LOCUS       sequence-159343-        9320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of WT L. seeligeri C2c2 CRISPR effector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9320)
  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 9320)
  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-Lse_C2c2_WT
FEATURES             Location/Qualifiers
     source          1..9320
                     /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(8856..8874)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8870..8917
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(9283..9288,9289..9305,9306..9311))
                     /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 tggatcagca tcaagacctt aatccatcac
     5401 cttggggtcc tgttcttctg cgactatatg tacaatcgcc gtgagaagaa aattattgaa
     5461 gtcaagacga tgcgtattac caaagtagaa gtggatcgca aaaaggtgct tatctcccgc
     5521 gataagaatg gtggaaaact ggtttacgaa aatgagatgc aggataatac ggagcagatc
     5581 atgcaccaca aaaagtcctc attttacaag tctgtcgtca ataaaacaat ttgccgtcca
     5641 gagcaaaaac aaatgaaaaa gttagtgcat gggcttttac aggaaaactc tcaagagaag
     5701 atcaaagtat cggatgttac caagcttaac atctcgaact ttcttaatca tcgcttcaaa
     5761 aaaagccttt actacttccc ggaaaattct ccggataaaa gcgaggagta ccgcattgag
     5821 attaatttaa gtcagttgct tgaagattcg ctgaagaagc agcaggggac ctttatttgc
     5881 tgggagagct tttcaaaaga catggagctg tacattaatt gggccgaaaa ttatatttcg
     5941 agcaaaacca aacttattaa gaaatctatt cgtaataatc gtatccagtc tacggaaagc
     6001 cgttccggac agttaatgga tcgctacatg aaagacatcc tgaacaagaa taagcctttt
     6061 gacattcagt cagtctcgga aaagtaccag ttggaaaaat taacttcggc actgaaggct
     6121 acctttaagg aggctaagaa gaatgacaag gagattaact ataaattaaa gagcacgctg
     6181 cagaaccatg aacgccagat catcgaggaa ttaaaagaga actccgaact taaccaattc
     6241 aacatcgaaa tccgtaagca tcttgagacc tacttcccca tcaaaaagac caaccgtaag
     6301 gtaggcgaca ttcgtaactt agagattggg gaaattcaga aaatcgtcaa ccatcgtttg
     6361 aagaataaga ttgtccaacg tatccttcag gaaggaaagc ttgcgtcgta cgagattgaa
     6421 tcaacggtaa actctaactc gttacagaag atcaagattg aggaggcgtt cgcacttaag
     6481 ttcattaatg cttgtctttt tgcctcgaac aatttacgta acatggttta cccagtctgc
     6541 aaaaaagaca ttttgatgat tggggagttt aagaactcct ttaaggaaat caagcataag
     6601 aagttcattc gtcagtggtc ccaattcttt agccaggaga tcactgtcga tgacattgag
     6661 ttggcctcat gggggttacg cggagcaatt gctccgattc gtaacgaaat cattcattta
     6721 aagaaacata gctggaagaa atttttcaac aatcccactt tcaaagtcaa aaaatctaag
     6781 atcattaacg gaaagaccaa ggatgttacc agtgaattcc tttataaaga aacactgttt
     6841 aaagattact tttattctga attggattct gtgcccgagt tgatcatcaa taagatggaa
     6901 tcttcgaaaa tcttggacta ttactcaagc gatcagctta accaggtctt cacaattccc
     6961 aatttcgagt tgagcttgtt gacatcggcc gttccttttg cgccatcctt taaacgtgta
     7021 tatcttaaag gttttgacta tcagaaccag gatgaagcac agcctgacta caatttgaag
     7081 ctgaatattt acaatgagaa agctttcaat agtgaagctt ttcaggctca atatagctta
     7141 ttcaaaatgg tttattatca agtgttcctt ccgcagttca ccactaataa cgatcttttt
     7201 aaaagctcag tcgatttcat tttgactttg aacaaagaac gcaaagggta cgcgaaggcc
     7261 tttcaggata ttcgcaaaat gaataaagac gaaaaacctt ctgagtacat gtcgtacatt
     7321 cagtcgcagc tgatgttgta ccagaagaag caggaagaga aagaaaaaat caaccatttt
     7381 gagaaattta tcaaccaagt gttcattaag gggtttaact cgtttattga gaaaaaccgt
     7441 ctgacttata tctgtcatcc aacaaagaat actgtcccgg agaatgataa cattgaaatc
     7501 ccatttcaca cggatatgga tgattcaaac atcgcattct ggctgatgtg caaattactg
     7561 gatgcgaaac aacttagcga attacgtaat gagatgatta agttctcctg ttctttacaa
     7621 tctaccgagg aaatttccac attcactaaa gctcgtgaag tgattggtct ggctttactt
     7681 aacggggaga aaggctgcaa tgactggaaa gagttatttg acgataaaga ggcatggaag
     7741 aaaaacatgt cactgtatgt gagcgaggaa cttcttcagt cattacccta tacccaggag
     7801 gatgggcaga cgccagttat caaccgttcc atcgatctgg ttaagaaata cggtacggag
     7861 accatcttag agaaattgtt tagttcatca gatgactata aagtctcagc gaaggacatc
     7921 gcaaagttac acgagtacga cgtaactgaa aagatcgcac aacaagagag tttacataag
     7981 cagtggatcg aaaagcctgg gcttgctcgc gactcggcat ggaccaaaaa ataccagaat
     8041 gtaattaatg atatttcgaa ttaccagtgg gctaagacta aggtggagct tacccaggtt
     8101 cgtcaccttc atcagttgac catcgattta ttgtcccgct tagctgggta tatgtctatt
     8161 gcagatcgcg atttccagtt ctccagtaat tatatcttgg aacgtgaaaa tagtgagtac
     8221 cgcgtaacga gctggatcct tttatctgaa aacaagaaca agaataagta taatgactat
     8281 gaactgtata atcttaagaa cgcttcaatc aaagtcagca gcaaaaatga ccctcagctt
     8341 aaagttgatt taaaacagtt gcgtttaacg ttggagtact tagagctttt cgacaaccgc
     8401 ttaaaggaga agcgtaataa tatttcgcac ttcaactacc ttaacggaca gcttggtaac
     8461 agcatcttgg aattattcga cgacgcccgc gatgtactgt cttatgatcg caaattaaag
     8521 aatgccgtct cgaaatcatt aaaagaaatc ctttcttcac acggtatgga agtaacgttt
     8581 aagccgcttt atcaaacaaa ccaccatttg aaaatcgaca agttgcaacc caaaaagatt
     8641 catcacttag gtgagaaaag tacggttagt tcgaatcaag tatctaatga atactgccag
     8701 ttagttcgta ctttgcttac catgaaataa taacattgga agtggataac ggatccgcga
     8761 tcgcggcgcg ccacctggtg gccggccggt accacgcgtg cgcgctgatc cggctgctaa
     8821 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     8881 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     8941 atatccacag gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg
     9001 aagcgagcag gactgggcgg cggccaaagc ggtcggacag tgctccgaga acgggtgcgc
     9061 atagaaattg catcaacgca tatagcgcta gcagcacgcc atagtgactg gcgatgctgt
     9121 cggaatggac gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta
     9181 cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg
     9241 gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga
     9301 taagctgtca aacatgagaa
//
