LOCUS       sequence-178225-        8098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for bzCas13b and crRNA with both HEPN domains 
            mutated. New spacers can be cloned by digesting with BsaI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8098)
  AUTHORS   Smargon AA, Cox DB, Pyzocha NK, Zheng K, Slaymaker IM, Gootenberg 
            JS, Abudayyeh OA, Essletzbichler P, Shmakov S, Makarova KS, Koonin 
            EV, Zhang F
  TITLE     Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase 
            Differentially Regulated by Accessory Proteins Csx27 and Csx28.
  JOURNAL   Mol Cell. 2017 Feb 16;65(4):618-630.e7. doi: 
            10.1016/j.molcel.2016.12.023. Epub 2017 Jan 5.
  PUBMED    28065598
REFERENCE   2  (bases 1 to 8098)
  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.2016.12.023"; journalName: "Mol Cell"; date: 
            "2017-02-16- 16"; volume: "65"; issue: "4"; pages: "618-630.e7"
COMMENT     Sequence Label: pBZCas13b-HEPN
FEATURES             Location/Qualifiers
     source          1..8098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(1921..1926,1927..1944,1945..1950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     promoter        complement(join(2100..2105,2106..2122,2123..2128))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      2240..2785
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     2686..2705
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2902..2919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3311..3413
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3414..4073
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3480..3499)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     5926..5945
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(6295..6329)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
ORIGIN
        1 cttttccaat ttcctttctt aattcttttt cacctttctt ttctaacttt tctttctttt
       61 tctcccattt atcgaaaaac tcatacaaaa tggaatgaat gtcattcata ctcagataag
      121 ccgtgggttg cccaccgaaa acaatagctt cttttggatt gctttcattg attggcacta
      181 tttcttctat aatgttctgg atagacggct tgctggcttt tctttgcttg agctgatggg
      241 cttttacggc tttatctact tctgaaacat attgttggtt aagcaatttc acttttatac
      301 caattttccc tgcgacaggc tgtttctctc tgtccaaaat actacccgcg ataggaaaat
      361 ctttatcatt aacggaaata ttttccttag gaaaatcata attggggtta ggaaaaattt
      421 cccaatagtg ttctctatct tcattggttt cggtattttt tataaacaat gcttttttat
      481 gttccaattc cgaaagcctt ccgaaaacgg tgattttttc ttttattctt cggtcagaaa
      541 tcagattttc tttagggcga gaatcgtgaa gataattccc taaatgcact tgaaaacgaa
      601 gcgtagggaa ctgagcaaac tcatctaaaa atcgaatggc aaaataattg aatttatctt
      661 cgtaacgctt gcgaatcact ggatgaatta cctgctcttc ttccattgtc cctacatcgc
      721 cattgttctc ttttaggtat tcattccaat cttcgatgaa agttttttgt acatcttcgc
      781 tcaaattctg atagaccaca tcgggtactt tgctcaattc atcaagcatc tgcatcatta
      841 gagtttcttt ggcataaacg gaaagctgtt cagcattttc tgcctcacca tagttgattt
      901 cagccgttct cacttttcgt ttcaattttt tgtaagccaa atgcgagaaa atttcgtgcg
      961 tagccataaa acaaatgctg ttttttccat ggctcaccgt tgcttcagat accgttgctt
     1021 catcgataac cgtagcctta aaccctgcta ttttagattt gaatgcgtgt atttcttgtt
     1081 tggtgagaaa caaagaaagc aggaagacta ctccgttttt cgaaattgga attttcaaat
     1141 cgccttcttc ttgctgagga tcacttttgg tattgtactt tgctttgctg gattctttta
     1201 ggctatcttt tttcttatcg atataaacat caaaagcatc attataaatc gctgctatta
     1261 aatcatccct tttatcactg tatttgaatg gagctaccaa ttctttctct cctctttctc
     1321 tttgatttct acgcttctcc tctatttttc ttgctgtgtc tcttagatat tctaatttct
     1381 tttggcataa aatatccaat tgtttttcta tgcttttttt caggatttct tttgttttat
     1441 cggtttttac ttttttcttt tttaccgtta aaaccgtgct tttcagcatc tcatccaata
     1501 cgccaaatat ttcatctgta atttctacct ctccgtgttc ctttgccgta taaaagtttg
     1561 ctaaatctct tacggctttt ataatgcctt taaaattctt tttaaaattt tcttttcgtt
     1621 cttttatagg aacttctttt ttatccaaaa gacgagccat agggaaataa tcagaaagca
     1681 atttcacata cctttcgtat tccactaatg aaatattttc cttaaagata gcatcaaaaa
     1741 aattctccga agtgtactct ttacctttta atctttttcc caattctcta aatacactat
     1801 ctgtattttc cattgctgca ttgaaataac ccgcaaaata cgatttatct tgtggtttaa
     1861 aagggttgta ataaatatta ttccccaaag atgttttgtt ttccatgata aagacctcct
     1921 aacatacgag ccggaagcat aaagtgtaaa atcccgcaag aggcccggca gtaccggcat
     1981 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
     2041 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
     2101 ttaaagctta tcgatgataa gctgtcaaac atgagaatta caacttatat cgtatggggc
     2161 tgacttcagg tgctacattt gaagagataa attgcactga aatctagaaa tattttatct
     2221 gattaataag atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa
     2281 acgaaaaaac cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga
     2341 accgaggtaa ctggcttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt
     2401 aaccggcgca tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt
     2461 ggtgcttttg catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag
     2521 cggtcggact gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg
     2581 gaactgagtg tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt
     2641 aaaccgaaag gcaggaacag gagagcgcac gagggagccg ccagggggaa acgcctggta
     2701 tctttatagt cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt
     2761 gtcagggggg cggagcctat ggaaaaacgg ctttgccgcg gccctctcac ttccctgtta
     2821 agtatcttcc tggcatcttc caggaaatct ccgccccgtt cgtaagccat ttccgctcgc
     2881 cgcagtcgaa cgaccgagcg tagcgagtca gtgagcgagg aagcggaata tatcctgtat
     2941 cacatattct gctgacgcac cggtgcagcc ttttttctcc tgccacatga agcacttcac
     3001 tgacaccctc atcagtgcca acatagtaag ccagtataca ctccgctagc gctgatgtcc
     3061 ggcggtgctt ttgccgttac gcaccacccc gtcagtagct gaacaggagg gacagctgat
     3121 agaaacagaa gccactggag cacctcaaaa acaccatcat acactaaatc agtaagttgg
     3181 cagcatcacc cgacgcactt tgcgccgaat aaatacctgt gacggaagat cacttcgcag
     3241 aataaataaa tcctggtgtc cctgttgata ccgggaagcc ctgggccaac ttttggcgaa
     3301 aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac
     3361 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
     3421 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     3481 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     3541 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     3601 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     3661 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     3721 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     3781 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     3841 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     3901 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     3961 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     4021 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taattttttt
     4081 aaggcagtta ttggtgccct taaacgcctg gttgctacgc ctgaataagt gataataagc
     4141 ggatgaatgg cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg
     4201 ttaaatagcc gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt
     4261 gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat
     4321 aattgacgat atgatcattt attctgcctc ccagagcctg ataaaaacgg ttagcgcttc
     4381 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4441 aacataatgg tgcagggcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     4501 ggactgttgg gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4561 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctaccggc agcggtgcgg
     4621 actgttgtaa ctcagaataa gaaatgaggc cgctcatggc gttgactctc agtcatagta
     4681 tcgtggtatc accggttggt tccactctct gttgcgggca acttcagcag cacgtagggg
     4741 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     4801 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     4861 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     4921 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg
     4981 agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc
     5041 gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc
     5101 ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac
     5161 aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc
     5221 ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
     5281 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa
     5341 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc
     5401 tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat
     5461 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     5521 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5581 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5641 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5701 tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag
     5761 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     5821 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     5881 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     5941 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     6001 gaggatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag
     6061 cgaagcgagc aggactgggc ggcggccaaa gcggtcggac agtgctccga gaacgggtgc
     6121 gcatagaaat tgcatcaacg catatagcgc tagcagcacg ccatagtgac tggcgatgct
     6181 gtcggaatgg acgatgttgt gattaccctc caaaatgaga gcagttccaa ctgagacctt
     6241 ccgccatggt ctcgttgtga ttaccctcca aaatgagagc agttccaact cagcgctagc
     6301 attataccta ggactgagct agctgtcaat tactttatca gtgcttcttt ttccttttta
     6361 aatacctcag caaaatactc ggcataggtt ttttcttttt ctattttgcc gtatttttct
     6421 tgacaatagt cccaaaattc ttttttaggt aattggtttg ctgcaaattt atttgcaata
     6481 taggttaaaa caaaagcttt ttgttctaaa tcggttgctt cttgttttag ttggttaatg
     6541 ttcacatctt caggttcagt atttaagttg aataccttat agctctccac atcttcattt
     6601 ttaagttgct ttaaaagtcc gttgagaatg tagtatttga aattttgatt atcccctttt
     6661 tttaatattt ccttatcttt taccttttcc aaaatatatt cctctataag gtggacttcc
     6721 ttcaacagtt cttcccttct cactttttca tattgctcta tttctctttc caccacatac
     6781 ggataatttt cctcttcctt actctctttg attagaaacg cctgccattc tatattttcc
     6841 tcatatttta agaaagcttg aaccctttgg tcgtattcat attttatgaa atctcctaca
     6901 ttttttagct ttacattttc taccgtaatt tttccgtcac acaactttaa atcaacggtt
     6961 ttgttccaaa tatagttagt atttcgctcg cccgtttgtc tggctctctc ctgatttcct
     7021 aagcgttcct cccgagattg atacaaatct tctaacgaaa actgatcgat gctatcctgc
     7081 ttgaataccg atttgaaaat atgttttgcc atcaaaagtg taaacacatc gtttttcttt
     7141 tgttggtaga ttttagtttt tcgttttcgg tctgcttgtt ttttttccaa ttctacaagc
     7201 ggataatttt cagtatcata aaatgtctga aaattctgat attccttgta atacctaaac
     7261 caatcggcaa aaagagcttc atttccttta aaagtttttc ctttgattat cgtaggtttt
     7321 tcatccataa agccccgctc taaaaagata ggatacccca atatagactg aacccgagca
     7381 tctaattctt tgtgagtata attttgtttt ttcaaaaatt taaaacactc attttctacc
     7441 tttttgaaga ctttattttc ggatttaaag ttttcggctt gttgcaccaa cccactgatg
     7501 tattccaacc gtttatctaa ataacaagtg taaaattggt acaaatattt ttgttgaaaa
     7561 tatcctccca acttaaaagg taaatgctga aatacttttt caggcaaaag atttttcaac
     7621 tcctctttac attgctcata atacgccaac gatttttgta acaagctgtg ttgctcccct
     7681 ttccattcgt ttttgaaatc ggtaggcata aagcgtttga tgtcattcgc caaccaaacc
     7741 gctacttttc ctttttctcg ataataaaga acttcttttc gtgcaggagc ttcagggtac
     7801 tttcgtttaa ggttatcttt taaatattgt ttatgatttc tatctctgac tttatttatc
     7861 tccctatttt tatcttgata agcaataaaa tcattatatt ctttttcacg aaccgtttgt
     7921 tcatctttta atttttgtag aatgttttgc tcttgcttca aatcttttat caatttctct
     7981 ttatcaatag cagtagaatt tccgtcttga taaggtttta atatcttggc attggtatct
     8041 ttatctatga tttgctggat ttgagcttga atttttccta cgattttctt ttctaact
//
