https://www.alphaknockout.com Mouse Trim41 Knockout Project (CRISPR/Cas9) Objective: To create a Trim41 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Trim41 gene (NCBI Reference Sequence: NM_145377 ; Ensembl: ENSMUSG00000040365 ) is located on Mouse chromosome 11. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000047145). Exon 1~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 1 starts from the coding region. Exon 1~3 covers 60.32% of the coding region. The size of effective KO region: ~7606 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 6 Legends Exon of mouse Trim41 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 813 bp section of Exon 1 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 231 bp section of Exon 3 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(813bp) | A(24.11% 196) | C(20.66% 168) | T(17.47% 142) | G(37.76% 307) Note: The 813 bp section of Exon 1 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(231bp) | A(23.38% 54) | C(25.54% 59) | T(15.15% 35) | G(35.93% 83) Note: The 231 bp section of Exon 3 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 813 1 813 813 100.0% chr11 - 48815842 48816654 813 browser details YourSeq 77 150 401 813 74.5% chr9 + 63680540 63680625 86 browser details YourSeq 76 54 163 813 81.0% chr14 + 106106964 106107064 101 browser details YourSeq 66 156 523 813 92.3% chr11 - 94989194 95212233 223040 browser details YourSeq 50 192 400 813 93.0% chr13 - 20580035 20580365 331 browser details YourSeq 48 301 465 813 72.3% chr5 - 27627385 27627499 115 browser details YourSeq 47 206 464 813 65.4% chr13 - 81012860 81012950 91 browser details YourSeq 46 133 490 813 62.8% chr12 + 31155126 31155258 133 browser details YourSeq 45 181 391 813 66.0% chr9 - 29830874 29830968 95 browser details YourSeq 43 291 491 813 62.5% chr11 - 42442394 42442465 72 browser details YourSeq 41 144 462 813 59.6% chr12 - 104780916 104781018 103 browser details YourSeq 37 301 487 813 57.5% chr10 + 24396983 24397032 50 browser details YourSeq 36 375 491 813 61.6% chr11 + 116225919 116225957 39 browser details YourSeq 35 148 312 813 59.0% chr10 + 60131381 60131440 60 browser details YourSeq 33 293 463 813 55.6% chr10 - 22610100 22610160 61 browser details YourSeq 32 447 491 813 91.9% chr1 + 17545720 17545764 45 browser details YourSeq 31 441 487 813 91.7% chr13 - 22064901 22064948 48 browser details YourSeq 31 447 491 813 74.3% chr1 + 94768813 94768848 36 browser details YourSeq 30 453 488 813 96.9% chr11 + 95749563 95749718 156 browser details YourSeq 27 374 400 813 100.0% chr3 + 25838297 25838323 27 Note: The 813 bp section 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 231 1 231 231 100.0% chr11 - 48809051 48809281 231 browser details YourSeq 24 1 25 231 100.0% chr4 + 150797301 150797422 122 browser details YourSeq 21 158 180 231 95.7% chr13 + 30574065 30574087 23 browser details YourSeq 20 45 64 231 100.0% chr7 - 130406947 130406966 20 browser details YourSeq 20 54 73 231 100.0% chr12 - 53762960 53762979 20 Note: The 231 bp section of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Trim41 tripartite motif-containing 41 [ Mus musculus (house mouse) ] Gene ID: 211007, updated on 12-Aug-2019 Gene summary Official Symbol Trim41 provided by MGI Official Full Name tripartite motif-containing 41 provided by MGI Primary source MGI:MGI:2384814 See related Ensembl:ENSMUSG00000040365 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 RINCK; R75223; AW552703; BC020156 Expression Ubiquitous expression in testis adult (RPKM 35.3), adrenal adult (RPKM 16.3) and 28 other tissues See more Orthologs human all Genomic context Location: 11; 11 B1.2 See Trim41 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (48806403..48818199, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (48619906..48630893, complement) Chromosome 11 - NC_000077.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Trim41 ENSMUSG00000040365 Description tripartite motif-containing 41 [Source:MGI Symbol;Acc:MGI:2384814] Gene Synonyms RINCK Location Chromosome 11: 48,806,404-48,817,353 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 157 orthologues, 73 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Trim41-201 ENSMUST00000047145.13 3432 630aa ENSMUSP00000037055.7 Protein coding CCDS36138 Q5NCC3 TSL:1 GENCODE basic APPRIS P1 Trim41-204 ENSMUST00000140800.1 723 161aa ENSMUSP00000121705.1 Protein coding - Q5NCC2 CDS 3' incomplete TSL:3 Trim41-203 ENSMUST00000138019.1 471 157aa ENSMUSP00000118789.1 Protein coding - F6X2H0 CDS 5' and 3' incomplete TSL:3 Trim41-202 ENSMUST00000131888.7 429 126aa ENSMUSP00000119707.1 Protein coding - Q5NCC4 CDS 5' incomplete TSL:3 Page 7 of 9 https://www.alphaknockout.com 30.95 kb Forward strand 48.80Mb 48.81Mb 48.82Mb Genes Rack1-206 >retained intron Rack1-204 >lncRNA Trim7-202 >protein coding (Comprehensive set... Rack1-205 >lncRNA Rack1-201 >protein coding Rack1-203 >retained intron Gm25296-201 >snoRNA Snord96a-201 >snoRNA Snord95-201 >snoRNA Rack1-202 >lncRNA Contigs AL645849.17 > Genes (Comprehensive set... < Trim41-201protein coding < Gm12184-201protein coding < Trim41-202protein coding < Trim41-204protein coding < Trim41-203protein coding Regulatory Build 48.80Mb 48.81Mb 48.82Mb Reverse strand 30.95 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000047145 < Trim41-201protein coding Reverse strand 10.95 kb ENSMUSP00000037... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57850 SSF57845 Concanavalin A-like lectin/glucanase domain superfamily SMART Zinc finger, RING-type B-box-type zinc finger SPRY-associated SPRY domain Prints Butyrophylin-like, SPRY domain Pfam PF15227 B-box-type zinc finger SPRY-associated SPRY domain PROSITE profiles Zinc finger, RING-type B-box-type zinc finger B30.2/SPRY domain PROSITE patterns Zinc finger, RING-type, conserved site PANTHER PTHR24103:SF638 PTHR24103 Gene3D Zinc finger, RING/FYVE/PHD-type 3.30.40.200 2.60.120.920 CDD cd16602 B-box-type zinc finger cd13741 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 630 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.