https://www.alphaknockout.com Mouse Birc7 Knockout Project (CRISPR/Cas9) Objective: To create a Birc7 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Birc7 gene (NCBI Reference Sequence: NM_001163247 ; Ensembl: ENSMUSG00000038840 ) is located on Mouse chromosome 2. 7 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 6 (Transcript: ENSMUST00000108875). Exon 1~6 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: Mice homozygous for a knock-out allele exhibit normal retinal morphology and function. Exon 1 starts from about 0.12% of the coding region. Exon 1~6 covers 100.0% of the coding region. The size of effective KO region: ~4181 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 Legends Exon of mouse Birc7 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon 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 2000 bp section downstream of stop codon 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(24.2% 484) | C(25.15% 503) | T(25.9% 518) | G(24.75% 495) Note: The 2000 bp section upstream of start codon 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(24.25% 485) | C(24.75% 495) | T(26.4% 528) | G(24.6% 492) Note: The 2000 bp section downstream of stop codon 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 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr2 + 180927148 180929147 2000 browser details YourSeq 82 700 978 2000 73.7% chr3 + 103456375 103456560 186 browser details YourSeq 76 688 823 2000 81.0% chr17 + 7684700 7684848 149 browser details YourSeq 67 653 784 2000 87.3% chr11 + 9151996 9152126 131 browser details YourSeq 64 689 827 2000 73.9% chr10 - 69157355 69157659 305 browser details YourSeq 62 730 997 2000 69.0% chr4 - 71823525 71823747 223 browser details YourSeq 49 707 811 2000 73.4% chr1 - 120223685 120223789 105 browser details YourSeq 48 689 982 2000 89.9% chr2 + 57434197 57434531 335 browser details YourSeq 46 700 761 2000 87.1% chr10 + 21633049 21633110 62 browser details YourSeq 44 428 545 2000 70.4% chr17 + 46839760 46839833 74 browser details YourSeq 43 707 982 2000 63.9% chr12 - 30490981 30491116 136 browser details YourSeq 41 707 811 2000 69.6% chr1 - 126144385 126144489 105 browser details YourSeq 41 682 759 2000 77.0% chr13 + 70052428 70052512 85 browser details YourSeq 36 458 545 2000 89.2% chr11 - 69440267 69440356 90 browser details YourSeq 36 681 721 2000 95.2% chr11 - 40311136 40311183 48 browser details YourSeq 36 693 738 2000 89.2% chr10 + 107129090 107129135 46 browser details YourSeq 35 897 945 2000 87.2% chr6 - 38086522 38086568 47 browser details YourSeq 35 524 580 2000 80.8% chr12 - 16793640 16793696 57 browser details YourSeq 34 525 580 2000 80.4% chr4 - 108175396 108175451 56 browser details YourSeq 34 949 985 2000 97.3% chr10 - 33985916 33985961 46 Note: The 2000 bp section upstream of start codon 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% chr2 + 180933329 180935328 2000 browser details YourSeq 27 347 375 2000 96.6% chr13 + 57871315 57871343 29 browser details YourSeq 26 822 849 2000 88.9% chr10 - 7781070 7781096 27 browser details YourSeq 25 1073 1100 2000 84.7% chr6 + 115327711 115327736 26 browser details YourSeq 24 1471 1496 2000 88.0% chr6 - 44552988 44553012 25 browser details YourSeq 23 134 156 2000 100.0% chr7 + 94410880 94410902 23 browser details YourSeq 23 510 532 2000 100.0% chr6 + 84230255 84230277 23 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Birc7 baculoviral IAP repeat-containing 7 (livin) [ Mus musculus (house mouse) ] Gene ID: 329581, updated on 10-Oct-2019 Gene summary Official Symbol Birc7 provided by MGI Official Full Name baculoviral IAP repeat-containing 7 (livin) provided by MGI Primary source MGI:MGI:2676458 See related Ensembl:ENSMUSG00000038840 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 KIAP; Livin; ML-IAP; E130019N06 Expression Low expression observed in reference dataset See more Orthologs human all Genomic context Location: 2; 2 H4 See Birc7 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (180928609..180934010) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (180663728..180668715) Chromosome 2 - NC_000068.7 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Birc7 ENSMUSG00000038840 Description baculoviral IAP repeat-containing 7 (livin) [Source:MGI Symbol;Acc:MGI:2676458] Gene Synonyms KIAP, Livin, ML-IAP Location Chromosome 2: 180,929,023-180,934,010 forward strand. GRCm38:CM000995.2 About this gene This gene has 2 transcripts (splice variants), 162 orthologues, 10 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Birc7-201 ENSMUST00000108875.1 1178 285aa ENSMUSP00000104503.1 Protein coding CCDS50840 A2AWP0 TSL:5 GENCODE basic APPRIS P1 Birc7-202 ENSMUST00000137859.1 1529 No protein - Retained intron - - TSL:1 24.99 kb Forward strand 180.92Mb 180.93Mb 180.94Mb Genes (Comprehensive set... Birc7-201 >protein coding Birc7-202 >retained intron Contigs AL954707.18 > Genes < Ythdf1-201protein coding < Nkain4-202protein coding (Comprehensive set... < Ythdf1-202protein coding < Nkain4-201protein coding < Ythdf1-203protein coding < Nkain4-207protein coding < Nkain4-205protein coding < Nkain4-206protein coding < Nkain4-204protein coding < Nkain4-203lncRNA Regulatory Build 180.92Mb 180.93Mb 180.94Mb Reverse strand 24.99 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000108875 4.99 kb Forward strand Birc7-201 >protein coding ENSMUSP00000104... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57924 SMART BIR repeat Zinc finger, RING-type Pfam BIR repeat PF13920 PROSITE profiles BIR repeat Zinc finger, RING-type PROSITE patterns BIR repeat Zinc finger, RING-type, conserved site PANTHER PTHR10044:SF163 PTHR10044 Gene3D 1.10.1170.10 Zinc finger, RING/FYVE/PHD-type CDD BIR repeat cd16713 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 285 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.