LOCUS       sequence-381461-        5858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes MmCas12m Delta-ZF (H549A, C552A) under a constitutive 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  AUTHORS   Wu WY, Mohanraju P, Liao C, Adiego-Perez B, Creutzburg SCA, Makarova
            KS, Keessen K, Lindeboom TA, Khan TS, Prinsen S, Joosten R, Yan WX, 
            Migur A, Laffeber C, Scott DA, Lebbink JHG, Koonin EV, Beisel CL, 
            van der Oost J
  TITLE     The miniature CRISPR-Cas12m effector binds DNA to block 
            transcription.
  JOURNAL   Mol Cell. 2022 Dec 1;82(23):4487-4502.e7. doi: 
            10.1016/j.molcel.2022.11.003. Epub 2022 Nov 24.
  PUBMED    36427491
REFERENCE   2  (bases 1 to 5858)
  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.2022.11.003"; journalName: "Mol Cell"; date: 
            "2022-12-1- 1"; volume: "82"; issue: "23"; pages: "4487-4502.e7"
COMMENT     Sequence Label: pCas-Cas12m-Delta-ZF
FEATURES             Location/Qualifiers
     source          1..5858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     386..405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        500..534
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     primer_bind     complement(2381..2398)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2551..2637
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2729..2756
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2775..2866
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3324..3779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3411..3430)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3621..3642
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4335..4994)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4909..4928
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4995..5097)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5489..5506)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5623..5858,1..310))
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(5703..5722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
       61 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
      121 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
      181 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
      241 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
      301 acgatctcaa gaagatcatc ttattaatca gataaaatat ttctaggctc atgagcccga
      361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      421 ctgtggcgct ggtgatgccg gccacgatgc gtccggcgta gaggatctgc tcatgtttga
      481 cagcttatca tcgatgcatc tgacagctag ctcagtccta ggtataatgc tagcgagctc
      541 aggaggacat catgacaaca atgacagtac atacaatggg agtacattat aagtggcaga
      601 tccccgaagt ccttcggcag caactgtggt tagcacataa cctgcgggaa gatctggtat
      661 cgcttcagtt agcatatgat gatgatttaa aggcaatttg gtcatcgtat cccgacgtag
      721 cacaggcaga agatacaatg gcagcagccg aagcagatgc agttgcactg tcggaacggg
      781 ttaaacaggc acggattgaa gcacggtcga aaaagatttc gacagaactg acacaacagt
      841 tacgggatgc aaaaaaacgg ttaaaagatg cccggcaagc acggcgggat gcaattgcag
      901 ttgtaaagga tgacgcagcc gaacggcgga aggcacggtc ggatcaatta gcagcagatc
      961 agaaggcact gtatgggcaa tattgccgag atggagatct gtattgggca tcgtttaata
     1021 cagtactgga tcaccataaa acagccgtta agcgaatcgc agcacaacgg gcctcaggga
     1081 aaccggcaac tcttcgacac caccggtttg acggatcggg aacaattgca gtacagctgc
     1141 agcggcaggc aggggcaccg ccgcggacac ccatggtctt agcagatgaa gcaggaaaat
     1201 atcggaatgt acttcatatc cccgggtgga ctgatcccga cgtatgggaa cagatgacac
     1261 ggtcacaatg ccggcaatcg gggcgggtta cagtacggat gcggtgcgga tcgacagatg
     1321 gacagcccca gtggattgat ctaccggtac aagtacatcg ctggttaccg gcagatgcag
     1381 atattacagg agcagaatta gttgtcacac gggttgcagg aatttatcgg gcaaaactgt
     1441 gtgttacagc acggattggg gatactgaac ccgttacatc ggggccgaca gtagcattac
     1501 atctgggatg gcgctcgaca gaagaaggaa cagcagtagc aacttggcgg tcagatgccc
     1561 ccctggatat tccctttgga ctacggacag taatgcgggt tgacgccgca gggacatcgg
     1621 ggattattgt agtacccgca acaattgaac ggcggctgac tcggactgaa aatattgcat
     1681 cgtcgcggtc gctggcacta gacgcactac gggataaggt tgtcgggtgg ctatcagata
     1741 acgatgcccc cacataccgg gatgccccgc tggaagcagc cactgttaag cagtggaagt
     1801 caccccagcg ctttgcctcg ctagcacatg catggaagga taatggaaca gaaatttcgg
     1861 atattttatg ggcatggttt tcgttagatc gcaaacaatg ggcacaacaa gaaaatgggc
     1921 ggcggaaagc cttagggcat cgggatgatt tatatcggca gattgcagca gtaatttcgg
     1981 atcaggcagg acatgtttta gttgatgata catcggtagc agaattatcg gcacgggcaa
     2041 tggaacggac tgaattaccc acagaagtac aacagaaaat tgatcggcgg cgggaccatg
     2101 cagcacccgg ggggctacgg gcatcggttg tagcagcaat gacacgcgat ggagtccccg
     2161 tcacaattgt tgccgccgca gactttactc ggactgcttc gcgcgccgga catgttaatc
     2221 ccgccgatga tcggtatctg tcgaatcccg tacggtgcga cggatgcggg gcaatgtatg
     2281 atcaagatcg gtcgttcgtt actctgatgc tgcgcgcagc aactgccccc tcgaacccct
     2341 agaagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     2401 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     2461 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     2521 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     2581 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     2641 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     2701 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     2761 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2821 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2881 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2941 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcagcaa actattaact
     3001 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3061 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3121 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3181 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3241 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3301 tcatatatac tttagattga tttacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3361 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3421 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3481 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     3541 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3601 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3661 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3721 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaattta
     3781 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     3841 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3901 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3961 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4021 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4081 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtcaggcat ttgagaagca
     4141 cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc
     4201 tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca
     4261 ttcatccgct tattatcact tattcaggcg tagcaaccag gcgtttaagg gcaccaataa
     4321 ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta
     4381 agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc
     4441 atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag
     4501 ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag
     4561 acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac
     4621 gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag
     4681 agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc
     4741 catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg
     4801 cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt
     4861 aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga
     4921 aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg
     4981 atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg
     5041 cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg
     5101 tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac accaggattt
     5161 atttattctg cgaagtgatc ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt
     5221 gatgctgcca acttactgat ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc
     5281 tgtttctatc agctgtccct cctgttcagc tactgacggg gtggtgcgta acggcaaaag
     5341 caccgccgga catcagcgct agcggagtgt atactggctt actatgttgg cactgatgag
     5401 ggtgtcagtg aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga
     5461 atatgtgata caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc
     5521 gactgcggcg agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga
     5581 agatacttaa cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc
     5641 ccctgacaag catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact
     5701 ataaagatac caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt
     5761 tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac
     5821 tcagttccgg gtaggcagtt cgctccaagc tggactgt
//
