LOCUS       sequence-217292-        9356 bp    DNA     circular SYN 14-APR-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 9356)
  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 9356)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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..9356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..130
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     214..233
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             321..343
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             363..392
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             402..1502
                     /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     1476..1499
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1557..1577
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5099..5117)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5113..5160
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5526..5531,5532..5548,5549..5554))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5581..5599)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5667..5771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5772..5840,5841..6632)
                     /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(5990..6009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6803..7391
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7292..7311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7545..7562
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7577..7716
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7642..7661)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7802..7824
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7818..8009)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
       61 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      121 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      181 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
      241 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
      301 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt
      361 ctcaccatca ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta
      421 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      481 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
      541 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
      601 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
      661 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
      721 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
      781 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
      841 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
      901 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
      961 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     1021 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1081 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1141 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1201 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1261 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1321 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     1381 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1441 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1501 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa
     1561 acctgtactt ccaatccaat gcaatggccg tcaaatccat caaagtgaaa cttcgtctcg
     1621 acgatatgcc ggagattcgg gccggtctat ggaaacttca taaggaagtc aatgcggggg
     1681 ttcgatatta cacggaatgg ctcagtcttc tccgtcaaga gaacttgtat cgaagaagtc
     1741 cgaatgggga cggagagcaa gaatgtgata agactgcaga agaatgcaaa gccgaattgt
     1801 tggagcggct gcgcgcgcgt caagtggaga atggacaccg tggtccggcg ggatcggacg
     1861 atgaattgct gcagttggcg cgtcaactct atgagttgtt ggttccgcag gcgataggtg
     1921 cgaaaggcga cgcgcagcaa attgcccgca aatttttgag ccccttggcc gacaaggacg
     1981 cagttggtgg gcttggaatc gcgaaggcgg ggaacaaacc gcggtgggtt cgcatgcgcg
     2041 aagcggggga accaggctgg gaagaggaga aggagaaggc tgagacgagg aaatctgcgg
     2101 atcggactgc ggatgttttg cgcgcgctcg cggattttgg gttaaagcca ctgatgcgcg
     2161 tatacaccga ttctgagatg tcatcggtgg agtggaaacc gcttcggaag ggacaagccg
     2221 ttcggacgtg ggatagggac atgttccaac aagctatcga acggatgatg tcgtgggagt
     2281 cgtggaatca gcgcgttggg caagagtacg cgaaactcgt agaacaaaaa aatcgatttg
     2341 agcagaagaa tttcgtcggc caggaacatc tggtccatct cgtcaatcag ttgcaacaag
     2401 atatgaaaga agcatcgccc ggactcgaat cgaaagagca aaccgcgcac tatgtgacgg
     2461 gacgggcatt gcgcggatcg gacaaggtat ttgagaagtg ggggaaactc gcccccgatg
     2521 cacctttcga tttgtacgac gccgaaatca agaatgtgca gagacgtaac acgagacgat
     2581 tcggatcaca tgacttgttc gcaaaattgg cagagccaga gtatcaggcc ctgtggcgcg
     2641 aagatgcttc gtttctcacg cgttacgcgg tgtacaacag catccttcgc aaactgaatc
     2701 acgccaaaat gttcgcgacg tttactttgc cggatgcaac ggcgcacccg atttggactc
     2761 gcttcgataa attgggtggg aatttgcacc agtacacctt tttgttcaac gaatttggag
     2821 aacgcaggca cgcgattcgt tttcacaagc tattgaaagt cgagaatggt gtcgcaagag
     2881 aagttgatga tgtcaccgtg cccatttcaa tgtcagagca attggataat ctgcttccca
     2941 gagatcccaa tgaaccgatt gcgctatatt ttcgagatta cggagccgaa cagcatttca
     3001 caggtgaatt tggtggcgcg aagatccagt gccgccggga tcagctggct catatgcacc
     3061 gacgcagagg ggcgagggat gtttatctca atgtcagcgt acgtgtgcag agtcagtctg
     3121 aggcgcgggg agaacgtcgc ccgccgtatg cggcagtatt tcgtctggtc ggggacaacc
     3181 atcgcgcgtt tgtccatttc gataaactat cggattatct tgcggaacat ccggatgatg
     3241 ggaagctcgg gtcggagggg ttgctttccg ggctgcgggt gatgagtgtc gatctcggcc
     3301 ttcgcacatc tgcatcgatt tccgtttttc gcgttgcccg gaaggacgag ttgaagccga
     3361 actcaaaagg tcgtgtaccg tttttctttc cgataaaagg gaatgacaat ctcgtcgcgg
     3421 ttcatgagcg atcacaactc ttgaagctgc ctggcgaaac ggagtcgaag gacctgcgtg
     3481 ctatccgaga agaacgccaa cggacattgc ggcagttgcg gacgcaactg gcgtatttgc
     3541 ggctgctcgt gcggtgtggg tcggaagatg tggggcggcg tgaacggagt tgggcaaagc
     3601 ttatcgagca gccggtggat gcggccaatc acatgacacc ggattggcgc gaggcttttg
     3661 aaaacgaact tcagaagctt aagtcactcc atggtatctg tagcgacaag gaatggatgg
     3721 atgctgtcta cgagagcgtt cgccgcgtgt ggcgtcacat gggcaaacag gttcgcgatt
     3781 ggcgaaagga cgtacgaagc ggagagcggc ccaagattcg cggctatgcg aaagacgtgg
     3841 tcggtggaaa ctcgattgag caaatcgagt atctggaacg tcagtacaag ttcctcaaga
     3901 gttggagctt ctttggtaag gtgtcgggac aagtgattcg tgcggagaag ggatctcgtt
     3961 ttgcgatcac gctgcgcgaa cacattgatc acgcgaagga agatcggctg aagaaattgg
     4021 cggatcgcat cattatggag gctctcggct atgtgtacgc gttggatgag cgcggcaaag
     4081 gaaagtgggt tgcgaagtat ccgccgtgcc agctcatcct gctggaggaa ttgagcgagt
     4141 accagttcaa taacgacagg cctccgagcg aaaacaacca gttgatgcaa tggagtcatc
     4201 gcggcgtgtt ccaggagttg ataaatcagg cccaagtcca tgatttactc gttgggacga
     4261 tgtatgcagc gttctcgtcg cgattcgacg cgcgaactgg ggcaccgggt atccgctgtc
     4321 gccgggttcc ggcgcgttgc acccaggagc acaatccaga accatttcct tggtggctga
     4381 acaagtttgt ggtggaacat acgttggatg cttgtcccct acgcgcagac gacctcatcc
     4441 caacgggtga aggagagatt tttgtctcgc cgttcagcgc ggaggagggg gactttcatc
     4501 agattcacgc cgacctgaat gcggcgcaaa atctgcagca gcgactctgg tctgattttg
     4561 atatcagtca aattcggttg cggtgtgatt ggggtgaagt ggacggtgaa ctcgttctga
     4621 tcccaaggct tacaggaaaa cgaacggcgg attcatatag caacaaggtg ttttatacca
     4681 atacaggtgt cacctattat gagcgagagc gggggaagaa gcggagaaag gttttcgcgc
     4741 aagagaaatt gtcggaggaa gaggcggagt tgctcgtgga agcagacgag gcgagggaga
     4801 aatcggtcgt tttgatgcgt gatccgtctg gcatcatcaa tcggggaaat tggaccaggc
     4861 aaaaggaatt ttggtcgatg gtgaaccagc ggatcgaagg atacttggtc aagcagattc
     4921 gctcgcgcgt tccattacaa gatagtgcgt gtgaaaacac gggggatatt taataacatt
     4981 ggaagtggat aacggatccg cgatcgcggc gcgccacctg gtggccggcc ggtaccacgc
     5041 gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     5101 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     5161 ctgaaaggag gaactatatc cggatatcca caggacgggt gtggtcgcca tgatcgcgta
     5221 gtcgatagtg gctccaagta gcgaagcgag caggactggg cggcggccaa agcggtcgga
     5281 cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac
     5341 gccatagtga ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac
     5401 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
     5461 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
     5521 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
     5581 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5641 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taacattgaa
     5761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6001 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6301 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6481 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     6961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7441 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     7501 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     7561 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     7621 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     7681 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     7741 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     7801 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     7861 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     7921 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     7981 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     8041 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     8101 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     8161 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     8221 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     8281 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     8341 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     8401 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     8461 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     8521 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     8581 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     8641 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     8701 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     8761 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
     8821 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     8881 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     8941 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     9001 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     9061 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     9121 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     9181 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     9241 ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta
     9301 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggt
//
