https://www.alphaknockout.com Mouse Tmem158 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Tmem158 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmem158 gene (NCBI Reference Sequence: NM_001002267 ; Ensembl: ENSMUSG00000054871 ) is located on Mouse chromosome 9. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000068140). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem158 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-271D21 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele are viable, fertile and developmentally normal, and exhibit a normal lifespan and no predisposition to spontaneous or chemically-induced tumors. Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~2003 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele A T 5' G gRNA region 3' 1 Targeting vector A T G Targeted allele A T G Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Tmem158 cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(6858bp) | A(24.64% 1690) | C(25.34% 1738) | T(25.37% 1740) | G(24.64% 1690) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 123260546 123263545 3000 browser details YourSeq 76 1433 1953 3000 84.6% chr1 + 119037138 119374415 337278 browser details YourSeq 71 1433 1541 3000 81.4% chr16 + 17183529 17183636 108 browser details YourSeq 69 1342 1935 3000 91.4% chr1 - 82818125 83209021 390897 browser details YourSeq 65 1434 1520 3000 91.4% chr2 - 130651862 130652116 255 browser details YourSeq 63 1433 1543 3000 86.3% chr2 - 130749185 130793832 44648 browser details YourSeq 63 1451 1550 3000 87.3% chr12 - 71031415 71031889 475 browser details YourSeq 58 1462 1559 3000 74.7% chr2 - 32143188 32143266 79 browser details YourSeq 57 1908 2376 3000 76.2% chr7 - 24504937 24505371 435 browser details YourSeq 56 1422 1522 3000 84.8% chr10 - 120984681 120984779 99 browser details YourSeq 56 1422 1519 3000 80.5% chr8 + 70126517 70126612 96 browser details YourSeq 56 1433 1520 3000 81.9% chr10 + 42237916 42238003 88 browser details YourSeq 54 1433 1516 3000 80.8% chr3 - 134199075 134199156 82 browser details YourSeq 54 1457 1528 3000 92.4% chr10 - 127492800 127492886 87 browser details YourSeq 54 1880 2007 3000 90.8% chr4 + 135333248 135333446 199 browser details YourSeq 53 1455 1579 3000 86.2% chr4 + 116015860 116015986 127 browser details YourSeq 52 1460 1529 3000 87.2% chr1 - 179892318 179892387 70 browser details YourSeq 52 1468 1551 3000 79.5% chr15 + 38411170 38411251 82 browser details YourSeq 51 1433 1520 3000 79.4% chr11 - 5565149 5565387 239 browser details YourSeq 50 1455 1522 3000 88.4% chr5 - 146828375 146828441 67 Note: The 3000 bp section upstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr9 - 123256688 123259687 3000 browser details YourSeq 86 2698 2867 3000 84.4% chr19 - 4418173 4418359 187 browser details YourSeq 82 2698 2873 3000 86.0% chr2 - 133023744 133023919 176 browser details YourSeq 74 2736 2891 3000 84.3% chr2 - 6013891 6014051 161 browser details YourSeq 70 2695 2811 3000 80.4% chr12 + 83442740 83581866 139127 browser details YourSeq 69 2701 2807 3000 87.3% chr3 + 9229182 9229295 114 browser details YourSeq 68 2698 2867 3000 76.2% chr15 - 76804401 76804583 183 browser details YourSeq 68 2716 2892 3000 84.8% chr12 - 98780820 98780992 173 browser details YourSeq 63 2693 2873 3000 89.8% chr11 - 119020561 119020749 189 browser details YourSeq 61 2579 2813 3000 65.7% chr4 + 126783551 126783654 104 browser details YourSeq 61 2694 2813 3000 89.7% chr15 + 83019956 83020076 121 browser details YourSeq 59 2696 2810 3000 87.5% chr12 - 111315241 111315353 113 browser details YourSeq 59 2707 2870 3000 88.4% chr4 + 128569706 128569961 256 browser details YourSeq 58 2703 2811 3000 79.0% chr14 - 118329974 118330126 153 browser details YourSeq 58 1014 1203 3000 92.8% chr1 - 154850072 154850274 203 browser details YourSeq 57 2735 2865 3000 81.4% chr1 - 160315605 160316004 400 browser details YourSeq 57 2564 2767 3000 92.6% chr15 + 63844594 64037528 192935 browser details YourSeq 57 2565 2771 3000 69.6% chr10 + 52330371 52330446 76 browser details YourSeq 56 2566 2765 3000 67.7% chr9 - 66190728 66190795 68 browser details YourSeq 56 2698 2869 3000 91.2% chr1 - 191636241 191636427 187 Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Tmem158 transmembrane protein 158 [ Mus musculus (house mouse) ] Gene ID: 72309, updated on 12-Aug-2019 Gene summary Official Symbol Tmem158 provided by MGI Official Full Name transmembrane protein 158 provided by MGI Primary source MGI:MGI:1919559 See related Ensembl:ENSMUSG00000054871 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ris1; 2310037P21Rik Orthologs human all Genomic context Location: 9; 9 F4 See Tmem158 in Genome Data Viewer Exon count: 1 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (123259057..123260789, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (123168175..123169907, complement) Chromosome 9 - NC_000075.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Tmem158 ENSMUSG00000054871 Description transmembrane protein 158 [Source:MGI Symbol;Acc:MGI:1919559] Gene Synonyms 2310037P21Rik, Ris1 Location Chromosome 9: 123,259,053-123,260,764 reverse strand. GRCm38:CM001002.2 About this gene This gene has 1 transcript (splice variant), 113 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmem158-201 ENSMUST00000068140.5 1712 286aa ENSMUSP00000069161.4 Protein coding CCDS23658 B2RTM1 TSL:NA GENCODE basic APPRIS P1 21.71 kb Forward strand 123.25Mb 123.26Mb 123.27Mb Contigs AC134248.3 > < AC164123.4 Genes (Comprehensive set... < Tmem158-201protein coding Regulatory Build 123.25Mb 123.26Mb 123.27Mb Reverse strand 21.71 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000068140 < Tmem158-201protein coding Reverse strand 1.71 kb ENSMUSP00000069... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... PANTHER Transmembrane protein 158 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 286 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.