LOCUS       sequence-381462-        7141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MmCas12m fused to His-tag for protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7141)
  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 7141)
  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-pML-1B_6xHis-Cas12m
FEATURES             Location/Qualifiers
     source          1..7141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2608..2627)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2619..3434
                     /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             2619..3434
                     /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     complement(2692..2711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3527..3982)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3664..3685)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3876..3895
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4019..4066)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4062..4080
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4133..4150)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6017..6037)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(6038..6055)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6074..6096
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6111..6135
                     /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)."
     primer_bind     complement(6135..6154)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6136..6154)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6238..6257)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6463..6540
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6541..7141,1..482)
                     /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     complement(6560..6579)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggtatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     2581 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2641 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2701 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2761 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2821 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2881 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2941 aggtattaga agaatatcct gattcaggtg aaaacattgt tgatgcgctg gcagtgttcc
     3001 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3061 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3121 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3181 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3241 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3301 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3361 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3421 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt
     3541 actagtttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3601 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3661 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3721 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3781 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3841 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3901 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3961 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     4021 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     4081 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt
     4141 ggtggtggtg ctcgagtgcg gccgcaagct tgtcgacggc gctcgaattc ggatccgtta
     4201 tccacttcca atgttattac taggggttcg agggggcagt tgctgcgcgc agcatcagag
     4261 taacgaacga ccgatcttga tcatacattg ccccgcatcc gtcgcaccgt acgggattcg
     4321 acagataccg atcatcggcg ggattaacat gtccgcagcg cgaatgagtc cgagtaaagt
     4381 ctgcggcggc aacaattgtg acggggactc catcgcgtgt cattgctgct acaaccgatg
     4441 cccgtagccc cccgggtgct gcatggtccc gccgccgatc aattttctgt tgtacttctg
     4501 tgggtaattc agtccgttcc attgcccgtg ccgataattc tgctaccgat gtatcatcaa
     4561 ctaaaacatg tcctgcctga tccgaaatta ctgctgcaat ctgccgatat aaatcatccc
     4621 gatgccctaa ggctttccgc cgcccatttt cttgttgtgc ccattgtttg cgatctaacg
     4681 aaaaccatgc ccataaaata tccgaaattt ctgttccatt atccttccat gcatgtgcta
     4741 gcgaggcaaa gcgctggggt gacttccact gcttaacagt ggctgcttcc agcggggcat
     4801 cccggtatgt gggggcatcg ttatctgata gccacccgac aaccttatcc cgtagtgcgt
     4861 ctagtgccag cgaccgcgac gatgcaatat tttcagtccg agtcagccgc cgttcaattg
     4921 ttgcgggtac tacaataatc cccgatgtcc ctgcggcgtc aacccgcatt actgtccgta
     4981 gtccaaaggg aatatccagg ggggcatctg accgccaagt tgctactgct gttccttctt
     5041 ctgtcgagcg ccatcccaga tgtaatgcta ctgtcggccc cgatgtaacg ggttcagtat
     5101 ccccaatccg tgctgtaaca cacagttttg cccgataaat tcctgcaacc cgtgtgacaa
     5161 ctaattctgc tcctgtaata tctgcatctg ccggtaacca gcgatgtact tgtaccggta
     5221 gatcaatcca ctggggctgt ccatctgtcg atccgcaccg catccgtact gtaacccgcc
     5281 ccgattgccg gcattgtgac cgtgtcatct gttcccatac gtcgggatca gtccacccgg
     5341 ggatatgaag tacattccga tattttcctg cttcatctgc taagaccatg ggtgtccgcg
     5401 gcggtgcccc tgcctgccgc tgcagctgta ctgcaattgt tcccgatccg tcaaaccggt
     5461 ggtgtcgaag agttgccggt ttccctgagg cccgttgtgc tgcgattcgc ttaacggctg
     5521 ttttatggtg atccagtact gtattaaacg atgcccaata cagatctcca tctcggcaat
     5581 attgcccata cagtgccttc tgatctgctg ctaattgatc cgaccgtgcc ttccgccgtt
     5641 cggctgcgtc atcctttaca actgcaattg catcccgccg tgcttgccgg gcatctttta
     5701 accgtttttt tgcatcccgt aactgttgtg tcagttctgt cgaaatcttt ttcgaccgtg
     5761 cttcaatccg tgcctgttta acccgttccg acagtgcaac tgcatctgct tcggctgctg
     5821 ccattgtatc ttctgcctgt gctacgtcgg gatacgatga ccaaattgcc tttaaatcat
     5881 catcatatgc taactgaagc gataccagat cttcccgcag gttatgtgct aaccacagtt
     5941 gctgccgaag gacttcgggg atctgccact tataatgtac tcccattgta tgtactgtca
     6001 ttgttgtcat tgcattggat tggaagtaca ggttttcatg gtgatggtga tggtgagaag
     6061 aacccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     6121 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
     6181 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
     6241 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
     6301 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
     6361 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
     6421 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     6481 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     6541 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     6601 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     6661 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     6721 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     6781 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     6841 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     6901 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     6961 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     7021 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     7081 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     7141 t
//
