LOCUS       sequence-214017-        9359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Expresses 10xHis-MBP fused to AaCas12b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9359)
  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 9359)
  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-AaCas12b
FEATURES             Location/Qualifiers
     source          1..9359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1495..1514
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1598..1617
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1598..1616
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1705..1727
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1747..1776
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1786..2886
                     /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     2860..2883
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2941..2961
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6483..6501)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6497..6544
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6910..6915,6916..6932,6933..6938))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6965..6983)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7051..7155
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7156..7224,7225..8016)
                     /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(7374..7393)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8187..8775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8676..8695
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8929..8946
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8961..9103
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9029..9048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9189..9211
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(9205..9359,1..37))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
       61 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
      121 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
      181 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
      241 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
      301 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
      361 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
      421 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
      481 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
      541 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
      601 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
      661 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
      721 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
      781 cgaggcggca taaatcgccg tgacgatcag cggtccagtg atcgaagtta ggctggtaag
      841 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
      901 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
      961 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     1021 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     1081 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     1141 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     1201 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     1261 ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct tatgcgactc
     1321 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     1381 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     1441 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     1501 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     1561 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tatagggaga
     1621 ccacaacggt ttccctctag tgccggctcc ggagagctct ttaattaagc ggccgccctg
     1681 caggactcga gttctagaaa taattttgtt taactttaag aaggagatat acatatgaaa
     1741 tcttctcacc atcaccatca ccatcaccat caccatggtt cttctatgaa aatcgaagaa
     1801 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
     1861 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
     1921 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
     1981 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
     2041 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
     2101 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
     2161 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
     2221 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac
     2281 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
     2341 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
     2401 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
     2461 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
     2521 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
     2581 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
     2641 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
     2701 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc
     2761 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     2821 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     2881 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag
     2941 gaaaacctgt acttccaatc caatgcaatg gccgtcaaat ccatcaaagt gaaacttcgt
     3001 ctcgacgata tgccggagat tcgggccggt ctatggaaac ttcataagga agtcaatgcg
     3061 ggggttcgat attacacgga atggctcagt cttctccgtc aagagaactt gtatcgaaga
     3121 agtccgaatg gggacggaga gcaagaatgt gataagactg cagaagaatg caaagccgaa
     3181 ttgttggagc ggctgcgcgc gcgtcaagtg gagaatggac accgtggtcc ggcgggatcg
     3241 gacgatgaat tgctgcagtt ggcgcgtcaa ctctatgagt tgttggttcc gcaggcgata
     3301 ggtgcgaaag gcgacgcgca gcaaattgcc cgcaaatttt tgagcccctt ggccgacaag
     3361 gacgcagttg gtgggcttgg aatcgcgaag gcggggaaca aaccgcggtg ggttcgcatg
     3421 cgcgaagcgg gggaaccagg ctgggaagag gagaaggaga aggctgagac gaggaaatct
     3481 gcggatcgga ctgcggatgt tttgcgcgcg ctcgcggatt ttgggttaaa gccactgatg
     3541 cgcgtataca ccgattctga gatgtcatcg gtggagtgga aaccgcttcg gaagggacaa
     3601 gccgttcgga cgtgggatag ggacatgttc caacaagcta tcgaacggat gatgtcgtgg
     3661 gagtcgtgga atcagcgcgt tgggcaagag tacgcgaaac tcgtagaaca aaaaaatcga
     3721 tttgagcaga agaatttcgt cggccaggaa catctggtcc atctcgtcaa tcagttgcaa
     3781 caagatatga aagaagcatc gcccggactc gaatcgaaag agcaaaccgc gcactatgtg
     3841 acgggacggg cattgcgcgg atcggacaag gtatttgaga agtgggggaa actcgccccc
     3901 gatgcacctt tcgatttgta cgacgccgaa atcaagaatg tgcagagacg taacacgaga
     3961 cgattcggat cacatgactt gttcgcaaaa ttggcagagc cagagtatca ggccctgtgg
     4021 cgcgaagatg cttcgtttct cacgcgttac gcggtgtaca acagcatcct tcgcaaactg
     4081 aatcacgcca aaatgttcgc gacgtttact ttgccggatg caacggcgca cccgatttgg
     4141 actcgcttcg ataaattggg tgggaatttg caccagtaca cctttttgtt caacgaattt
     4201 ggagaacgca ggcacgcgat tcgttttcac aagctattga aagtcgagaa tggtgtcgca
     4261 agagaagttg atgatgtcac cgtgcccatt tcaatgtcag agcaattgga taatctgctt
     4321 cccagagatc ccaatgaacc gattgcgcta tattttcgag attacggagc cgaacagcat
     4381 ttcacaggtg aatttggtgg cgcgaagatc cagtgccgcc gggatcagct ggctcatatg
     4441 caccgacgca gaggggcgag ggatgtttat ctcaatgtca gcgtacgtgt gcagagtcag
     4501 tctgaggcgc ggggagaacg tcgcccgccg tatgcggcag tatttcgtct ggtcggggac
     4561 aaccatcgcg cgtttgtcca tttcgataaa ctatcggatt atcttgcgga acatccggat
     4621 gatgggaagc tcgggtcgga ggggttgctt tccgggctgc gggtgatgag tgtcgatctc
     4681 ggccttcgca catctgcatc gatttccgtt tttcgcgttg cccggaagga cgagttgaag
     4741 ccgaactcaa aaggtcgtgt accgtttttc tttccgataa aagggaatga caatctcgtc
     4801 gcggttcatg agcgatcaca actcttgaag ctgcctggcg aaacggagtc gaaggacctg
     4861 cgtgctatcc gagaagaacg ccaacggaca ttgcggcagt tgcggacgca actggcgtat
     4921 ttgcggctgc tcgtgcggtg tgggtcggaa gatgtggggc ggcgtgaacg gagttgggca
     4981 aagcttatcg agcagccggt ggatgcggcc aatcacatga caccggattg gcgcgaggct
     5041 tttgaaaacg aacttcagaa gcttaagtca ctccatggta tctgtagcga caaggaatgg
     5101 atggatgctg tctacgagag cgttcgccgc gtgtggcgtc acatgggcaa acaggttcgc
     5161 gattggcgaa aggacgtacg aagcggagag cggcccaaga ttcgcggcta tgcgaaagac
     5221 gtggtcggtg gaaactcgat tgagcaaatc gagtatctgg aacgtcagta caagttcctc
     5281 aagagttgga gcttctttgg taaggtgtcg ggacaagtga ttcgtgcgga gaagggatct
     5341 cgttttgcga tcacgctgcg cgaacacatt gatcacgcga aggaagatcg gctgaagaaa
     5401 ttggcggatc gcatcattat ggaggctctc ggctatgtgt acgcgttgga tgagcgcggc
     5461 aaaggaaagt gggttgcgaa gtatccgccg tgccagctca tcctgctgga ggaattgagc
     5521 gagtaccagt tcaataacga caggcctccg agcgaaaaca accagttgat gcaatggagt
     5581 catcgcggcg tgttccagga gttgataaat caggcccaag tccatgattt actcgttggg
     5641 acgatgtatg cagcgttctc gtcgcgattc gacgcgcgaa ctggggcacc gggtatccgc
     5701 tgtcgccggg ttccggcgcg ttgcacccag gagcacaatc cagaaccatt tccttggtgg
     5761 ctgaacaagt ttgtggtgga acatacgttg gatgcttgtc ccctacgcgc agacgacctc
     5821 atcccaacgg gtgaaggaga gatttttgtc tcgccgttca gcgcggagga gggggacttt
     5881 catcagattc acgccgacct gaatgcggcg caaaatctgc agcagcgact ctggtctgat
     5941 tttgatatca gtcaaattcg gttgcggtgt gattggggtg aagtggacgg tgaactcgtt
     6001 ctgatcccaa ggcttacagg aaaacgaacg gcggattcat atagcaacaa ggtgttttat
     6061 accaatacag gtgtcaccta ttatgagcga gagcggggga agaagcggag aaaggttttc
     6121 gcgcaagaga aattgtcgga ggaagaggcg gagttgctcg tggaagcaga cgaggcgagg
     6181 gagaaatcgg tcgttttgat gcgtgatccg tctggcatca tcaatcgggg aaattggacc
     6241 aggcaaaagg aattttggtc gatggtgaac cagcggatcg aaggatactt ggtcaagcag
     6301 attcgctcgc gcgttccatt acaagatagt gcgtgtgaaa acacggggga tatttaataa
     6361 cattggaagt ggataacgga tccgcgatcg cggcgcgcca cctggtggcc ggccggtacc
     6421 acgcgtgcgc gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     6481 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     6541 tttgctgaaa ggaggaacta tatccggata tccacaggac gggtgtggtc gccatgatcg
     6601 cgtagtcgat agtggctcca agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt
     6661 cggacagtgc tccgagaacg ggtgcgcata gaaattgcat caacgcatat agcgctagca
     6721 gcacgccata gtgactggcg atgctgtcgg aatggacgat atcccgcaag aggcccggca
     6781 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     6841 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     6901 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga
     6961 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     7021 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7081 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataacat
     7141 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7201 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7261 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7321 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7381 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     7441 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7501 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7561 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     7621 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     7681 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     7741 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     7801 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     7861 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     7921 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     7981 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8041 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8101 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8161 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     8221 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     8281 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     8341 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     8401 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     8461 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     8521 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     8581 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     8641 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     8701 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     8761 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     8821 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     8881 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     8941 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     9001 tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     9061 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     9121 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     9181 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     9241 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     9301 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagc
//
