttatca
     6181 aaaatgaaga gtattttgag ttcgtgcaga ataggaataa ctaataacat tggaagtgga
     6241 taacggatcc gcgatcgcgg cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct
     6301 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     6361 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     6421 ggaactatat ccggatatcc acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt
     6481 ggctccaagt agcgaagcga gcaggactgg gcggcggcca aagcggtcgg acagtgctcc
     6541 gagaacgggt gcgcatagaa attgcatcaa cgcatatagc gctagcagca cgccatagtg
     6601 actggcgatg ctgtcggaat ggacgatatc ccgcaagagg cccggcagta ccggcataac
     6661 caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga gcgcattgtt
     6721 agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac taccgcatta
     6781 aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag ggcctcgtga
     6841 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6901 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataacattga aaaaggaaga
     7021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     7081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     7141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     7201 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     7261 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     7321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     7381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     7441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     7501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     7561 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     7621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     7681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     7741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     7801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7921 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     8041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     8101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     8161 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     8221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     8281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     8341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     8401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     8461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     8521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     8581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     8641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     8701 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     8761 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     8821 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     8881 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     8941 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     9001 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     9061 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     9121 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     9181 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     9241 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     9301 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     9361 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     9421 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     9481 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     9541 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     9601 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     9661 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     9721 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     9781 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     9841 aattgattgg ctccaattct tggagtggtg aa
//
