LOCUS       sequence-358156-        8536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Wild-type Cas12c2 (Yan et al., 2019) in a plasmid containing a SC101
            origin and lac promoter for in vivo interference assay..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8536)
  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 8536)
  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: pCJH021_SC101_pLAC_Cas12c2_Kan
FEATURES             Location/Qualifiers
     source          1..8536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(661..1611)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      1659..1881
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(2425..3240)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2425..3240)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3148..3167
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3232..3251
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3425..4507)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4469..4488
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4508..4585)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4791..4810
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    4878..4894
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    4901..4917
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
ORIGIN
        1 tgcggcaaga ccatgcatgc ggacgaaaac gcggcggtta acattggtgg caagtttatc
       61 gcggacattg agaaataagc tgcaaggagg actttggcag gtgctctcct gagtaggaca
      121 aatccgccgc cctagaccta gggtacgggt tttgctgccc gcaaacgggc tgttctggtg
      181 ttgctagttt gttatcagaa tcgcagatcc ggcttcagcc ggtttgccgg ctgaaagcac
      241 tatttcttcc agaattgcca tgattttttc cccacgggag gcgtcactgg ctcccgtgtt
      301 gtcggcagct ttgattcgat aagcagcatc gcctgtttca ggctgtctat gtgtgactgt
      361 tgagctgtaa caagttgtct caggtgttca atttcatgtt ctagttgctt tgttttactg
      421 gtttcacctg ttctattagg tgttacatgc tgttcatctg ttacattgtc gatctgttca
      481 tggtgaacag ctttgaatgc accaaaaact cgtaaaagct ctgatgtatc tatctttttt
      541 acaccgtttt catctgtgca tatggacagt tttccctttg atatgtaacg gtgaacagtt
      601 gttctacttt tgtttgttag tcttgatgct tcactgatag atacaagagc cataagaacc
      661 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
      721 ccatgagaac gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc
      781 aaaactggtg agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt
      841 atgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
      901 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
      961 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
     1021 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
     1081 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
     1141 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
     1201 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
     1261 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
     1321 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
     1381 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
     1441 ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
     1501 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
     1561 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
     1621 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     1681 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     1741 tgtaaattct gctataccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     1801 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     1861 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     1921 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     1981 cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga
     2041 gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg
     2101 gtaaatggca ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag
     2161 aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg tctgctatgt ggtgctatct
     2221 gactttttgc tgttcagcag ttcctgccct ctgattttcc agtctgacca cttcggatta
     2281 tcccgtgaca ggtcattcag actggctaat gcacccagta aggcagcggt atcatcaaca
     2341 ggcttacccg tcttactgtc cctagtgtca gatttcttac tttattcagg cgattacgtg
     2401 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     2461 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     2521 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2581 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2641 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     2701 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2761 caaaccgtta ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa
     2821 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2881 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2941 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3001 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     3061 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     3121 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     3181 atccatgttg gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat
     3241 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     3301 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt gtcgaataaa
     3361 tcgaactttt gctgagttga aggatcagca cctgcccgaa ccagagactt atctcaggtt
     3421 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3481 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3541 gtgatacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3601 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     3661 tataacatga gctgtcctcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     3721 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3781 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     3841 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3901 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3961 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcctcat
     4021 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4081 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4141 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4201 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4261 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4321 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4381 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     4441 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4501 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4561 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     4621 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     4681 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     4741 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     4801 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     4861 gcgtagagga tcgagaattg tgagcggata acaattgaca ttgtgagcgg ataacaagat
     4921 actgagcacc gtgttaactt taagaaggag atataccatg accaaacaca gcatcccgct
     4981 gcatgcgttc cgtaacagcg gcgcggatgc gcgtaagtgg aaaggtcgta ttgcgctgct
     5041 ggcgaagcgt ggcaaagaaa ccatgcgtac cctgcagttt ccgctggaga tgagcgaacc
     5101 ggaggcggcg gcgatcaaca ccaccccgtt tgcggtggcg tacaacgcga ttgaaggcac
     5161 cggcaaaggc accctgttcg actattgggc gaagctgcac ctggcgggtt ttcgtttctt
     5221 tccgagcggt ggcgcggcga ccatcttccg tcagcaagcg gtttttgagg atgcgagctg
     5281 gaacgcggcg ttctgccagc aaagcggcaa agactggccg tggctggtgc cgagcaagct
     5341 gtacgaacgt ttcaccaaag cgccgcgtga ggttgcgaag aaagatggta gcaagaaaag
     5401 catcgaattt acccaggaga acgtggcgaa cgagagccat gtgagcctgg ttggcgcgag
     5461 cattaccgac aagaccccgg aagatcaaaa ggagttcttt ctgaaaatgg cgggtgcgct
     5521 ggcggaaaag ttcgatagct ggaaaagcgc gaacgaggac cgtatcgtgg cgatgaaggt
     5581 tattgacgaa tttctgaaaa gcgagggtct gcacctgccg agcctggaaa acatcgcggt
     5641 gaagtgcagc gttgaaacca aaccggataa cgcgaccgtg gcgtggcatg atgcgccgat
     5701 gagcggcgtg cagaacctgg cgatcggtgt tttcgcgacc tgcgcgagcc gtatcgacaa
     5761 catttacgat ctgaacggtg gcaagctgag caaactgatt caagaaagcg cgaccacccc
     5821 gaacgttacc gcgctgagct ggctgttcgg caaaggtctg gagtattttc gtaccaccga
     5881 catcgatacc attatgcagg actttaacat cccggcgagc gcgaaggaaa gcattaaacc
     5941 gctggttgag agcgcgcaag cgatcccgac catgaccgtt ctgggcaaga aaaactacgc
     6001 gccgttccgt ccgaactttg gtggcaagat cgatagctgg attgcgaact atgcgagccg
     6061 tctgatgctg ctgaacgaca tcctggaaca gattgagccg ggtttcgaac tgccgcaagc
     6121 gctgctggat aacgaaaccc tgatgagcgg catcgacatg accggtgatg aactgaaaga
     6181 gctgattgaa gcggtgtacg cgtgggttga tgcggcgaaa caaggcctgg cgaccctgct
     6241 gggtcgtggt ggcaacgtgg acgatgcggt tcagaccttc gaacaattta gcgcgatgat
     6301 ggataccctg aacggcaccc tgaacaccat cagcgcgcgt tatgtgcgtg cggttgagat
     6361 ggcgggtaaa gatgaagcgc gtctggagaa gctgatcgaa tgcaaattcg acattccgaa
     6421 gtggtgcaaa agcgtgccga agctggttgg tatcagcggt ggcctgccga aggtggagga
     6481 agagattaaa gttatgaacg cggcgtttaa agacgtgcgt gcgcgtatgt tcgttcgttt
     6541 tgaagagatt gcggcgtatg tggcgagcaa gggtgcgggt atggacgttt atgatgcgct
     6601 ggaaaaacgt gaactggagc agattaagaa actgaaaagc gcggtgccgg agcgtgcgca
     6661 catccaagcg taccgtgcgg tgctgcatcg tattggtcgt gcggttcaga actgcagcga
     6721 aaagaccaaa caactgttca gcagcaaggt gatcgagatg ggcgttttta aaaacccgag
     6781 ccacctgaac aacttcatct ttaaccagaa aggtgcgatt taccgtagcc cgttcgatcg
     6841 tagccgtcac gcgccgtatc aactgcatgc ggacaagctg ctgaaaaacg attggctgga
     6901 gctgctggcg gaaattagcg cgaccctgat ggcgagcgag agcaccgaac agatggagga
     6961 cgcgctgcgt ctggaacgta cccgtctgca gctgcaactg agcggcctgc cggattggga
     7021 gtatccggcg agcctggcga agccggacat cgaagttgag attcagaccg cgctgaagat
     7081 gcaactggcg aaagacaccg tgaccagcga tgttctgcag cgtgcgttca acctgtatag
     7141 cagcgtgctg agcggtctga cctttaaact gctgcgtcgt agcttcagcc tgaagatgcg
     7201 ttttagcgtt gcggatacca cccaactgat ctacgtgccg aaggtttgcg actgggcgat
     7261 tccgaaacag tatctgcaag cggaaggcga gatcggtatt gcggcgcgtg tggttaccga
     7321 aagcagcccg gcgaaaatgg tgaccgaagt tgagatgaag gagccgaaag cgctgggcca
     7381 cttcatgcag caagcgccgc atgactggta ctttgatgcg agcctgggtg gcacccaggt
     7441 ggcgggtcgt atcgttgaga agggcaaaga agtgggcaag gagcgtaaac tggttggcta
     7501 ccgtatgcgt ggtaacagcg cgtataagac cgtgctggac aaaagcctgg ttggcaacac
     7561 cgaactgagc cagtgcagca tgatcattga gattccgtac acccaaaccg tggacgcgga
     7621 tttccgtgcg caggttcaag cgggtctgcc gaaggtgagc atcaacctgc cggttaaaga
     7681 aaccattacc gcgagcaaca aggacgagca gatgctgttc gatcgttttg ttgcgattga
     7741 cctgggtgaa cgtggcctgg gttacgcggt ttttgatgcg aaaaccctgg aactgcaaga
     7801 gagcggtcac cgtccgatca aggcgattac caacctgctg aaccgtaccc accattatga
     7861 gcagcgtccg aaccagcgtc aaaagttcca agcgaaattt aacgttaacc tgagcgaact
     7921 gcgtgagaac accgtgggtg acgtttgcca tcagatcaac cgtatttgcg cgtactataa
     7981 cgcgttcccg gtgctggaat acatggttcc ggaccgtctg gataagcaac tgaaaagcgt
     8041 gtacgagagc gttaccaacc gttatatctg gagcagcacc gatgcgcaca agagcgcgcg
     8101 tgtgcagttt tggctgggtg gcgaaacctg ggagcatccg tatctgaaga gcgcgaaaga
     8161 caagaaaccg ctggttctga gcccgggtcg tggtgcgagc ggcaaaggca ccagccaaac
     8221 ctgcagctgc tgcggccgta acccgttcga cctgatcaag gatatgaaac cgcgtgcgaa
     8281 gattgcggtg gttgacggca aggcgaaact ggaaaacagc gagctgaaac tgtttgaacg
     8341 taacctggag agcaaggacg atatgctggc gcgtcgtcac cgtaacgaac gtgcgggtat
     8401 ggagcagccg ctgaccccgg gtaactacac cgtggatgaa atcaaagcgc tgctgcgtgc
     8461 gaacctgcgt cgtgcgccga agaaccgtcg taccaaagac accaccgtga gcgagtatca
     8521 ctgcgttttc agcgat
//
