https://www.alphaknockout.com Mouse Fam32a Knockout Project (CRISPR/Cas9) Objective: To create a Fam32a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fam32a gene (NCBI Reference Sequence: NM_026455 ; Ensembl: ENSMUSG00000003039 ) is located on Mouse chromosome 8. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000003123). Exon 1~4 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 about 0.3% of the coding region. Exon 1~4 covers 100.0% of the coding region. The size of effective KO region: ~2461 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 Legends Exon of mouse Fam32a 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. 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 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(30.15% 603) | C(21.7% 434) | T(26.4% 528) | G(21.75% 435) 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(25.95% 519) | C(21.15% 423) | T(27.5% 550) | G(25.4% 508) 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% chr8 + 72217788 72219787 2000 browser details YourSeq 210 1320 1582 2000 95.0% chr18 - 10297676 10297923 248 browser details YourSeq 205 901 1567 2000 87.4% chrX + 37100994 37101335 342 browser details YourSeq 204 1376 1595 2000 97.3% chr2 + 34382064 34382299 236 browser details YourSeq 202 1379 1618 2000 93.6% chr5 - 122573697 122573928 232 browser details YourSeq 200 1376 1587 2000 98.1% chr13 - 99762377 99762588 212 browser details YourSeq 200 1366 1591 2000 94.5% chr12 + 8768336 8768557 222 browser details YourSeq 198 1368 1582 2000 98.1% chr19 + 44163008 44163236 229 browser details YourSeq 196 1366 1575 2000 99.1% chr6 - 145509646 145509857 212 browser details YourSeq 195 1381 1588 2000 98.6% chr5 - 144029473 144029685 213 browser details YourSeq 195 1382 1584 2000 98.6% chr18 - 77748616 77748828 213 browser details YourSeq 195 1364 1573 2000 97.7% chr13 - 104282176 104282532 357 browser details YourSeq 194 1379 1592 2000 94.1% chr5 + 123311726 123311929 204 browser details YourSeq 193 1365 1582 2000 96.7% chr5 - 135363335 135363553 219 browser details YourSeq 193 1366 1579 2000 96.1% chr1 - 182074988 182075199 212 browser details YourSeq 192 930 1572 2000 89.3% chrX - 166177108 166177482 375 browser details YourSeq 192 1378 1592 2000 97.6% chr7 + 141224648 141224871 224 browser details YourSeq 191 1382 1575 2000 99.5% chr11 - 120341380 120341576 197 browser details YourSeq 191 1382 1577 2000 99.0% chr10 + 122840163 122840359 197 browser details YourSeq 190 1386 1666 2000 97.1% chr3 - 122004320 122004778 459 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% chr8 + 72222249 72224248 2000 browser details YourSeq 817 1 1179 2000 88.4% chr5 + 47975334 47976724 1391 browser details YourSeq 786 1 1122 2000 88.7% chr17 - 8360830 8362105 1276 browser details YourSeq 715 55 1175 2000 89.4% chr7 - 39444306 39445783 1478 browser details YourSeq 319 675 1149 2000 87.8% chr11 + 17259834 17260494 661 browser details YourSeq 172 1540 2000 2000 93.9% chr6 - 52289339 52470897 181559 browser details YourSeq 147 1526 1845 2000 93.0% chr4 - 144418691 144419212 522 browser details YourSeq 144 1307 1686 2000 85.6% chr13 + 64442823 64443090 268 browser details YourSeq 135 1532 1680 2000 95.2% chr12 - 76827744 76827890 147 browser details YourSeq 135 1424 1775 2000 83.1% chr14 + 52107259 52107526 268 browser details YourSeq 133 1535 1686 2000 94.6% chr15 + 79660255 79660405 151 browser details YourSeq 132 1131 1686 2000 80.0% chr1 - 75822230 75822411 182 browser details YourSeq 131 1535 1686 2000 90.6% chr11 - 67950606 67950753 148 browser details YourSeq 131 1535 1686 2000 91.9% chr10 - 120611723 120611871 149 browser details YourSeq 130 1535 1687 2000 94.0% chr9 - 21514542 21514694 153 browser details YourSeq 130 1540 1686 2000 93.0% chr11 - 86415235 86415378 144 browser details YourSeq 130 1535 1686 2000 92.5% chr5 + 146577039 146577188 150 browser details YourSeq 129 1539 1686 2000 92.4% chr4 - 8757140 8757284 145 browser details YourSeq 129 1540 1697 2000 92.4% chr3 - 8867456 8867611 156 browser details YourSeq 129 1539 1686 2000 91.5% chr6 + 84086434 84086575 142 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: Fam32a family with sequence similarity 32, member A [ Mus musculus (house mouse) ] Gene ID: 67922, updated on 10-Oct-2019 Gene summary Official Symbol Fam32a provided by MGI Official Full Name family with sequence similarity 32, member A provided by MGI Primary source MGI:MGI:1915172 See related Ensembl:ENSMUSG00000003039 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 Otag12; OTAG-12; AW822030; 2510049I19Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 38.9), CNS E14 (RPKM 36.4) and 28 other tissues See more Orthologs human all Genomic context Location: 8; 8 B3.3 See Fam32a in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (72219745..72225034) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (74743629..74747674) Chromosome 8 - NC_000074.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Fam32a ENSMUSG00000003039 Description family with sequence similarity 32, member A [Source:MGI Symbol;Acc:MGI:1915172] Gene Synonyms 2510049I19Rik Location Chromosome 8: 72,219,730-72,224,418 forward strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 205 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fam32a-201 ENSMUST00000003123.9 2564 112aa ENSMUSP00000003123.8 Protein coding CCDS52595 Q9CR80 TSL:1 GENCODE basic APPRIS P1 Fam32a-202 ENSMUST00000212117.1 3387 No protein - Retained intron - - TSL:1 24.69 kb Forward strand 72.21Mb 72.22Mb 72.23Mb Genes (Comprehensive set... Fam32a-201 >protein coding Fam32a-202 >retained intron Contigs < AC158898.7 Genes < Cib3-202retained intron < Gm11034-201protein coding (Comprehensive set... < Cib3-203protein coding < Cib3-201protein coding Regulatory Build 72.21Mb 72.22Mb 72.23Mb Reverse strand 24.69 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000003123 4.69 kb Forward strand Fam32a-201 >protein coding ENSMUSP00000003... MobiDB lite Low complexity (Seg) Pfam Protein FAM32A PANTHER PTHR13282:SF10 Protein FAM32A All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 10 20 30 40 50 60 70 80 90 100 112 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.