LOCUS       sequence-358155-        8536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Wild-type Cas12c_4 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: pCJH019_SC101_pLAC_Cas12c(4)_Kan
FEATURES             Location/Qualifiers
     source          1..8536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(718..1668)
                     /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      1716..1938
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(2482..3297)
                     /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(2482..3297)
                     /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     3205..3224
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3289..3308
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3482..4564)
                     /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     4526..4545
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4565..4642)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4848..4867
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    4935..4951
                     /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    4958..4974
                     /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 aagaacagac ggaccaagga caccaccgtg tccgagtacc actgcgtgtt cagcgattgc
       61 ggcaagacca tgcatgccga cgaaaacgcc gccgtgaaca tcggcggaaa gtttatcgcc
      121 gatatcgaga agtgagctgc aaggaggact ttggcaggtg ctctcctgag taggacaaat
      181 ccgccgccct agacctaggg tacgggtttt gctgcccgca aacgggctgt tctggtgttg
      241 ctagtttgtt atcagaatcg cagatccggc ttcagccggt ttgccggctg aaagcactat
      301 ttcttccaga attgccatga ttttttcccc acgggaggcg tcactggctc ccgtgttgtc
      361 ggcagctttg attcgataag cagcatcgcc tgtttcaggc tgtctatgtg tgactgttga
      421 gctgtaacaa gttgtctcag gtgttcaatt tcatgttcta gttgctttgt tttactggtt
      481 tcacctgttc tattaggtgt tacatgctgt tcatctgtta cattgtcgat ctgttcatgg
      541 tgaacagctt tgaatgcacc aaaaactcgt aaaagctctg atgtatctat cttttttaca
      601 ccgttttcat ctgtgcatat ggacagtttt ccctttgata tgtaacggtg aacagttgtt
      661 ctacttttgt ttgttagtct tgatgcttca ctgatagata caagagccat aagaacctca
      721 gatccttccg tatttagcca gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca
      781 tgagaacgaa ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa
      841 actggtgagc tgaatttttg cagttaaagc atcgtgtagt gtttttctta gtccgttatg
      901 taggtaggaa tctgatgtaa tggttgttgg tattttgtca ccattcattt ttatctggtt
      961 gttctcaagt tcggttacga gatccatttg tctatctagt tcaacttgga aaatcaacgt
     1021 atcagtcggg cggcctcgct tatcaaccac caatttcata ttgctgtaag tgtttaaatc
     1081 tttacttatt ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc
     1141 aagcattaac atgaacttaa attcatcaag gctaatctct atatttgcct tgtgagtttt
     1201 cttttgtgtt agttctttta ataaccactc ataaatcctc atagagtatt tgttttcaaa
     1261 agacttaaca tgttccagat tatattttat gaattttttt aactggaaaa gataaggcaa
     1321 tatctcttca ctaaaaacta attctaattt ttcgcttgag aacttggcat agtttgtcca
     1381 ctggaaaatc tcaaagcctt taaccaaagg attcctgatt tccacagttc tcgtcatcag
     1441 ctctctggtt gctttagcta atacaccata agcattttcc ctactgatgt tcatcatctg
     1501 agcgtattgg ttataagtga acgataccgt ccgttctttc cttgtagggt tttcaatcgt
     1561 ggggttgagt agtgccacac agcataaaat tagcttggtt tcatgctccg ttaagtcata
     1621 gcgactaatc gctagttcat ttgctttgaa aacaactaat tcagacatac atctcaattg
     1681 gtctaggtga ttttaatcac tataccaatt gagatgggct agtcaatgat aattactagt
     1741 ccttttcctt tgagttgtgg gtatctgtaa attctgctag acctttgctg gaaaacttgt
     1801 aaattctgct ataccctctg taaattccgc tagacctttg tgtgtttttt ttgtttatat
     1861 tcaagtggtt ataatttata gaataaagaa agaataaaaa aagataaaaa gaatagatcc
     1921 cagccctgtg tataactcac tactttagtc agttccgcag tattacaaaa ggatgtcgca
     1981 aacgctgttt gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccct
     2041 cgcaagctcg ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg cacctgagtc
     2101 gctgtctttt tcgtgacatt cagttcgctg cgctcacggc tctggcagtg aatgggggta
     2161 aatggcacta caggcgcctt ttatggattc atgcaaggaa actacccata atacaagaaa
     2221 agcccgtcac gggcttctca gggcgtttta tggcgggtct gctatgtggt gctatctgac
     2281 tttttgctgt tcagcagttc ctgccctctg attttccagt ctgaccactt cggattatcc
     2341 cgtgacaggt cattcagact ggctaatgca cccagtaagg cagcggtatc atcaacaggc
     2401 ttacccgtct tactgtccct agtgtcagat ttcttacttt attcaggcga ttacgtgtta
     2461 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2521 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2581 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2641 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2701 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
     2761 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2821 accgttattc attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg
     2881 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2941 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     3001 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     3061 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     3121 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     3181 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3241 catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac
     3301 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     3361 atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtc gaataaatcg
     3421 aacttttgct gagttgaagg atcagcacct gcccgaacca gagacttatc tcaggttgcg
     3481 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3541 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     3601 atacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     3661 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     3721 aacatgagct gtcctcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     3781 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     3841 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     3901 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     3961 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4021 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcctcatggg
     4081 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4141 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4201 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4261 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     4321 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     4381 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     4441 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     4501 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     4561 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     4621 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     4681 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     4741 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     4801 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     4861 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     4921 tagaggatcg agaattgtga gcggataaca attgacattg tgagcggata acaagatact
     4981 gagcaccgtg ttaactttaa gaaggagata taccatgacc aagcacagca tccctctgca
     5041 cgccttcaga aactctggcg ccgatgccag aaagtggaag ggcagaattg ccctgctggc
     5101 caagaggggc aaagaaacca tgcggaccct gcagttccct ctggaaatga gcgaacctga
     5161 ggccgctgcc atcaacacca caccttttgc cgtggcctac aacgccatcg aaggcacagg
     5221 caagggcacc ctgtttgact actgggccaa actgcacctg gccggcttca gattctttcc
     5281 atctggcgga gccgccacca tctttaggca gcaggccgtg tttgaggacg cctcttggaa
     5341 tgccgccttc tgtcagcaga gcggcaagga ttggccttgg ctggtgccta gcaagctgta
     5401 cgagcggttc acaaaggccc ctagagaggt ggccaagaag gacggcagca agaagtccat
     5461 cgagttcacc caagagaacg tggccaacga gagccacgtg tcacttgtgg gcgccagcat
     5521 caccgataag acccctgagg accagaaaga atttttcctg aagatggcag gcgccctggc
     5581 cgagaagttc gattcttgga agtccgccaa cgaggaccgg atcgtggcca tgaaagtgat
     5641 cgacgagttc ctgaagtccg agggcctgca tctgcccagc ctggaaaaca ttgccgtgaa
     5701 gtgcagcgtg gaaacaaagc ccgacaatgc cacagtggcc tggcacgatg ctcctatgag
     5761 cggagtgcag aatctggcca tcggagtgtt tgccacctgt gccagccgga tcgacaacat
     5821 ctacgacctg aacggcggca agctgtccaa gctgattcaa gagagcgcca ccacaccaaa
     5881 cgtgacagcc ctgtcttggc tgttcggcaa aggcctggaa tacttccgga ccaccgacat
     5941 cgacaccatc atgcaggact tcaacatccc cgccagcgcc aaagagtcca tcaagcctct
     6001 ggtggaaagc gcccaggcta tccctaccat gacagtgctg ggcaagaaga actacgcccc
     6061 tttcagaccc aactttggcg gcaagatcga cagctggatc gccaactacg ccagcagact
     6121 gatgctgctg aacgacatcc tggaacagat cgagcccggc ttcgaactgc ctcaggctct
     6181 gctggacaac gagacactga tgagcggcat cgacatgacc ggcgacgagc tgaaagaact
     6241 gatcgaggcc gtgtacgcct gggttgacgc cgctaaacaa ggactggcca cactgcttgg
     6301 cagaggcggc aatgtggatg atgccgtgca gaccttcgag cagttcagcg ccatgatgga
     6361 caccctgaac ggaaccctga acaccatcag cgccagatat gtgcgcgccg tggaaatggc
     6421 cggaaaggat gaagcccggc tggaaaagct gatcgagtgc aagttcgaca tccccaagtg
     6481 gtgcaagagc gtgccaaagc tcgtgggcat ttctggcggc ctgcctaagg tggaagagga
     6541 aatcaaagtg atgaacgccg ccttcatgga cgtgcgggcc agaatgttcg tcagattcga
     6601 agagatcgcc gcctacgtgg cctctaaagg cgccggaatg gatgtgtacg acgccctgga
     6661 aaagagagag ctggaacagc tcaagaaact caagagcgcc gtgcctgaga gagcccacat
     6721 ccaggcttat agagccgtgc tgcacagaat cggacgggct gtgcagaact gcagcgaaaa
     6781 gaccaagcag ctgttctcca gcaaagtgat tgagatgggc gtgttcaaga accccagcca
     6841 cctgaacaac ttcatcttca accagaaggg cgccatctac agaagcccct tcgacagatc
     6901 tcggcacgcc ccatatcagc tgcacgccga taagctgctg aagaacgact ggatggaact
     6961 gctggccgaa atcagcacca cactgatggc cagcgagtcc accgagcaga tggaagatgc
     7021 cctgagactg gaacggacca gactgcaact gcagctgtct ggcctgcctg actgggagta
     7081 tcctgcctct ctggccaagc ctgacatcga ggtggaaatc cagaccgctc tgaagatgca
     7141 gctggccaaa gacaccgtga ccagcgacgt cctgcagcgg accttcaatc tgtacagctc
     7201 tgtgctgagc ggcctgacct tcaagctgct gcggagaagc ttcagcctga aaatgcggtt
     7261 cagcgtggcc gacaccacac agctgatcta cgtgcccaag gactgcgact gggctatccc
     7321 caagcagtat ctgcaggccg agggcgagat tgggatcgcc gctagagtgg tcacagagtc
     7381 tagccccgcc aagatggtca ccgaagtgga aatgaaggaa cccaaggctc tgggccactt
     7441 catgcagcag gctccccacg actggtactt cgatgcctct ctcggaggaa ctcaggtggc
     7501 cggcagaatc gtggaaaagg gaaaagaagt cggcaaagag cggaagctcg tcggctacag
     7561 aatgagaggc aacagcgcct acaagaccgt gctggataag agcctcgtgg gaaacaccga
     7621 gctgagccag tgctctatga tcatcgagat cccttacaca cagaccgtgg acgccgactt
     7681 cagagcacag gttcaagccg gcctgccaaa agtgtctatc aacctgcctg tgaaagagac
     7741 aatcaccgcc agcaacaagg atgagcagat gctgttcgat cgctttgtgg ccatcgacct
     7801 gggcgagaga ggactgggat acgccgtgtt cgatgccaag acactggaac tgcaagagtc
     7861 cggccaccgg cctatcaagg ccatcaccaa cctgctgaac cggacccatc actacgagca
     7921 gaggcccaac cagagacaga agttccaggc caagttcaac gtgaacctga gcgagctgcg
     7981 cgagaatact gtgggcgacg tgtgccacca gatcaacaga atctgcgcct actacaacgc
     8041 tttccccgtg ctcgagtaca tggtgcccga cagactggac aaacagctga agtctgtgta
     8101 cgagagcgtg accaaccggt acatctggtc cagcacagac gcccacaaaa gcgctcgcgt
     8161 gcagttttgg ctcggcggag aaacatggga gcacccctac ctgaaaagcg ccaaggacaa
     8221 gaaacccctg gtgctgtctc ctggaagagg cgcttctggc aagggaacca gccagacctg
     8281 tagctgctgt ggcagaaacc ctttcgacct gatcaaggac atgaagccca gggccaagat
     8341 cgccgtggtg gatggaaaag ccaagctgga aaattccgag ctgaagctgt ttgagcggaa
     8401 ccgcgagagc aaggacgaca tgctggctcg gagacacaga aacgagcgcg ctggaatgga
     8461 acagcctctg acacccggca actacaccgt ggatgagatc aaggccctgc tgagagccaa
     8521 cctgagaagg gcccct
//
