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Mouse Farp2 Knockout Project (CRISPR/Cas9)

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Mouse Farp2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Farp2 Knockout Project (CRISPR/Cas9) Objective: To create a Farp2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Farp2 gene (NCBI Reference Sequence: NM_145519 ; Ensembl: ENSMUSG00000034066 ) is located on Mouse chromosome 1. 27 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 27 (Transcript: ENSMUST00000120301). Exon 2 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: Mice homozygous for a knock-out allele exhibit slight increase in bone volumetrics and reduced osteoclast differentiation from BMDMs cultured with M-CSF and RANKL Exon 2 starts from the coding region. Exon 2 covers 5.73% of the coding region. The size of effective KO region: ~211 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 27 Legends Exon of mouse Farp2 Knockout region Page 2 of 9 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 Exon 2 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. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 2 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(26.2% 524) | C(19.05% 381) | T(31.5% 630) | G(23.25% 465) Note: The 2000 bp section upstream of Exon 2 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(26.7% 534) | C(20.05% 401) | T(29.3% 586) | G(23.95% 479) Note: The 2000 bp section downstream of Exon 2 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 9 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% chr1 + 93526598 93528597 2000 browser details YourSeq 132 659 842 2000 92.9% chr5 + 82699598 82699793 196 browser details YourSeq 132 660 831 2000 92.4% chr5 + 34752826 34753394 569 browser details YourSeq 131 646 824 2000 86.9% chr3 + 154916026 154916187 162 browser details YourSeq 131 1646 1807 2000 88.7% chr12 + 86934071 86934223 153 browser details YourSeq 129 1593 1811 2000 85.6% chr11 - 97216204 97216349 146 browser details YourSeq 129 659 820 2000 95.9% chr16 + 14476439 14476632 194 browser details YourSeq 128 1657 1811 2000 89.8% chr10 - 55201119 55201267 149 browser details YourSeq 127 1648 1807 2000 89.2% chr2 - 93901462 93901620 159 browser details YourSeq 126 659 831 2000 92.7% chr1 - 150644661 150644864 204 browser details YourSeq 126 659 820 2000 95.8% chr12 + 4240646 4240822 177 browser details YourSeq 125 1673 1811 2000 95.0% chr5 - 108560804 108560942 139 browser details YourSeq 125 1648 1811 2000 88.3% chr11 - 105344045 105344205 161 browser details YourSeq 123 659 829 2000 94.4% chr13 - 6294824 6295020 197 browser details YourSeq 122 659 833 2000 88.9% chr9 - 20072804 20072974 171 browser details YourSeq 122 659 829 2000 94.9% chr7 - 37281547 37281763 217 browser details YourSeq 122 1654 1861 2000 88.6% chr4 + 133149758 133150224 467 browser details YourSeq 121 659 829 2000 94.9% chr6 - 68042678 68042882 205 browser details YourSeq 120 659 820 2000 88.8% chr13 - 75415285 75415429 145 browser details YourSeq 119 1673 1811 2000 92.9% chr9 + 100826834 100826972 139 Note: The 2000 bp section upstream of Exon 2 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% chr1 + 93528781 93530780 2000 browser details YourSeq 83 193 334 2000 90.9% chr10 + 130133411 130133552 142 browser details YourSeq 82 193 334 2000 91.8% chr19 - 22341656 22341806 151 browser details YourSeq 80 228 334 2000 96.6% chr7 + 100224511 100224627 117 browser details YourSeq 74 234 338 2000 95.3% chr12 - 9029091 9029205 115 browser details YourSeq 74 239 336 2000 92.0% chr16 + 93789635 93789742 108 browser details YourSeq 73 234 338 2000 94.1% chr10 + 47847103 47847217 115 browser details YourSeq 70 234 334 2000 94.0% chr18 - 24505947 24506057 111 browser details YourSeq 70 234 337 2000 89.9% chr18 + 63721762 63721862 101 browser details YourSeq 70 234 334 2000 92.9% chr12 + 111771509 111771619 111 browser details YourSeq 69 228 338 2000 89.1% chr9 + 108410414 108410534 121 browser details YourSeq 68 234 731 2000 73.8% chr16 + 17020972 17021157 186 browser details YourSeq 67 234 334 2000 96.0% chr4 - 133356414 133356526 113 browser details YourSeq 67 221 331 2000 93.6% chr12 + 19271140 19271265 126 browser details YourSeq 66 234 334 2000 93.6% chr16 - 51566885 51566995 111 browser details YourSeq 62 258 334 2000 97.1% chr3 - 138194871 138194952 82 browser details YourSeq 62 258 336 2000 90.8% chr14 - 28517116 28517195 80 browser details YourSeq 62 257 338 2000 95.7% chr15 + 76786759 76786854 96 browser details YourSeq 62 234 335 2000 93.2% chr1 + 91617880 91617996 117 browser details YourSeq 61 258 333 2000 91.8% chr3 - 34514635 34514717 83 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Farp2 FERM, RhoGEF and pleckstrin domain protein 2 [ Mus musculus (house mouse) ] Gene ID: 227377, updated on 12-Aug-2019 Gene summary Official Symbol Farp2 provided by MGI Official Full Name FERM, RhoGEF and pleckstrin domain protein 2 provided by MGI Primary source MGI:MGI:2385126 See related Ensembl:ENSMUSG00000034066 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 Fir; AI465173; BC009153; mKIAA0793; D030026M03Rik Expression Ubiquitous expression in large intestine adult (RPKM 5.0), colon adult (RPKM 4.4) and 28 other tissues See more Orthologs human all Genomic context Location: 1; 1 D See Farp2 in Genome Data Viewer Exon count: 27 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (93512104..93621976) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (95408681..95518553) Chromosome 1 - NC_000067.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Farp2 ENSMUSG00000034066 Description FERM, RhoGEF and pleckstrin domain protein 2 [Source:MGI Symbol;Acc:MGI:2385126] Gene Synonyms D030026M03Rik, Fir Location Chromosome 1: 93,512,079-93,621,976 forward strand. GRCm38:CM000994.2 About this gene This gene has 2 transcripts (splice variants), 206 orthologues, 10 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Farp2-201 ENSMUST00000120301.7 3937 1065aa ENSMUSP00000112725.1 Protein coding CCDS15191 Q91VS8 TSL:1 GENCODE basic APPRIS P1 Farp2-202 ENSMUST00000122402.2 2816 795aa ENSMUSP00000113790.1 Protein coding - D3Z4C0 TSL:1 GENCODE basic Page 7 of 9 https://www.alphaknockout.com 129.90 kb Forward strand 93.52Mb 93.54Mb 93.56Mb 93.58Mb 93.60Mb 93.62Mb Genes Sept2-215 >protein coding Gm37250-201 >TEC (Comprehensive set... Sept2-216 >protein coding Sept2-214 >protein coding Sept2-201 >protein coding Sept2-209 >retained intron Sept2-212 >protein coding Farp2-201 >protein coding Farp2-202 >protein coding Contigs < AC131316.8 Genes < Stk25-204retained intron (Comprehensive set... < Stk25-205retained intron < Stk25-202retained intron < Stk25-201protein coding < Stk25-203protein coding < Stk25-206protein coding Regulatory Build 93.52Mb 93.54Mb 93.56Mb 93.58Mb 93.60Mb 93.62Mb Reverse strand 129.90 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 processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000120301 109.90 kb Forward strand Farp2-201 >protein coding ENSMUSP00000112... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily FERM superfamily, second domain Dbl homology (DH) domain superfamily SSF50729 Ubiquitin-like domain superfamily SMART Band 4.1 domain FERM adjacent (FA) Dbl homology (DH) domain FERM, C-terminal PH-like domain Pleckstrin homology domain Prints Band 4.1 domain Ezrin/radixin/moesin-like Pfam FERM, N-terminal FERM, C-terminal PH-like domain Pleckstrin homology domain FERM central domain FERM adjacent (FA) Dbl homology (DH) domain PROSITE profiles FERM domain Dbl homology (DH) domain Pleckstrin homology domain PROSITE patterns FERM conserved site PANTHER PTHR45858:SF4 PTHR45858 Gene3D FERM/acyl-CoA-binding protein superfamily Dbl homology (DH) domain superfamily 3.10.20.90 PH-like domain superfamily CDD cd17190 FERM central domain Dbl homology (DH) domain cd13235 FARP1/FARP2/FRMD7, FERM domain C-lobe cd01220 All sequence SNPs/i..
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