LOCUS       sequence-358153-        9626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial protein expression plasmid of wild-type Cas12c_4. This is 
            a R965H version of the Cas12c in Harrington et al., 2020..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9626)
  AUTHORS   Huang CJ, Adler BA, Doudna JA
  TITLE     A naturally DNase-free CRISPR-Cas12c enzyme silences gene 
            expression.
  JOURNAL   Mol Cell. 2022 Jun 2;82(11):2148-2160.e4. doi: 
            10.1016/j.molcel.2022.04.020.
  PUBMED    35659325
REFERENCE   2  (bases 1 to 9626)
  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.2022.04"; journalName: "Mol Cell"; date: 
            "2022-06-2- 2"; volume: "82"; issue: "11"; pages: "2148-2160.e4"
COMMENT     Sequence Label: pCJH002_10xHis-MBP-TEVcs-Cas12c(4)_Amp
FEATURES             Location/Qualifiers
     source          1..9626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     761..780
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     864..883
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        864..882
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             971..993
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1013..1042
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1052..2152
                     /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     2126..2149
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2207..2227
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6019..6037)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6033..6080
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6446..6451,6452..6468,6469..6474))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6501..6519)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6587..6691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6692..6760,6761..7552)
                     /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(6910..6929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7723..8311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8212..8231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8465..8482
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8497..8636
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8562..8581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8722..8744
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8738..8929)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
       61 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
      121 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
      181 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
      241 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
      301 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
      361 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctacagcga aagcggtcct
      421 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
      481 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
      541 tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc
      601 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
      661 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
      721 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
      781 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
      841 tcgagatctc gatccagcga aattaatacg actcactata gggagaccac aacggtttcc
      901 ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg actcgagttc
      961 tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt ctcaccatca
     1021 ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta aactggtaat
     1081 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     1141 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     1201 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     1261 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     1321 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     1381 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     1441 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     1501 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     1561 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     1621 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     1681 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     1741 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     1801 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     1861 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     1921 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     1981 agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat
     2041 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     2101 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     2161 ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa acctgtactt
     2221 ccaatccaat gcaatgacca agcacagcat ccctctgcac gccttcagaa actctggcgc
     2281 cgatgccaga aagtggaagg gcagaattgc cctgctggcc aagaggggca aagaaaccat
     2341 gcggaccctg cagttccctc tggaaatgag cgaacctgag gccgctgcca tcaacaccac
     2401 accttttgcc gtggcctaca acgccatcga aggcacaggc aagggcaccc tgtttgacta
     2461 ctgggccaaa ctgcacctgg ccggcttcag attctttcca tctggcggag ccgccaccat
     2521 ctttaggcag caggccgtgt ttgaggacgc ctcttggaat gccgccttct gtcagcagag
     2581 cggcaaggat tggccttggc tggtgcctag caagctgtac gagcggttca caaaggcccc
     2641 tagagaggtg gccaagaagg acggcagcaa gaagtccatc gagttcaccc aagagaacgt
     2701 ggccaacgag agccacgtgt cacttgtggg cgccagcatc accgataaga cccctgagga
     2761 ccagaaagaa tttttcctga agatggcagg cgccctggcc gagaagttcg attcttggaa
     2821 gtccgccaac gaggaccgga tcgtggccat gaaagtgatc gacgagttcc tgaagtccga
     2881 gggcctgcat ctgcccagcc tggaaaacat tgccgtgaag tgcagcgtgg aaacaaagcc
     2941 cgacaatgcc acagtggcct ggcacgatgc tcctatgagc ggagtgcaga atctggccat
     3001 cggagtgttt gccacctgtg ccagccggat cgacaacatc tacgacctga acggcggcaa
     3061 gctgtccaag ctgattcaag agagcgccac cacaccaaac gtgacagccc tgtcttggct
     3121 gttcggcaaa ggcctggaat acttccggac caccgacatc gacaccatca tgcaggactt
     3181 caacatcccc gccagcgcca aagagtccat caagcctctg gtggaaagcg cccaggctat
     3241 ccctaccatg acagtgctgg gcaagaagaa ctacgcccct ttcagaccca actttggcgg
     3301 caagatcgac agctggatcg ccaactacgc cagcagactg atgctgctga acgacatcct
     3361 ggaacagatc gagcccggct tcgaactgcc tcaggctctg ctggacaacg agacactgat
     3421 gagcggcatc gacatgaccg gcgacgagct gaaagaactg atcgaggccg tgtacgcctg
     3481 ggttgacgcc gctaaacaag gactggccac actgcttggc agaggcggca atgtggatga
     3541 tgccgtgcag accttcgagc agttcagcgc catgatggac accctgaacg gaaccctgaa
     3601 caccatcagc gccagatatg tgcgcgccgt ggaaatggcc ggaaaggatg aagcccggct
     3661 ggaaaagctg atcgagtgca agttcgacat ccccaagtgg tgcaagagcg tgccaaagct
     3721 cgtgggcatt tctggcggcc tgcctaaggt ggaagaggaa atcaaagtga tgaacgccgc
     3781 cttcatggac gtgcgggcca gaatgttcgt cagattcgaa gagatcgccg cctacgtggc
     3841 ctctaaaggc gccggaatgg atgtgtacga cgccctggaa aagagagagc tggaacagct
     3901 caagaaactc aagagcgccg tgcctgagag agcccacatc caggcttata gagccgtgct
     3961 gcacagaatc ggacgggctg tgcagaactg cagcgaaaag accaagcagc tgttctccag
     4021 caaagtgatt gagatgggcg tgttcaagaa ccccagccac ctgaacaact tcatcttcaa
     4081 ccagaagggc gccatctaca gaagcccctt cgacagatct cggcacgccc catatcagct
     4141 gcacgccgat aagctgctga agaacgactg gatggaactg ctggccgaaa tcagcaccac
     4201 actgatggcc agcgagtcca ccgagcagat ggaagatgcc ctgagactgg aacggaccag
     4261 actgcaactg cagctgtctg gcctgcctga ctgggagtat cctgcctctc tggccaagcc
     4321 tgacatcgag gtggaaatcc agaccgctct gaagatgcag ctggccaaag acaccgtgac
     4381 cagcgacgtc ctgcagcgga ccttcaatct gtacagctct gtgctgagcg gcctgacctt
     4441 caagctgctg cggagaagct tcagcctgaa aatgcggttc agcgtggccg acaccacaca
     4501 gctgatctac gtgcccaagg actgcgactg ggctatcccc aagcagtatc tgcaggccga
     4561 gggcgagatt gggatcgccg ctagagtggt cacagagtct agccccgcca agatggtcac
     4621 cgaagtggaa atgaaggaac ccaaggctct gggccacttc atgcagcagg ctccccacga
     4681 ctggtacttc gatgcctctc tcggaggaac tcaggtggcc ggcagaatcg tggaaaaggg
     4741 aaaagaagtc ggcaaagagc ggaagctcgt cggctacaga atgagaggca acagcgccta
     4801 caagaccgtg ctggataaga gcctcgtggg aaacaccgag ctgagccagt gctctatgat
     4861 catcgagatc ccttacacac agaccgtgga cgccgacttc agagcacagg ttcaagccgg
     4921 cctgccaaaa gtgtctatca acctgcctgt gaaagagaca atcaccgcca gcaacaagga
     4981 tgagcagatg ctgttcgatc gctttgtggc catcgacctg ggcgagagag gactgggata
     5041 cgccgtgttc gatgccaaga cactggaact gcaagagtcc ggccaccggc ctatcaaggc
     5101 catcaccaac ctgctgaacc ggacccatca ctacgagcag aggcccaacc agagacagaa
     5161 gttccaggcc aagttcaacg tgaacctgag cgagctgcgc gagaatactg tgggcgacgt
     5221 gtgccaccag atcaacagaa tctgcgccta ctacaacgct ttccccgtgc tcgagtacat
     5281 ggtgcccgac agactggaca aacagctgaa gtctgtgtac gagagcgtga ccaaccggta
     5341 catctggtcc agcacagacg cccacaaaag cgctcgcgtg cagttttggc tcggcggaga
     5401 aacatgggag cacccctacc tgaaaagcgc caaggacaag aaacccctgg tgctgtctcc
     5461 tggaagaggc gcttctggca agggaaccag ccagacctgt agctgctgtg gcagaaaccc
     5521 tttcgacctg atcaaggaca tgaagcccag ggccaagatc gccgtggtgg atggaaaagc
     5581 caagctggaa aattccgagc tgaagctgtt tgagcggaac cgcgagagca aggacgacat
     5641 gctggctcgg agacacagaa acgagcgcgc tggaatggaa cagcctctga cacccggcaa
     5701 ctacaccgtg gatgagatca aggccctgct gagagccaac ctgagaaggg cccctaagaa
     5761 cagacggacc aaggacacca ccgtgtccga gtaccactgc gtgttcagcg attgcggcaa
     5821 gaccatgcat gccgacgaaa acgccgccgt gaacatcggc ggaaagttta tcgccgatat
     5881 cgagaagtga tgataacatt ggaagtggat aacggatccg cgatcgcggc gcgccacctg
     5941 gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc taacaaagcc cgaaaggaag
     6001 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     6061 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatcca caggacgggt
     6121 gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta gcgaagcgag caggactggg
     6181 cggcggccaa agcggtcgga cagtgctccg agaacgggtg cgcatagaaa ttgcatcaac
     6241 gcatatagcg ctagcagcac gccatagtga ctggcgatgc tgtcggaatg gacgatatcc
     6301 cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg
     6361 gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag
     6421 caatttaact gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga
     6481 gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     6541 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6601 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6661 aatgcttcaa taacattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6721 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6781 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6841 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6901 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     6961 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     7021 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7081 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7141 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7201 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     7261 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7321 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7381 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7441 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7501 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7561 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7621 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7681 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7741 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7801 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7861 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcaac
     7921 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7981 gtgtattacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     8041 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     8101 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     8161 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     8221 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     8281 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     8341 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     8401 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     8461 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     8521 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     8581 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     8641 gacacccgcc aacaccagct gacgagccct gacgggcttg tctgctcccg gcatccgctt
     8701 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     8761 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     8821 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     8881 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     8941 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     9001 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     9061 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     9121 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     9181 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     9241 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     9301 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     9361 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgccagaga
     9421 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     9481 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     9541 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     9601 acgcaacgcg gggaggcaga caaggt
//
