https://www.alphaknockout.com

Mouse Fbxl7 Knockout Project (CRISPR/Cas9)

Objective: To create a Fbxl7 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fbxl7 (NCBI Reference Sequence: NM_176959 ; Ensembl: ENSMUSG00000043556 ) is located on Mouse 15. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000059204). Exon 3~4 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 3 starts from about 8.69% of the coding region. Exon 3~4 covers 91.38% of the coding region. The size of effective KO region: ~9966 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

Legends Exon of mouse Fbxl7 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 612 bp section 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 734 bp section of Exon 4 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.

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

Sequence 12

Summary: Full Length(612bp) | A(19.93% 122) | C(32.68% 200) | T(21.73% 133) | G(25.65% 157)

Note: The 612 bp section 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(734bp) | A(21.25% 156) | C(29.16% 214) | T(23.3% 171) | G(26.29% 193)

Note: The 734 bp section 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 612 1 612 612 100.0% chr15 - 26552440 26553051 612 browser details YourSeq 33 121 176 612 92.5% chr14 - 46456993 46457351 359 browser details YourSeq 28 322 352 612 96.7% chr11 + 54910289 54910322 34 browser details YourSeq 27 426 460 612 96.7% chr3 + 28745530 28745571 42 browser details YourSeq 26 247 273 612 100.0% chr11 - 84096950 84096983 34 browser details YourSeq 25 109 134 612 100.0% chr14 - 25310076 25310109 34 browser details YourSeq 21 28 48 612 100.0% chr4 - 120734438 120734458 21 browser details YourSeq 21 416 440 612 92.0% chr16 + 17971537 17971561 25 browser details YourSeq 20 96 115 612 100.0% chr1 - 87639648 87639667 20 browser details YourSeq 20 275 294 612 100.0% chr12 + 54879157 54879176 20

Note: The 612 bp section 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 734 1 734 734 100.0% chr15 - 26543088 26543821 734 browser details YourSeq 21 484 504 734 100.0% chr12 + 68537909 68537929 21 browser details YourSeq 20 61 80 734 100.0% chr5 - 119946572 119946591 20 browser details YourSeq 20 522 541 734 100.0% chr19 - 43664326 43664345 20 browser details YourSeq 20 460 479 734 100.0% chr1 + 53247635 53247654 20

Note: The 734 bp section of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Fbxl7 F-box and leucine-rich repeat protein 7 [ Mus musculus (house mouse) ] Gene ID: 448987, updated on 10-Oct-2019

Gene summary

Official Symbol Fbxl7 provided by MGI Official Full Name F-box and leucine-rich repeat protein 7 provided by MGI Primary source MGI:MGI:3052506 See related Ensembl:ENSMUSG00000043556 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as FBL7; Fbl6; AL023057; D230018M15Rik Expression Broad expression in limb E14.5 (RPKM 5.8), ovary adult (RPKM 5.0) and 23 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 B1 See Fbxl7 in Genome Data Viewer Exon count: 5

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (26540454..26895564, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (26470214..26825319, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 2 transcripts

Gene: Fbxl7 ENSMUSG00000043556

Description F-box and leucine-rich repeat protein 7 [Source:MGI Symbol;Acc:MGI:3052506] Gene Synonyms D230018M15Rik, FBL7, Fbl6 Location Chromosome 15: 26,540,459-26,895,580 reverse strand. GRCm38:CM001008.2 About this gene This gene has 2 transcripts (splice variants), 222 orthologues, 17 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fbxl7-201 ENSMUST00000059204.10 4632 491aa ENSMUSP00000061305.9 Protein coding CCDS37052 Q5BJ29 TSL:1 GENCODE basic APPRIS P1

Fbxl7-202 ENSMUST00000226377.1 2490 No protein - Retained intron - - -

375.12 kb Forward strand 26.6Mb 26.7Mb 26.8Mb 26.9Mb Gm49267-201 >lncRNA Gm49268-201 >TEC (Comprehensive set...

Contigs < AC107453.14 < AC102847.3 < AC163394.4 Genes (Comprehensive set... < Fbxl7-201protein coding

< 9430068D22Rik-201TEC < Gm6330-202lncRNA < Fbxl7-202retained intron

< Gm49265-201TEC < Gm6330-201transcribed processed pseudogene

< Gm49266-201TEC

Regulatory Build

26.6Mb 26.7Mb 26.8Mb 26.9Mb Reverse strand 375.12 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene processed transcript RNA gene

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Transcript: ENSMUST00000059204

< Fbxl7-201protein coding

Reverse strand 355.12 kb

ENSMUSP00000061... MobiDB lite Low complexity (Seg) Superfamily F-box-like domain superfamily

SSF52047 SMART F-box domain Leucine-rich repeat, cysteine-containing subtype Pfam F-box domain Leucine-rich repeat PROSITE profiles F-box domain PANTHER PTHR13318

PTHR13318:SF50 Gene3D Leucine-rich repeat domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 491

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

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