https://www.alphaknockout.com Mouse Fam76b Knockout Project (CRISPR/Cas9) Objective: To create a Fam76b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fam76b gene (NCBI Reference Sequence: NM_176836 ; Ensembl: ENSMUSG00000037808 ) is located on Mouse chromosome 9. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000059579). Exon 3~8 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 3 starts from about 15.04% of the coding region. Exon 3~8 covers 66.47% of the coding region. The size of effective KO region: ~7248 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 3 4 5 6 7 8 10 Legends Exon of mouse Fam76b 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 900 bp section upstream 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 8 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(900bp) | A(32.33% 291) | C(14.0% 126) | T(31.78% 286) | G(21.89% 197) Note: The 900 bp section upstream 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.15% 583) | C(17.75% 355) | T(33.0% 660) | G(20.1% 402) Note: The 2000 bp section downstream of Exon 8 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 900 1 900 900 100.0% chr9 + 13828826 13829725 900 browser details YourSeq 38 614 754 900 95.2% chr1 + 78219240 78222261 3022 browser details YourSeq 28 607 635 900 100.0% chr1 + 127383886 127383915 30 browser details YourSeq 27 592 632 900 89.7% chr3 - 104554532 104554571 40 browser details YourSeq 27 594 634 900 83.0% chrX + 93898887 93898927 41 browser details YourSeq 25 611 635 900 100.0% chr4 + 117518181 117518205 25 browser details YourSeq 25 608 633 900 100.0% chr1 + 113422448 113422474 27 browser details YourSeq 24 611 635 900 100.0% chr18 - 66954471 66954496 26 browser details YourSeq 23 612 634 900 100.0% chr5 - 132489220 132489242 23 browser details YourSeq 23 614 636 900 100.0% chr4 - 106279271 106279293 23 browser details YourSeq 23 607 630 900 100.0% chr3 - 36820498 36820522 25 browser details YourSeq 23 614 636 900 100.0% chr9 + 44676534 44676556 23 browser details YourSeq 23 614 637 900 100.0% chr1 + 127401810 127401847 38 browser details YourSeq 23 612 636 900 96.0% chr1 + 41032027 41032051 25 browser details YourSeq 22 613 634 900 100.0% chr5 - 26827831 26827852 22 browser details YourSeq 22 612 633 900 100.0% chr19 - 37283356 37283377 22 browser details YourSeq 22 99 121 900 100.0% chr1 - 144199066 144199093 28 browser details YourSeq 22 614 635 900 100.0% chr1 - 34476588 34476609 22 browser details YourSeq 22 612 633 900 100.0% chr14 + 75472471 75472492 22 browser details YourSeq 22 614 635 900 100.0% chr12 + 51498071 51498092 22 Note: The 900 bp section upstream of Exon 3 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 2000 1 2000 2000 100.0% chr9 + 13836974 13838973 2000 browser details YourSeq 114 1344 1543 2000 90.8% chr13 - 37317616 37317829 214 browser details YourSeq 111 1050 1543 2000 91.8% chr7 - 29416645 29417205 561 browser details YourSeq 102 1345 1493 2000 90.6% chr9 - 60858892 60859066 175 browser details YourSeq 98 1347 1547 2000 92.4% chr19 + 26975745 26975948 204 browser details YourSeq 93 1365 1489 2000 93.0% chrX - 112295199 112295528 330 browser details YourSeq 93 1349 1543 2000 87.9% chr9 - 122296070 122296277 208 browser details YourSeq 93 1356 1493 2000 91.2% chr6 + 57538372 57538532 161 browser details YourSeq 91 1371 1493 2000 85.5% chr5 - 111858721 111858833 113 browser details YourSeq 91 1348 1503 2000 88.3% chr4 - 53626403 53626577 175 browser details YourSeq 90 1345 1543 2000 93.4% chr18 - 24954529 24954729 201 browser details YourSeq 90 1338 1547 2000 91.0% chr9 + 72089314 72089538 225 browser details YourSeq 89 1345 1471 2000 92.5% chr5 + 139123047 139123193 147 browser details YourSeq 89 1345 1493 2000 85.8% chr16 + 36096387 36096553 167 browser details YourSeq 88 1349 1484 2000 87.9% chr13 - 43725202 43725343 142 browser details YourSeq 88 1348 1470 2000 84.9% chr11 - 66954549 66954650 102 browser details YourSeq 88 1345 1543 2000 90.9% chr8 + 66425350 66425556 207 browser details YourSeq 88 1352 1493 2000 88.5% chr5 + 137325863 137326009 147 browser details YourSeq 87 1344 1543 2000 85.5% chr6 - 117425710 117425891 182 browser details YourSeq 87 1349 1543 2000 93.2% chr4 - 14542203 14542412 210 Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Fam76b family with sequence similarity 76, member B [ Mus musculus (house mouse) ] Gene ID: 72826, updated on 14-Aug-2019 Gene summary Official Symbol Fam76b provided by MGI Official Full Name family with sequence similarity 76, member B provided by MGI Primary source MGI:MGI:1920076 See related Ensembl:ENSMUSG00000037808 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 C78303; 2810485I05Rik Expression Ubiquitous expression in CNS E14 (RPKM 10.9), whole brain E14.5 (RPKM 9.9) and 26 other tissues See more Orthologs human all Genomic context Location: 9; 9 A1 See Fam76b in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (13827194..13854687) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (13632171..13650966) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Fam76b ENSMUSG00000037808 Description family with sequence similarity 76, member B [Source:MGI Symbol;Acc:MGI:1920076] Gene Synonyms 2810485I05Rik Location Chromosome 9: 13,827,716-13,854,987 forward strand. GRCm38:CM001002.2 About this gene This gene has 5 transcripts (splice variants), 196 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fam76b- ENSMUST00000059579.11 3702 339aa ENSMUSP00000062642.5 Protein coding CCDS22819 Q80XP8 TSL:1 201 GENCODE basic APPRIS P1 Fam76b- ENSMUST00000213416.1 540 74aa ENSMUSP00000149169.1 Protein coding - A0A1L1SQS5 CDS 5' 205 incomplete TSL:2 Fam76b- ENSMUST00000156680.1 3760 233aa ENSMUSP00000115751.1 Nonsense mediated - D6RG49 TSL:5 204 decay Fam76b- ENSMUST00000129861.1 723 No - Retained intron - - TSL:2 202 protein Fam76b- ENSMUST00000130106.1 697 No - Retained intron - - TSL:3 203 protein Page 7 of 9 https://www.alphaknockout.com 47.27 kb Forward strand 13.82Mb 13.83Mb 13.84Mb 13.85Mb 13.86Mb Genes (Comprehensive set... Fam76b-204 >nonsense mediated decay Fam76b-201 >protein coding Fam76b-202 >retained intron Fam76b-203 >retained intron Fam76b-205 >protein coding Contigs < CT010488.9 Genes < Cep57-206nonsense mediated decay (Comprehensive set... < Cep57-201protein coding < Cep57-209protein coding < Cep57-208protein coding < Cep57-203protein coding < Cep57-205nonsense mediated decay < Cep57-211protein coding < Cep57-212protein coding < Cep57-210retained intron < Cep57-207nonsense mediated decay Regulatory Build 13.82Mb 13.83Mb 13.84Mb 13.85Mb 13.86Mb Reverse strand 47.27 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000059579 18.83 kb Forward strand Fam76b-201 >protein coding ENSMUSP00000062... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam FAM76 protein PANTHER FAM76 protein FAM76B All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 339 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.