https://www.alphaknockout.com

Mouse Fbxw5 Knockout Project (CRISPR/Cas9)

Objective: To create a Fbxw5 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fbxw5 (NCBI Reference Sequence: NM_013908 ; Ensembl: ENSMUSG00000015095 ) is located on Mouse 2. 9 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000015239). Exon 6~9 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 6 starts from about 39.33% of the coding region. Exon 6~9 covers 44.33% of the coding region. The size of effective KO region: ~1244 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 4 5 6 7 8 9

Legends Exon of mouse Fbxw5 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 6 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(21.65% 433) | C(27.6% 552) | T(21.25% 425) | G(29.5% 590)

Note: The 2000 bp section upstream of Exon 6 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(26.2% 524) | C(23.9% 478) | T(22.65% 453) | G(27.25% 545)

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.

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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 + 25501720 25503719 2000 browser details YourSeq 25 1864 1896 2000 75.0% chr1 - 135538664 135538691 28 browser details YourSeq 25 719 747 2000 81.5% chr17 + 65660685 65660711 27 browser details YourSeq 25 644 680 2000 74.1% chr1 + 158215866 158215896 31 browser details YourSeq 21 42 62 2000 100.0% chr8 + 59897941 59897961 21

Note: The 2000 bp section upstream of Exon 6 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 + 25504964 25506963 2000 browser details YourSeq 48 1878 1949 2000 87.5% chr12 + 36088815 36088926 112 browser details YourSeq 46 1882 1944 2000 91.3% chr4 - 119384352 119384436 85 browser details YourSeq 44 1878 1943 2000 92.4% chr5 - 92844590 92844663 74 browser details YourSeq 44 1879 1943 2000 88.9% chr2 - 120258795 120258861 67 browser details YourSeq 44 1878 1944 2000 95.9% chr5 + 120100798 120100888 91 browser details YourSeq 43 1884 1944 2000 94.0% chr1 - 125806665 125806746 82 browser details YourSeq 40 1875 1942 2000 95.6% chr4 - 135955325 135955419 95 browser details YourSeq 38 1876 1941 2000 95.3% chr14 - 66710265 66710348 84 browser details YourSeq 37 1903 1942 2000 97.5% chr1 - 18050995 18051054 60 browser details YourSeq 36 1903 1944 2000 95.3% chr6 - 51531020 51531081 62 browser details YourSeq 36 1881 1943 2000 95.0% chr17 - 30961110 30961193 84 browser details YourSeq 35 1905 1944 2000 94.9% chr4 + 152539609 152539668 60 browser details YourSeq 34 1883 1945 2000 92.5% chr5 + 52883073 52883158 86 browser details YourSeq 33 1903 1944 2000 90.3% chr5 - 38377649 38377709 61 browser details YourSeq 33 1905 1944 2000 92.4% chr2 - 143919821 143919880 60 browser details YourSeq 33 1878 1939 2000 97.2% chr14 - 25632744 25632807 64 browser details YourSeq 32 1905 1945 2000 90.3% chr1 - 160187146 160187206 61 browser details YourSeq 32 1908 1944 2000 94.5% chr7 + 27394701 27394758 58 browser details YourSeq 31 1909 1961 2000 74.3% chr10 - 128029473 128029516 44

Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Fbxw5 F-box and WD-40 domain protein 5 [ Mus musculus (house mouse) ] Gene ID: 30839, updated on 12-Aug-2019

Gene summary

Official Symbol Fbxw5 provided by MGI Official Full Name F-box and WD-40 domain protein 5 provided by MGI Primary source MGI:MGI:1354731 See related Ensembl:ENSMUSG00000015095 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 Fbw5; AI159739 Expression Ubiquitous expression in testis adult (RPKM 58.6), adrenal adult (RPKM 19.2) and 26 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 A3 See Fbxw5 in Genome Data Viewer Exon count: 9

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (25500750..25505470)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (25356298..25360990)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 9 transcripts

Gene: Fbxw5 ENSMUSG00000015095

Description F-box and WD-40 domain protein 5 [Source:MGI Symbol;Acc:MGI:1354731] Gene Synonyms Fbw5 Location Chromosome 2: 25,500,750-25,505,471 forward strand. GRCm38:CM000995.2 About this gene This gene has 9 transcripts (splice variants), 189 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fbxw5- ENSMUST00000015239.9 2388 573aa ENSMUSP00000015239.3 Protein CCDS15778 Q9QXW2 TSL:1 201 coding GENCODE basic APPRIS P1

Fbxw5- ENSMUST00000124375.1 739 246aa ENSMUSP00000117676.1 Protein - A0A0A0MQH6 CDS 5' and 3' 203 coding incomplete TSL:5

Fbxw5- ENSMUST00000126601.7 986 No - lncRNA - - TSL:5 204 protein

Fbxw5- ENSMUST00000148845.1 870 No - lncRNA - - TSL:2 209 protein

Fbxw5- ENSMUST00000124258.7 787 No - lncRNA - - TSL:3 202 protein

Fbxw5- ENSMUST00000129104.1 785 No - lncRNA - - TSL:2 205 protein

Fbxw5- ENSMUST00000142004.7 653 No - lncRNA - - TSL:5 208 protein

Fbxw5- ENSMUST00000135456.1 600 No - lncRNA - - TSL:2 206 protein

Fbxw5- ENSMUST00000135511.1 567 No - lncRNA - - TSL:5 207 protein

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24.72 kb Forward strand 25.495Mb 25.500Mb 25.505Mb 25.510Mb 25.515Mb (Comprehensive set... Fbxw5-201 >protein coding

Fbxw5-202 >lncRNA Fbxw5-209 >lncRNA

Fbxw5-204 >lncRNA

Fbxw5-208 >lncRNA

Fbxw5-207 >lncRNA

Fbxw5-205 >lncRNA

Fbxw5-206 >lncRNA

Fbxw5-203 >protein coding

Contigs AL732557.4 > AL732590.7 > Genes < Lcn12-201protein coding < C8g-201protein coding (Comprehensive set...

< Lcn12-202protein coding < C8g-202protein coding

< C8g-205lncRNA

< C8g-206lncRNA

< C8g-204lncRNA

< C8g-207lncRNA

< C8g-203lncRNA

Regulatory Build

25.495Mb 25.500Mb 25.505Mb 25.510Mb 25.515Mb Reverse strand 24.72 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

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Transcript: ENSMUST00000015239

4.72 kb Forward strand

Fbxw5-201 >protein coding

ENSMUSP00000015... Low complexity (Seg) Superfamily WD40-repeat-containing domain superfamily

F-box-like domain superfamily SMART F-box domain WD40 repeat

Pfam F-box domain WD40 repeat PROSITE profiles F-box domain WD40-repeat-containing domain

WD40 repeat PANTHER F-box/WD repeat-containing protein 5

Gene3D 1.20.1280.50

WD40/YVTN repeat-like-containing domain superfamily

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 573

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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