https://www.alphaknockout.com

Mouse Fbxo31 Knockout Project (CRISPR/Cas9)

Objective: To create a Fbxo31 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fbxo31 (NCBI Reference Sequence: NM_133765 ; Ensembl: ENSMUSG00000052934 ) is located on Mouse 8. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000059018). Exon 3~8 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 3 starts from about 24.39% of the coding region. Exon 3~8 covers 61.21% of the coding region. The size of effective KO region: ~6358 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 3 4 5 6 7 8 9

Legends Exon of mouse Fbxo31 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 3 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 1659 bp section downstream of Exon 8 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(18.55% 371) | C(24.6% 492) | T(28.9% 578) | G(27.95% 559)

Note: The 2000 bp section upstream of Exon 3 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(1659bp) | A(19.35% 321) | C(27.49% 456) | T(25.38% 421) | G(27.79% 461)

Note: The 1659 bp section downstream of Exon 8 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% chr8 - 121560463 121562462 2000 browser details YourSeq 265 898 1232 2000 91.5% chr10 + 77855388 77855715 328 browser details YourSeq 261 907 1234 2000 91.2% chr7 + 101904057 101904826 770 browser details YourSeq 258 895 1232 2000 91.6% chr15 - 51976632 51977236 605 browser details YourSeq 257 891 1234 2000 88.5% chr2 - 129145287 129145641 355 browser details YourSeq 257 906 1234 2000 92.5% chr8 + 122562602 122562981 380 browser details YourSeq 256 904 1234 2000 92.7% chr17 - 27835456 27835797 342 browser details YourSeq 247 904 1234 2000 92.5% chr8 + 77560157 77560670 514 browser details YourSeq 233 790 1234 2000 89.4% chr9 - 88555361 88555737 377 browser details YourSeq 226 915 1233 2000 93.2% chr15 + 99386182 99639220 253039 browser details YourSeq 226 899 1234 2000 93.5% chr1 + 86539578 86539931 354 browser details YourSeq 217 913 1205 2000 91.0% chr9 - 58267850 58268365 516 browser details YourSeq 210 884 1232 2000 87.4% chr2 + 71352805 71353120 316 browser details YourSeq 210 898 1234 2000 90.4% chr16 + 11275693 11276096 404 browser details YourSeq 209 914 1234 2000 91.3% chr6 - 116040781 116041380 600 browser details YourSeq 209 914 1205 2000 91.4% chr7 + 29545957 29546377 421 browser details YourSeq 208 921 1228 2000 91.3% chr2 + 36020356 36020950 595 browser details YourSeq 203 893 1233 2000 93.7% chr11 + 84835457 84835818 362 browser details YourSeq 195 944 1234 2000 89.9% chr15 - 82116317 82116742 426 browser details YourSeq 188 899 1234 2000 90.5% chr16 - 20358701 20359242 542

Note: The 2000 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 1659 1 1659 1659 100.0% chr8 - 121552446 121554104 1659 browser details YourSeq 156 1121 1318 1659 91.1% chr3 + 131357198 131357406 209 browser details YourSeq 155 1133 1331 1659 89.2% chrX - 52170727 52170920 194 browser details YourSeq 153 1138 1325 1659 93.3% chr19 + 3960583 4273961 313379 browser details YourSeq 152 1132 1318 1659 92.4% chr5 - 129921005 129921211 207 browser details YourSeq 152 1138 1326 1659 89.6% chr11 + 52372274 52372460 187 browser details YourSeq 151 1134 1325 1659 91.3% chr10 + 117658227 117658424 198 browser details YourSeq 151 1133 1318 1659 92.3% chr1 + 24687533 24687719 187 browser details YourSeq 150 1131 1323 1659 91.9% chr10 + 69427871 69790327 362457 browser details YourSeq 149 1140 1329 1659 91.3% chr13 - 65092683 65092883 201 browser details YourSeq 149 1137 1323 1659 89.9% chr5 + 135292975 135293159 185 browser details YourSeq 149 1142 1326 1659 92.1% chr18 + 73892645 73892852 208 browser details YourSeq 148 1046 1318 1659 85.0% chr13 - 42190080 42190316 237 browser details YourSeq 148 1134 1318 1659 92.6% chrX + 50489466 50489650 185 browser details YourSeq 148 1139 1323 1659 91.6% chr11 + 97632434 97632619 186 browser details YourSeq 147 1134 1318 1659 89.6% chr17 - 12947009 12947188 180 browser details YourSeq 147 1142 1323 1659 92.0% chr13 - 106934518 106934707 190 browser details YourSeq 147 1134 1318 1659 91.7% chrX + 20340349 20442566 102218 browser details YourSeq 147 1136 1318 1659 91.1% chr15 + 100243802 100371385 127584 browser details YourSeq 147 1138 1323 1659 91.1% chr13 + 113001744 113060380 58637

Note: The 1659 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and 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

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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

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49.37 kb Forward strand 121.54Mb 121.55Mb 121.56Mb 121.57Mb 121.58Mb 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

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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.

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