LOCUS       sequence-159341-        9437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of L. buccalis C2c2 CRISPR effector (double 
            HEPN nuclease 1 and 2 inactive mutant)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9437)
  AUTHORS   East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian
            R, Doudna JA
  TITLE     Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA 
            processing and RNA detection.
  JOURNAL   Nature. 2016 Sep 26. doi: 10.1038/nature19802.
  PUBMED    27669025
REFERENCE   2  (bases 1 to 9437)
  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"; journalName: "Nature. 
            2016 Sep 26. doi: 10.1038/nature19802."
COMMENT     Sequence Label: p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A
FEATURES             Location/Qualifiers
     source          1..9437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2014..2156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2082..2101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2242..2264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2258..2449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     3907..3926
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4010..4029
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4010..4028
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4176
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4186..5286
                     /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     5260..5283
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5341..5361
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(8973..8991)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8987..9034
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(9400..9405,9406..9422,9423..9428))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2041 tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc
     2101 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     2161 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     2221 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     2281 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     2341 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     2401 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     2461 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     2521 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     2581 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     2641 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     2701 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     2761 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     2821 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     2881 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     2941 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     3001 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     3061 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     3121 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     3181 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt
     3241 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     3301 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     3361 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     3421 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     3481 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     3541 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     3601 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     3661 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
     3721 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     3781 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     3841 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     3901 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     3961 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
     4021 actataggga gaccacaacg gtttccctct agtgccggct ccggagagct ctttaattaa
     4081 gcggccgccc tgcaggactc gagttctaga aataattttg tttaacttta agaaggagat
     4141 atacatatga aatcttctca ccatcaccat caccatggtt cttctatgaa aatcgaagaa
     4201 ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt
     4261 aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa
     4321 gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac
     4381 gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg
     4441 ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt
     4501 gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac
     4561 ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag
     4621 agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac
     4681 gggggttatg cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat
     4741 aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg
     4801 aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg
     4861 accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta
     4921 acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca
     4981 ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg
     5041 ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg
     5101 aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccactat ggaaaacgcc
     5161 cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt
     5221 actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg
     5281 cagactaatt cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag
     5341 gaaaacctgt acttccaatc caatgcaatg aaagtgacga aggtaggagg catttcgcat
     5401 aagaagtaca cgtccgaagg ccgcttagtg aagtcagaat cggaagaaaa tcgcacagac
     5461 gaacgtctgt cggcgttgct taatatgcgc cttgacatgt atatcaagaa tcccagcagc
     5521 acggaaacca aggaaaatca aaaacgcatt gggaaattaa agaaattctt ctcaaacaaa
     5581 atggtctatc ttaaagacaa taccttgagt ttgaagaatg ggaaaaagga gaacattgat
     5641 cgtgagtatt ctgagactga catccttgag agcgatgtcc gtgacaagaa aaacttcgcc
     5701 gtgttgaaaa agatctatct gaatgaaaac gtgaactcgg aggaattgga agtttttcgt
     5761 aacgacatta agaagaaact gaacaaaatc aacagcctga agtactcatt tgaaaagaat
     5821 aaggcgaatt atcaaaagat taatgagaat aacatcgaga aggttgaagg taagtcaaag
     5881 cgtaacatta tttacgatta ttatcgtgag tcagcgaaac gtgacgctta tgtaagcaat
     5941 gtgaaagaag cctttgataa gctttacaag gaagaggaca ttgcaaaact tgttcttgaa
     6001 attgagaacc ttacgaagtt agagaaatac aagattcgcg agttctacca cgaaattatt
     6061 ggacgtaaga atgacaagga aaactttgca aaaatcatct acgaagaaat ccagaatgtt
     6121 aataacatga aagagttgat cgagaaggta ccggacatga gtgagttgaa aaagagccaa
     6181 gtattttaca agtattactt agacaaagaa gagttgaacg acaagaacat caaatacgcg
     6241 ttttgtcatt tcgtggaaat cgaaatgagt cagttgctga agaactacgt atataagcgc
     6301 ttaagtaata tctcgaatga caaaattaag cgtatctttg aataccagaa cttgaaaaaa
     6361 ttgatcgaaa ataagctgtt aaacaaactt gacacgtacg tccgtaattg tggaaagtat
     6421 aattattatt tgcaagacgg cgaaattgcc acttcagatt tcatcgcccg caaccgtcag
     6481 aatgaagcgt ttcttcgcaa catcattggg gtgtcatctg tggcctactt ttctcttcgc
     6541 aacattcttg aaacggagaa cgagaatgat attactgggc gtatgcgcgg caaaacagtt
     6601 aagaacaata aaggtgaaga gaagtacgtg tccggagaag ttgataagat ctataatgaa
     6661 aataagaaga acgaggttaa ggagaactta aaaatgttct attcgtacga tttcaatatg
     6721 gacaacaaga atgaaatcga agatttcttc gccaacatcg acgaggcgat ttcttccatc
     6781 gctcacggta ttgtcgcctt caacttggaa ttagaaggta aggatatctt tgcgttcaag
     6841 aacattgcgc catccgaaat ctcaaagaag atgtttcaga atgagattaa cgagaaaaaa
     6901 ctgaaattga agatctttcg tcaactgaac tctgccaacg tgttccgcta tctcgaaaag
     6961 tataaaattc tgaattacct taaacgtaca cgcttcgagt ttgtcaataa aaatatccca
     7021 ttcgtcccgt ctttcaccaa attatattcg cgcattgatg acctgaagaa tagtcttggg
     7081 atttactgga aaactccgaa aacaaacgac gacaataaga ctaaggagat tattgatgcc
     7141 caaatctatt tgcttaaaaa catctattac ggggagttcc tgaattattt catgtcgaac
     7201 aatggtaatt tctttgagat ttctaaagaa atcatcgaat tgaacaagaa cgataaacgc
     7261 aacttaaaga ctgggtttta caagctgcaa aagtttgaag acatccagga gaagattcca
     7321 aaggaatact tggcgaatat ccagtccctg tacatgatta atgccggtaa tcaggacgaa
     7381 gaagaaaagg acacttatat tgatttcatt caaaagatct tcttaaaggg atttatgacg
     7441 tatcttgcta ataacggtcg tttaagtctg atttacatcg gctcggatga agaaacaaat
     7501 acgtcattag cagaaaagaa gcaagagttt gacaagttct tgaagaagta cgagcagaac
     7561 aataatatca agatccccta tgagatcaat gaattcctgc gtgagatcaa actgggaaac
     7621 atcctgaagt atactgagcg tttaaacatg ttctacctta tcttaaagct tttgaatcac
     7681 aaggagctga caaatctgaa gggtagtctt gaaaaatatc agtctgccaa taaggaagaa
     7741 gcgttctctg accaattgga gttaattaac ctgcttaacc ttgacaacaa ccgcgtgacg
     7801 gaagacttcg aattagaggc cgacgagatt ggaaaatttc ttgatttcaa tggcaacaaa
     7861 gttaaggata acaaggaact gaaaaagttc gatacaaaca agatctactt tgacggcgag
     7921 aacattatca aacaccgtgc cttctacaat attaagaaat atggcatgtt aaacttactg
     7981 gagaaaattg ccgacaaggc tggatacaag atctcgatcg aagagctgaa gaaatactcc
     8041 aataaaaaga atgagatcga gaagaaccat aagatgcagg aaaatctgca ccgcaaatac
     8101 gctcgtcccc gtaaagacga gaagtttaca gatgaggact atgaaagtta caagcaagct
     8161 attgagaata ttgaggagta cacccacctt aagaacaagg tagaattcaa tgagctgaat
     8221 ttactgcagg gcctgttgct gcgcatttta catcgtttag tcggatatac ctcaatttgg
     8281 gaacgcgatc tgcgcttccg ccttaaaggt gagttcccag aaaaccaata catcgaagag
     8341 atcttcaact ttgaaaataa gaagaacgtg aagtacaaag ggggtcagat tgtagagaaa
     8401 tacattaaat tctacaagga attacatcaa aatgatgaag ttaagatcaa caagtacagt
     8461 tccgcgaata tcaaggtgtt gaagcaagaa aagaaggacc tttatattgc taattacatc
     8521 gccgcattca attatattcc tcacgccgag atctcactgc tggaagtcct tgaaaatttg
     8581 cgtaaattgc tgtcctacga tcgcaaactg aaaaatgccg taatgaaatc agtagttgat
     8641 atccttaagg agtatggttt tgtagccaca ttcaaaatcg gggcggacaa gaagatcggt
     8701 attcagacac tggagagcga aaaaatcgtg catcttaaga atcttaagaa gaagaagtta
     8761 atgactgacc gcaattccga ggaactttgc aaattggtga agattatgtt tgaatacaaa
     8821 atggaagaga aaaagtctga aaactaataa cattggaagt ggataacgga tccgcgatcg
     8881 cggcgcgcca cctggtggcc ggccggtacc acgcgtgcgc gctgatccgg ctgctaacaa
     8941 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     9001 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggata
     9061 tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca agtagcgaag
     9121 cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg ggtgcgcata
     9181 gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg
     9241 aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     9301 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     9361 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     9421 gctgtcaaac atgagaa
//
