LOCUS       sequence-217356-        9869 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 9869)
  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 9869)
  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..9869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             175..197
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             217..246
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             256..1356
                     /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     1330..1353
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1411..1431
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5466..5484)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5480..5527
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5893..5898,5899..5915,5916..5921))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5948..5966)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6034..6138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6139..6207,6208..6999)
                     /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(6357..6376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7170..7758
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7659..7678
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7912..7929
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7944..8083
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8009..8028)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8169..8191
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8185..8376)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     9834..9853
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
       61 gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag tgccggctcc
      121 ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa taattttgtt
      181 taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca ccatcaccat
      241 caccatggtt cttctatgaa aatcgaagaa ggtaaactgg taatctggat taacggcgat
      301 aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa
      361 gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc
      421 gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc
      481 ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg
      541 gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg
      601 ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg
      661 ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg
      721 tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc
      781 aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc
      841 ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa
      901 gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac
      961 atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca
     1021 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag
     1081 ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat
     1141 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat
     1201 ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg
     1261 cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt
     1321 cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa caacaacaac
     1381 aataacaata acaacaacct cgggatcgag gaaaacctgt acttccaatc caatgcaatg
     1441 tcaatttatc aagaatttgt taataaatat agtttaagta aaactctaag atttgagtta
     1501 atcccacagg gtaaaacact tgaaaacata aaagcaagag gtttgatttt agatgatgag
     1561 aaaagagcta aagactacaa aaaggctaaa caaataattg ataaatatca tcagtttttt
     1621 atagaggaga tattaagttc ggtttgtatt agcgaagatt tattacaaaa ctattctgat
     1681 gtttatttta aacttaaaaa gagtgatgat gataatctac aaaaagattt taaaagtgca
     1741 aaagatacga taaagaaaca aatatctgaa tatataaagg actcagagaa atttaagaat
     1801 ttgtttaatc aaaaccttat cgatgctaaa aaagggcaag agtcagattt aattctatgg
     1861 ctaaagcaat ctaaggataa tggtatagaa ctatttaaag ccaatagtga tatcacagat
     1921 atagatgagg cgttagaaat aatcaaatct tttaaaggtt ggacaactta ttttaagggt
     1981 tttcatgaaa atagaaaaaa tgtttatagt agcaatgata ttcctacatc tattatttat
     2041 aggatagtag atgataattt gcctaaattt ctagaaaata aagctaagta tgagagttta
     2101 aaagacaaag ctccagaagc tataaactat gaacaaatta aaaaagattt ggcagaagag
     2161 ctaacctttg atattgacta caaaacatct gaagttaatc aaagagtttt ttcacttgat
     2221 gaagtttttg agatagcaaa ctttaataat tatctaaatc aaagtggtat tactaaattt
     2281 aatactatta ttggtggtaa atttgtaaat ggtgaaaata caaagagaaa aggtataaat
     2341 gaatatataa atctatactc acagcaaata aatgataaaa cactcaaaaa atataaaatg
     2401 agtgttttat ttaagcaaat tttaagtgat acagaatcta aatcttttgt aattgataag
     2461 ttagaagatg atagtgatgt agttacaacg atgcaaagtt tttatgagca aatagcagct
     2521 tttaaaacag tagaagaaaa atctattaaa gaaacactat ctttattatt tgatgattta
     2581 aaagctcaaa aacttgattt gagtaaaatt tattttaaaa atgataaatc tcttactgat
     2641 ctatcacaac aagtttttga tgattatagt gttattggta cagcggtact agaatatata
     2701 actcaacaaa tagcacctaa aaatcttgat aaccctagta agaaagagca agaattaata
     2761 gccaaaaaaa ctgaaaaagc aaaatactta tctctagaaa ctataaagct tgccttagaa
     2821 gaatttaata agcatagaga tatagataaa cagtgtaggt ttgaagaaat acttgcaaac
     2881 tttgcggcta ttccgatgat atttgatgaa atagctcaaa acaaagacaa tttggcacag
     2941 atatctatca aatatcaaaa tcaaggtaaa aaagacctac ttcaagctag tgcggaagat
     3001 gatgttaaag ctatcaagga tcttttagat caaactaata atctcttaca taaactaaaa
     3061 atatttcata ttagtcagtc agaagataag gcaaatattt tagacaagga tgagcatttt
     3121 tatctagtat ttgaggagtg ctactttgag ctagcgaata tagtgcctct ttataacaaa
     3181 attagaaact atataactca aaagccatat agtgatgaga aatttaagct caattttgag
     3241 aactcgactt tggctaatgg ttgggataaa aataaagagc ctgacaatac ggcaatttta
     3301 tttatcaaag atgataaata ttatctgggt gtgatgaata agaaaaataa caaaatattt
     3361 gatgataaag ctatcaaaga aaataaaggc gagggttata aaaaaattgt ttataaactt
     3421 ttacctggcg caaataaaat gttacctaag gttttctttt ctgctaaatc tataaaattt
     3481 tataatccta gtgaagatat acttagaata agaaatcatt ccacacatac aaaaaatggt
     3541 agtcctcaaa aaggatatga aaaatttgag tttaatattg aagattgccg aaaatttata
     3601 gatttttata aacagtctat aagtaagcat ccggagtgga aagattttgg atttagattt
     3661 tctgatactc aaagatataa ttctatagat gaattttata gagaagttga aaatcaaggc
     3721 tacaaactaa cttttgaaaa tatatcagag agctatattg atagcgtagt taatcagggt
     3781 aaattgtacc tattccaaat ctataataaa gatttttcag cttatagcaa agggcgacca
     3841 aatctacata ctttatattg gaaagcgctg tttgatgaga gaaatcttca agatgtggtt
     3901 tataagctaa atggtgaggc agagcttttt tatcgtaaac aatcaatacc taaaaaaatc
     3961 actcacccag ctaaagaggc aatagctaat aaaaacaaag ataatcctaa aaaagagagt
     4021 gtttttgaat atgatttaat caaagataaa cgctttactg aagataagtt tttctttcac
     4081 tgtcctatta caatcaattt taaatctagt ggagctaata agtttaatga tgaaatcaat
     4141 ttattgctaa aagaaaaagc aaatgatgtt catatattaa gtatagatag aggtgaaaga
     4201 catttagctt actatacttt ggtagatggt aaaggcaata tcatcaaaca agatactttc
     4261 aacatcattg gtaatgatag aatgaaaaca aactaccatg ataagcttgc tgcaatagag
     4321 aaagataggg attcagctag gaaagactgg aaaaagataa ataacatcaa agagatgaaa
     4381 gagggctatc tatctcaggt agttcatgaa atagctaagc tagttataga gtataatgct
     4441 attgtggttt ttgaggattt aaattttgga tttaaaagag ggcgtttcaa ggtagagaag
     4501 caggtctatc aaaagttaga aaaaatgcta attgagaaac taaactatct agttttcaaa
     4561 gataatgagt ttgataaaac tgggggagtg cttagagctt atcagctaac agcacctttt
     4621 gagactttta aaaagatggg taaacaaaca ggtattatct actatgtacc agctggtttt
     4681 acttcaaaaa tttgtcctgt aactggtttt gtaaatcagt tatatcctaa gtatgaaagt
     4741 gtcagcaaat ctcaagagtt ctttagtaag tttgacaaga tttgttataa ccttgataag
     4801 ggctattttg agtttagttt tgattataaa aactttggtg acaaggctgc caaaggcaag
     4861 tggactatag ctagctttgg gagtagattg attaacttta gaaattcaga taaaaatcat
     4921 aattgggata ctcgagaagt ttatccaact aaagagttgg agaaattgct aaaagattat
     4981 tctatcgaat atgggcatgg cgaatgtatc aaagcagcta tttgcggtga gagcgacaaa
     5041 aagttttttg ctaagctaac tagtgtccta aatactatct tacaaatgcg taactcaaaa
     5101 acaggtactg agttagatta tctaatttca ccagtagcag atgtaaatgg caatttcttt
     5161 gattcgcgac aggcgccaaa aaatatgcct caagatgctg atgccaatgg tgcttatcat
     5221 attgggctaa aaggtctgat gctactaggt aggatcaaaa ataatcaaga gggcaaaaaa
     5281 ctcaatttgg ttatcaaaaa tgaagagtat tttgagttcg tgcagaatag gaataactaa
     5341 taacattgga agtggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt
     5401 accacgcgtg cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     5461 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     5521 ttttttgctg aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga
     5581 tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc
     5641 ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta
     5701 gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg
     5761 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
     5821 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
     5881 ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga
     5941 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6001 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6121 cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6361 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     6421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6661 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     6721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6841 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7141 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7201 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7261 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     7321 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7381 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7441 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7501 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7561 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7621 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7681 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7741 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7801 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     7861 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     7921 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     7981 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     8041 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     8101 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     8161 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     8221 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     8281 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     8341 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     8401 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     8461 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     8521 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     8581 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     8641 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     8701 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     8761 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     8821 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg
     8881 gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac
     8941 agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
     9001 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg
     9061 aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc
     9121 gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga
     9181 gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg
     9241 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc
     9301 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
     9361 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     9421 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     9481 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     9541 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     9601 gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc
     9661 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     9721 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     9781 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     9841 tcggcgatat aggcgccagc aaccgcacc
//
