LOCUS       sequence-181518-        9811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for Cas13a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9811)
  AUTHORS   East-Seletsky A, O'Connell MR, Burstein D, Knott GJ, Doudna JA
  TITLE     RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas 
            Enzymes.
  JOURNAL   Mol Cell. 2017 May 4;66(3):373-383.e3. doi: 
            10.1016/j.molcel.2017.04.008.
  PUBMED    28475872
REFERENCE   2  (bases 1 to 9811)
  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: 
            "10.1016/j.molcel.2017.04"; journalName: "Mol Cell"; date: 
            "2017-05-4- 4"; volume: "66"; issue: "3"; pages: "373-383.e3"
COMMENT     Sequence Label: p2CT-His-MBP-Hhe_Cas13a_WT
FEATURES             Location/Qualifiers
     source          1..9811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..23
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(17..208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1666..1685
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1769..1788
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1769..1787
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1876..1898
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1918..1935
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1945..3045
                     /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     3019..3042
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3100..3120
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7109..7127)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7123..7170
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7536..7541,7542..7558,7559..7564))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7591..7609)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7677..7781
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7782..7850,7851..8642)
                     /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(8000..8019)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8813..9401
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9302..9321
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9555..9572
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9587..9726
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9652..9671)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
       61 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
      121 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
      181 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
      241 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
      301 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
      361 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
      421 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
      481 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
      541 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
      601 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
      661 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
      721 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
      781 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
      841 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
      901 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
      961 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     1021 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     1081 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     1141 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     1201 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     1261 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     1321 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     1381 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     1441 tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag
     1501 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     1561 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     1621 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     1681 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     1741 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg
     1801 tttccctcta gtgccggctc cggagagctc tttaattaag cggccgccct gcaggactcg
     1861 agttctagaa ataattttgt ttaactttaa gaaggagata tacatatgaa atcttctcac
     1921 catcaccatc accatggttc ttctatgaaa atcgaagaag gtaaactggt aatctggatt
     1981 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
     2041 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
     2101 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
     2161 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
     2221 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
     2281 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
     2341 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
     2401 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
     2461 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
     2521 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
     2581 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
     2641 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
     2701 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
     2761 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     2821 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     2881 gcgaaagatc cacgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg
     2941 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     3001 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     3061 aacaacaaca ataacaataa caacaacctc gggatcgagg aaaacctgta cttccaatcc
     3121 aatgcaatga agttgacgcg tcgccgcatc agtggcaatt cagtcgacca gaagattaca
     3181 gctgcgtttt atcgtgatat gtcgcaaggg ttattatatt atgattcgga ggacaatgac
     3241 tgtacggaca aggtcatcga aagtatggat ttcgaacgct cttggcgtgg acgcattctg
     3301 aagaacggcg aggatgataa gaatccgttt tacatgttcg ttaaaggttt agttggttcc
     3361 aacgataaga tcgtgtgcga acctatcgac gtcgactcag accctgataa cttagatatc
     3421 ttgattaaca aaaatctgac ggggttcgga cgcaacttga aagcacctga cagtaacgat
     3481 accttagaaa atcttatccg caaaatccag gccggcattc ccgaagaaga ggttcttccg
     3541 gagctgaaga aaattaaaga gatgatccag aaagacatcg ttaaccgcaa agagcagttg
     3601 cttaaatcca tcaaaaacaa ccgtattcct ttttcattgg aaggttccaa gctggtccct
     3661 tccaccaaaa agatgaagtg gcttttcaag ttgattgacg ttcctaataa aaccttcaat
     3721 gaaaaaatgt tggagaagta ctgggagatc tacgactatg acaagcttaa ggcgaatatc
     3781 acgaaccgcc tggacaaaac ggataagaag gcacgttcaa tttcgcgtgc ggtaagcgag
     3841 gagttacgtg agtaccataa aaacttgcgc acaaactata atcgcttcgt atctggagac
     3901 cgtccagcag ccgggcttga caatgggggg agtgctaaat ataatcccga taaggaggag
     3961 ttcttattgt ttcttaaaga agttgagcag tacttcaaga aatacttccc agtcaagagc
     4021 aagcattcga acaagtccaa agacaaatca ttggttgata agtacaaaaa ttattgcagt
     4081 tacaaagtgg tcaagaaaga ggtcaaccgc agcatcatca atcaattggt ggctggactt
     4141 attcagcagg gtaagcttct gtattacttc tactataacg atacctggca ggaggatttc
     4201 ctgaattcct acggtcttag ttatattcag gttgaggagg catttaaaaa gagtgtaatg
     4261 accagtcttt cgtggggaat caaccgtctg acctcgttct ttatcgacga ctcgaacact
     4321 gtgaagtttg atgatatcac aacgaaaaag gctaaggagg cgattgagtc taattatttc
     4381 aacaaattac gcacatgctc ccgtatgcag gaccacttca aagagaaatt ggccttcttt
     4441 tacccggtgt atgtgaagga caagaaggat cgcccggatg atgatatcga aaacttaatt
     4501 gtactggtta agaacgccat tgagagcgtg agctatttgc gcaatcgcac gtttcacttt
     4561 aaagagagct ccttattgga gctgcttaaa gagttagacg ataaaaattc tggtcaaaat
     4621 aaaatcgact atagcgtagc cgctgaattt attaaacgcg atatcgagaa cttgtatgac
     4681 gtcttccgtg aacagatccg ctcacttggt attgctgagt actacaaggc ggacatgatt
     4741 tcggactgtt ttaagacatg cgggttagaa ttcgcgcttt attcgccaaa aaattcgttg
     4801 atgcccgctt ttaagaacgt ttataaacgc ggagcaaatc ttaacaaggc gtatattcgc
     4861 gataaagggc caaaggaaac aggagaccag ggtcagaact cgtacaaagc tttggaggaa
     4921 tatcgtgagc ttacatggta tattgaggtg aagaataacg accagtcgta caacgcctac
     4981 aagaatttgc ttcaactgat ttattatcat gcgtttcttc cggaggttcg tgaaaatgag
     5041 gctcttatca ctgacttcat taaccgcact aaagagtgga atcgtaagga aacagaggag
     5101 cgtctgaaca ctaaaaacaa taaaaaacac aagaatttcg acgagaatga cgatattacg
     5161 gtaaatacgt atcgttacga atctattccg gattaccagg gggaatccct tgatgattat
     5221 cttaaggtgt tacagcgcaa gcaaatggcg cgtgcgaagg aggtaaatga gaaggaggag
     5281 ggcaacaata actatatcca attcattcgt gacgtcgtag tatgggcgtt cggggcctac
     5341 ttggaaaaca agctgaagaa ttataagaac gaactgcaac caccgttgag caaagagaac
     5401 attggcttaa atgatacctt aaaggaattg tttcccgaag aaaaagtaaa gtcaccattt
     5461 aatatcaagt gccgtttctc cattagtaca tttatcgaca acaaaggtaa gtccaccgat
     5521 aatacctcgg ccgaggccgt caagactgat ggcaaggagg acgaaaagga caaaaagaat
     5581 attaaacgca aagacttgct ttgcttttac ctgttcttac gcttgcttga tgaaaacgaa
     5641 atttgcaaat tacaacacca atttattaaa taccgttgca gtttaaaaga gcgtcgtttt
     5701 ccaggtaacc gcaccaaact ggagaaggaa actgaacttt tggccgagct ggaagaactg
     5761 atggaactgg tacgttttac gatgccatca attcctgaaa tctctgcaaa ggcggaatca
     5821 ggttatgaca caatgattaa gaagtatttt aaagacttca tcgagaaaaa agtctttaag
     5881 aatccgaaga cgtccaacct gtactaccac tccgactcta aaactccggt cacccgcaaa
     5941 tatatggctc tgctgatgcg ttctgcgcct ttgcatctct acaaggatat cttcaagggc
     6001 tactatttga tcactaagaa ggagtgtttg gagtatatta aattaagcaa catcattaaa
     6061 gactatcaaa actcattgaa tgaattgcac gagcagctgg agcgcattaa attaaagagt
     6121 gagaaacaaa acgggaaaga ctctctgtat cttgacaaaa aggacttcta taaagtaaag
     6181 gagtacgtcg aaaatttaga acaagtggcg tgttacaagc acttgcagca caaaatcaac
     6241 ttcgaaagct tataccgtat ttttcgtatt cacgtggaca tcgctgcgcg tatggtcggt
     6301 tatacccaag actgggagcg tgatatgcac tttcttttca aggcgttggt ctataatggc
     6361 gtacttgagg agcgccgttt tgaagcaatc tttaacaata acgatgacaa caacgacggg
     6421 cgtatcgtaa aaaagatcca gaataactta aataacaaga accgcgaatt agtatctatg
     6481 ttatgttgga ataaaaagct gaacaagaac gaatttggtg ctattatttg gaagcgtaac
     6541 cctatcgccc atctgaacca ctttacacaa acggaacaaa actcaaagtc gagccttgaa
     6601 tctctgatca actcgctgcg catcctgtta gcgtacgacc gtaaacgtca aaacgctgtg
     6661 acaaagacta tcaatgacct tctgttaaac gactatcaca tccgcattaa gtgggaaggg
     6721 cgcgtggatg aagggcagat ctactttaat atcaaggaaa aggaagacat cgagaacgag
     6781 cctattatcc acttgaaaca cctgcataaa aaggactgct acatttacaa gaacagctat
     6841 atgttcgaca agcaaaagga gtggatttgt aatggcatta aagaagaagt ttatgacaag
     6901 tccattctta agtgtatcgg aaacttgttc aagtttgact atgaggacaa aaacaaatcc
     6961 tcagcaaatc ctaagcacac ctagtaaatt ggaagtggat aacggatccg cgatcgtggc
     7021 gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc
     7081 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     7141 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca
     7201 caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag
     7261 caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa
     7321 ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg
     7381 gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
     7441 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
     7501 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
     7561 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     7621 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     7681 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     7741 aaccctgata aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc
     7801 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     7861 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     7921 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     7981 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     8041 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     8101 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     8161 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     8221 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     8281 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     8341 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     8401 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     8461 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     8521 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     8581 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     8641 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     8701 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     8761 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     8821 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     8881 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     8941 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     9001 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     9061 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     9121 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     9181 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     9241 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     9301 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     9361 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     9421 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     9481 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     9541 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga acagcgcctg atgcggtatt
     9601 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     9661 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     9721 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     9781 gcatccgctt acagacaagc tgtgaccgtc t
//
