https://www.alphaknockout.com Mouse Fbxo31 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Fbxo31 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fbxo31 gene (NCBI Reference Sequence: NM_133765 ; Ensembl: ENSMUSG00000052934 ) is located on Mouse chromosome 8. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000059018). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Fbxo31 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-330D20 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 3 starts from about 24.39% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 5812 bp, and the size of intron 4 for 3'-loxP site insertion: 828 bp. The size of effective cKO region: ~1040 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 3 4 5 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Fbxo31 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7540bp) | A(19.72% 1487) | C(25.56% 1927) | T(25.6% 1930) | G(29.12% 2196) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr8 - 121560713 121563712 3000 browser details YourSeq 265 2148 2482 3000 91.5% chr10 + 77855388 77855715 328 browser details YourSeq 261 2157 2484 3000 91.2% chr7 + 101904057 101904826 770 browser details YourSeq 258 2145 2482 3000 91.6% chr15 - 51976632 51977236 605 browser details YourSeq 257 2141 2484 3000 88.5% chr2 - 129145287 129145641 355 browser details YourSeq 257 2156 2484 3000 92.5% chr8 + 122562602 122562981 380 browser details YourSeq 256 2154 2484 3000 92.7% chr17 - 27835456 27835797 342 browser details YourSeq 247 2154 2484 3000 92.5% chr8 + 77560157 77560670 514 browser details YourSeq 233 2040 2484 3000 89.4% chr9 - 88555361 88555737 377 browser details YourSeq 226 2165 2483 3000 93.2% chr15 + 99386182 99639220 253039 browser details YourSeq 226 2149 2484 3000 93.5% chr1 + 86539578 86539931 354 browser details YourSeq 217 2163 2455 3000 91.0% chr9 - 58267850 58268365 516 browser details YourSeq 210 2134 2482 3000 87.4% chr2 + 71352805 71353120 316 browser details YourSeq 210 2148 2484 3000 90.4% chr16 + 11275693 11276096 404 browser details YourSeq 209 2164 2484 3000 91.3% chr6 - 116040781 116041380 600 browser details YourSeq 209 2164 2455 3000 91.4% chr7 + 29545957 29546377 421 browser details YourSeq 208 2171 2478 3000 91.3% chr2 + 36020356 36020950 595 browser details YourSeq 203 2143 2483 3000 93.7% chr11 + 84835457 84835818 362 browser details YourSeq 195 2194 2484 3000 89.9% chr15 - 82116317 82116742 426 browser details YourSeq 188 2149 2484 3000 90.5% chr16 - 20358701 20359242 542 Note: The 3000 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 3000 1 3000 3000 100.0% chr8 - 121556673 121559672 3000 browser details YourSeq 302 1923 2262 3000 95.5% chr11 + 103756979 103757502 524 browser details YourSeq 298 1945 2286 3000 94.9% chr2 - 154470050 154470632 583 browser details YourSeq 295 1944 2289 3000 96.6% chr11 - 115735319 115736005 687 browser details YourSeq 293 1945 2285 3000 96.3% chr4 - 32941980 32960488 18509 browser details YourSeq 292 1949 2360 3000 93.0% chr7 + 127653774 127971594 317821 browser details YourSeq 288 1944 2259 3000 96.2% chr11 + 120837418 120837944 527 browser details YourSeq 287 1944 2270 3000 94.2% chr4 + 132958049 132958402 354 browser details YourSeq 285 1963 2307 3000 93.9% chr19 - 16158066 16158403 338 browser details YourSeq 285 1951 2286 3000 95.0% chr4 + 152155814 152156351 538 browser details YourSeq 285 1948 2262 3000 95.6% chr15 + 31514777 31515364 588 browser details YourSeq 281 1941 2262 3000 95.5% chr17 - 15614046 15614487 442 browser details YourSeq 280 1979 2593 3000 90.6% chr15 + 84099597 84100089 493 browser details YourSeq 275 1948 2262 3000 94.2% chr7 + 141397010 141397549 540 browser details YourSeq 272 1949 2262 3000 94.5% chr2 + 119206080 119206649 570 browser details YourSeq 268 1957 2288 3000 94.7% chr1 + 133021993 133022582 590 browser details YourSeq 242 1972 2522 3000 93.8% chr11 - 116568178 116568965 788 browser details YourSeq 236 2020 2284 3000 96.5% chrX + 8235621 8236309 689 browser details YourSeq 230 2010 2354 3000 96.8% chr9 - 110317513 110318361 849 browser details YourSeq 216 1986 2262 3000 95.4% chr7 - 30128905 30129591 687 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Fbxo31 F-box protein 31 [ Mus musculus (house mouse) ] Gene ID: 76454, updated on 12-Aug-2019 Gene summary Official Symbol Fbxo31 provided by MGI Official Full Name F-box protein 31 provided by MGI Primary source MGI:MGI:1354708 See related Ensembl:ENSMUSG00000052934 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 Fbx14; Fbxo14; 1110003O08Rik; 2310046N15Rik Expression Ubiquitous expression in adrenal adult (RPKM 24.3), cortex adult (RPKM 21.5) and 28 other tissues See more Orthologs human all Genomic context Location: 8; 8 E1 See Fbxo31 in Genome Data Viewer Exon count: 10 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (121549443..121578864, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (124075879..124102706, complement) Chromosome 8 - NC_000074.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Fbxo31 ENSMUSG00000052934 Description F-box protein 31 [Source:MGI Symbol;Acc:MGI:1354708] Gene Synonyms 1110003O08Rik, 2310046N15Rik, Fbx14, Fbxo14 Location Chromosome 8: 121,549,440-121,578,806 reverse strand. GRCm38:CM001001.2 About this gene This gene has 5 transcripts (splice variants), 263 orthologues, 1 paralogue, 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 Fbxo31- ENSMUST00000059018.13 4358 507aa ENSMUSP00000057573.5 Protein CCDS52693 Q3TQF0 TSL:1 201 coding GENCODE basic APPRIS P2 Fbxo31- ENSMUST00000212985.1 606 202aa ENSMUSP00000148763.1 Protein - A0A1D5RMG0 CDS 5' and 3' 205 coding incomplete TSL:5 APPRIS ALT2 Fbxo31- ENSMUST00000180539.1 845 No - Retained - - TSL:1 202 protein intron Fbxo31- ENSMUST00000180979.1 919 No - lncRNA - - TSL:3 203 protein Fbxo31- ENSMUST00000181663.1 799 No - lncRNA - - TSL:3 204 protein Page 6 of 8 https://www.alphaknockout.com 49.37 kb Forward strand 121.54Mb 121.55Mb 121.56Mb 121.57Mb 121.58Mb Genes Gm20388-201 >protein coding (Comprehensive set... Gm27045-201 >lncRNA Gm17786-201 >processed pseudogene 1700030M09Rik-201 >lncRNA 1700030M09Rik-202 >lncRNA Contigs AC122205.6 > AC182458.2 > Genes (Comprehensive set... < 1700018B08Rik-201protein coding< Fbxo31-201protein coding < 1700018B08Rik-204nonsense mediated decay < Fbxo31-203lncRNA < Fbxo31-205protein coding < 1700018B08Rik-203nonsense mediated decay < Fbxo31-202retained intron < Fbxo31-204lncRNA < 1700018B08Rik-202protein coding Regulatory Build 121.54Mb 121.55Mb 121.56Mb 121.57Mb 121.58Mb Reverse strand 49.37 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000059018 < Fbxo31-201protein coding Reverse strand 29.37 kb ENSMUSP00000057... MobiDB lite Low complexity (Seg) Superfamily F-box-like domain superfamily SMART F-box domain Pfam F-box domain PROSITE profiles F-box domain PANTHER PTHR10706 F-box only protein 31 Gene3D 1.20.1280.50 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 507 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.