https://www.alphaknockout.com Mouse Stradb Knockout Project (CRISPR/Cas9) Objective: To create a Stradb knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stradb gene (NCBI Reference Sequence: NM_172656 ; Ensembl: ENSMUSG00000026027 ) is located on Mouse chromosome 1. 12 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000027185). Exon 4~10 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 4 starts from about 7.5% of the coding region. Exon 4~10 covers 77.91% of the coding region. The size of effective KO region: ~7849 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 4 5 6 7 8 9 10 12 Legends Exon of mouse Stradb 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 2000 bp section upstream of Exon 4 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 423 bp section downstream of Exon 10 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(2000bp) | A(27.4% 548) | C(20.4% 408) | T(32.75% 655) | G(19.45% 389) Note: The 2000 bp section upstream of Exon 4 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(423bp) | A(31.21% 132) | C(22.93% 97) | T(28.13% 119) | G(17.73% 75) Note: The 423 bp section downstream of Exon 10 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 2000 1 2000 2000 100.0% chr1 + 58983290 58985289 2000 browser details YourSeq 121 497 865 2000 80.6% chr17 + 32175484 32175711 228 browser details YourSeq 117 673 1133 2000 78.6% chr18 + 4123418 4123691 274 browser details YourSeq 106 666 870 2000 82.4% chr11 + 68736030 68736206 177 browser details YourSeq 104 697 865 2000 80.9% chr13 + 93401175 93401332 158 browser details YourSeq 103 680 867 2000 84.1% chr11 - 98406234 98406377 144 browser details YourSeq 102 689 865 2000 91.2% chr4 - 129389570 129438964 49395 browser details YourSeq 96 668 863 2000 80.0% chr11 + 74555590 74555775 186 browser details YourSeq 92 680 865 2000 81.0% chr8 + 83680469 83680641 173 browser details YourSeq 88 693 860 2000 89.7% chr6 - 58866646 58866812 167 browser details YourSeq 88 672 862 2000 81.5% chr6 + 124707485 124707654 170 browser details YourSeq 87 663 863 2000 76.5% chr11 - 20029168 20029331 164 browser details YourSeq 87 492 861 2000 91.5% chr4 + 134103241 134103620 380 browser details YourSeq 86 674 861 2000 81.0% chr17 - 24137883 24138022 140 browser details YourSeq 85 687 871 2000 78.6% chr12 + 31460423 31460597 175 browser details YourSeq 85 743 866 2000 84.5% chr11 + 105096258 105096370 113 browser details YourSeq 85 664 870 2000 83.1% chr10 + 19282779 19282979 201 browser details YourSeq 83 746 872 2000 86.5% chr14 - 56805313 56805431 119 browser details YourSeq 83 672 870 2000 83.4% chr5 + 145360883 145361053 171 browser details YourSeq 83 685 862 2000 82.6% chr12 + 80646109 80646274 166 Note: The 2000 bp section upstream of Exon 4 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 423 1 423 423 100.0% chr1 + 58993139 58993561 423 browser details YourSeq 41 194 378 423 93.7% chr4 - 111863550 111863921 372 browser details YourSeq 40 337 378 423 100.0% chr12 - 52773756 52773851 96 browser details YourSeq 31 189 248 423 64.8% chr8 - 108899336 108899372 37 browser details YourSeq 29 206 249 423 76.5% chr5 + 24465189 24465228 40 browser details YourSeq 22 307 330 423 95.9% chr6 + 112646770 112646793 24 browser details YourSeq 22 394 418 423 95.9% chr14 + 107073782 107073811 30 browser details YourSeq 21 360 380 423 100.0% chrX - 99009096 99009116 21 browser details YourSeq 21 313 335 423 95.7% chr10 + 94834931 94834953 23 browser details YourSeq 20 335 354 423 100.0% chr7 - 11402342 11402361 20 browser details YourSeq 20 335 354 423 100.0% chr7 - 11518712 11518731 20 browser details YourSeq 20 325 344 423 100.0% chr11 - 92503383 92503402 20 browser details YourSeq 20 310 329 423 100.0% chr15 + 24501559 24501578 20 browser details YourSeq 20 404 423 423 100.0% chr12 + 37639971 37639990 20 Note: The 423 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Stradb STE20-related kinase adaptor beta [ Mus musculus (house mouse) ] Gene ID: 227154, updated on 10-Oct-2019 Gene summary Official Symbol Stradb provided by MGI Official Full Name STE20-related kinase adaptor beta provided by MGI Primary source MGI:MGI:2144047 See related Ensembl:ENSMUSG00000026027 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 Papk; ILPIP; ILPIPA; Syradb; Als2cr2; D1Ucla2; PRO1038; AA792893; B830008M19 Expression Ubiquitous expression in CNS E18 (RPKM 16.2), heart adult (RPKM 13.5) and 28 other tissues See more Orthologs human all Genomic context Location: 1 C1.3; 1 29.2 cM See Stradb in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (58973522..58995122) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (59030415..59051966) Chromosome 1 - NC_000067.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Stradb ENSMUSG00000026027 Description STE20-related kinase adaptor beta [Source:MGI Symbol;Acc:MGI:2144047] Gene Synonyms Als2cr2, D1Ucla2, PRO1038 Location Chromosome 1: 58,973,522-58,995,715 forward strand. GRCm38:CM000994.2 About this gene This gene has 7 transcripts (splice variants), 208 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stradb- ENSMUST00000027185.10 2960 418aa ENSMUSP00000027185.4 Protein coding CCDS14981 Q8K4T3 TSL:1 201 GENCODE basic APPRIS P1 Stradb- ENSMUST00000114296.7 1348 209aa ENSMUSP00000109935.1 Protein coding - Q8K4T3 TSL:1 202 GENCODE basic Stradb- ENSMUST00000152318.1 505 129aa ENSMUSP00000137790.1 Protein coding - M0QWE6 CDS 5' 206 incomplete TSL:5 Stradb- ENSMUST00000153990.7 2534 104aa ENSMUSP00000137724.1 Nonsense mediated - M0QW98 TSL:1 207 decay Stradb- ENSMUST00000123301.7 2070 189aa ENSMUSP00000138036.1 Nonsense mediated - A0A0R4J299 TSL:1 203 decay Stradb- ENSMUST00000147637.7 3076 No - Retained intron - - TSL:1 205 protein Stradb- ENSMUST00000123965.7 1978 No - Retained intron - - TSL:1 204 protein Page 7 of 9 https://www.alphaknockout.com 42.19 kb Forward strand 58.97Mb 58.98Mb 58.99Mb 59.00Mb Genes (Comprehensive set... Stradb-201 >protein coding Stradb-207 >nonsense mediated decay Stradb-203 >nonsense mediated decay Stradb-202 >protein coding Stradb-205 >retained intron Stradb-206 >protein coding Stradb-204 >retained intron Contigs AC169382.2 > Genes < Trak2-201protein coding < C2cd6-204protein coding (Comprehensive set... < Trak2-203protein coding < C2cd6-203protein coding < Trak2-204retained intron < AC169382.1-201lncRNA Regulatory Build 58.97Mb 58.98Mb 58.99Mb 59.00Mb Reverse strand 42.19 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000027185 22.19 kb Forward strand Stradb-201 >protein coding ENSMUSP00000027... Low complexity (Seg) Superfamily Protein kinase-like domain superfamily Pfam Protein kinase domain PROSITE profiles Protein kinase domain PANTHER PTHR24361 PTHR24361:SF382 Gene3D 3.30.200.20 CDD cd08226 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop lost missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 418 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.