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Mouse Pcgf5 Knockout Project (CRISPR/Cas9)

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Mouse Pcgf5 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Pcgf5 Knockout Project (CRISPR/Cas9) Objective: To create a Pcgf5 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pcgf5 gene (NCBI Reference Sequence: NM_029508 ; Ensembl: ENSMUSG00000024805 ) is located on Mouse chromosome 19. 9 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 9 (Transcript: ENSMUST00000071267). 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: Bone marrow cells from mice homozygous for a conditional allele exhibit normal hematopoietic and progenitor cell function. Exon 2 starts from the coding region. Exon 2 covers 15.82% of the coding region. The size of effective KO region: ~281 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 9 Legends Exon of mouse Pcgf5 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. 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 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. 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(28.05% 561) | C(19.45% 389) | T(26.35% 527) | G(26.15% 523) 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(27.2% 544) | C(18.1% 362) | T(33.6% 672) | G(21.1% 422) 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% chr19 + 36410126 36412125 2000 browser details YourSeq 57 795 915 2000 75.0% chr10 + 18385700 18385803 104 browser details YourSeq 52 790 929 2000 85.3% chr13 - 47335560 47335698 139 browser details YourSeq 52 734 910 2000 71.7% chr12 - 110103548 110103682 135 browser details YourSeq 52 790 915 2000 86.3% chr1 + 135229447 135229567 121 browser details YourSeq 50 792 926 2000 89.9% chr5 + 135834590 135834723 134 browser details YourSeq 49 794 929 2000 93.0% chr15 - 67038323 67038459 137 browser details YourSeq 45 790 929 2000 94.3% chr1 + 185219056 185219196 141 browser details YourSeq 44 792 929 2000 80.0% chr11 - 82655651 82655779 129 browser details YourSeq 44 790 976 2000 94.0% chr5 + 141557549 141557760 212 browser details YourSeq 44 790 931 2000 87.8% chr15 + 64353103 64353242 140 browser details YourSeq 43 790 862 2000 81.2% chr12 + 17078903 17078976 74 browser details YourSeq 42 790 918 2000 83.4% chr2 + 13805597 13805720 124 browser details YourSeq 41 802 927 2000 67.4% chr11 + 102236473 102236551 79 browser details YourSeq 40 790 913 2000 93.1% chr12 - 105855471 105855593 123 browser details YourSeq 39 897 965 2000 78.3% chr2 - 173937234 173937302 69 browser details YourSeq 39 790 926 2000 93.4% chr11 + 88318793 88318931 139 browser details YourSeq 38 892 938 2000 91.4% chr12 - 22943341 22943391 51 browser details YourSeq 38 790 915 2000 97.7% chr11 - 109518281 109518406 126 browser details YourSeq 38 790 914 2000 90.3% chr5 + 25741984 25742106 123 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% chr19 + 36412238 36414237 2000 browser details YourSeq 109 222 344 2000 92.5% chr1 + 14328095 14328215 121 browser details YourSeq 107 224 334 2000 98.2% chr3 - 121949930 121950040 111 browser details YourSeq 105 222 334 2000 96.5% chr11 + 88957136 88957248 113 browser details YourSeq 104 223 344 2000 91.0% chr2 + 129849121 129849241 121 browser details YourSeq 103 222 334 2000 95.6% chr2 + 32998548 32998660 113 browser details YourSeq 103 225 344 2000 94.1% chr11 + 102410790 102410909 120 browser details YourSeq 102 226 334 2000 97.3% chr4 - 24437602 24437714 113 browser details YourSeq 102 227 344 2000 91.5% chr14 - 88154118 88154234 117 browser details YourSeq 102 222 346 2000 92.0% chr18 + 38181201 38181486 286 browser details YourSeq 101 231 343 2000 94.7% chr5 - 134633843 134633955 113 browser details YourSeq 100 222 344 2000 91.1% chr2 - 86554482 86554606 125 browser details YourSeq 100 222 334 2000 94.7% chr12 + 15510622 15510735 114 browser details YourSeq 99 222 334 2000 92.8% chr7 + 116296888 116296999 112 browser details YourSeq 98 227 346 2000 94.7% chrX - 94974135 94974947 813 browser details YourSeq 95 230 334 2000 95.3% chr3 + 18169988 18170092 105 browser details YourSeq 95 224 344 2000 91.4% chr18 + 36804108 36804230 123 browser details YourSeq 94 240 344 2000 95.3% chr11 + 49770666 49770771 106 browser details YourSeq 93 231 334 2000 95.2% chr11 - 88095966 88096073 108 browser details YourSeq 92 227 346 2000 92.1% chrX - 94883710 94913287 29578 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: Pcgf5 polycomb group ring finger 5 [ Mus musculus (house mouse) ] Gene ID: 76073, updated on 24-Oct-2019 Gene summary Official Symbol Pcgf5 provided by MGI Official Full Name polycomb group ring finger 5 provided by MGI Primary source MGI:MGI:1923505 See related Ensembl:ENSMUSG00000024805 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 AI324127; 0610009F02Rik; 1110054A01Rik; 5830406C17Rik; 5830443C21Rik; 9530023M17Rik Expression Ubiquitous expression in placenta adult (RPKM 8.9), heart adult (RPKM 3.9) and 26 other tissues See more Orthologs human all Genomic context Location: 19; 19 C2 See Pcgf5 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (36348329..36460970) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (36453557..36530694) Chromosome 19 - NC_000085.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Pcgf5 ENSMUSG00000024805 Description polycomb group ring finger 5 [Source:MGI Symbol;Acc:MGI:1923505] Gene Synonyms 0610009F02Rik, 1110054A01Rik, 5830406C17Rik, 5830443C21Rik, 9530023M17Rik Location Chromosome 19: 36,348,309-36,460,970 forward strand. GRCm38:CM001012.2 About this gene This gene has 11 transcripts (splice variants), 205 orthologues, 7 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pcgf5-211 ENSMUST00000225920.1 6443 236aa ENSMUSP00000153206.1 Protein coding CCDS29772 Q3UK78 GENCODE basic APPRIS P2 Pcgf5-201 ENSMUST00000062389.5 1566 236aa ENSMUSP00000058730.5 Protein coding CCDS29772 Q3UK78 TSL:5 GENCODE basic APPRIS P2 Pcgf5-202 ENSMUST00000071267.13 1508 236aa ENSMUSP00000071245.6 Protein coding CCDS29772 Q3UK78 TSL:1 GENCODE basic APPRIS P2 Pcgf5-210 ENSMUST00000225411.1 1626 256aa ENSMUSP00000153464.1 Protein coding - Q3UK78 GENCODE basic APPRIS ALT1 Pcgf5-203 ENSMUST00000224679.1 1269 256aa ENSMUSP00000153681.1 Protein coding - Q3UK78 GENCODE basic APPRIS ALT1 Pcgf5-204 ENSMUST00000224772.1 1266 255aa ENSMUSP00000153066.1 Protein coding - B7ZP24 GENCODE basic APPRIS ALT1 Pcgf5-207 ENSMUST00000224971.1 599 125aa ENSMUSP00000153342.1 Protein coding - A0A286YDQ6 CDS 3' incomplete Pcgf5-206 ENSMUST00000224859.1 430 132aa ENSMUSP00000153211.1 Protein coding - A0A286YD06 CDS 5' incomplete Pcgf5-209 ENSMUST00000225185.1 3941 No protein - Retained intron - - - Pcgf5-208 ENSMUST00000225050.1 506 No protein - Retained intron - - - Pcgf5-205 ENSMUST00000224805.1 1824 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 132.66 kb Forward strand 36.34Mb 36.36Mb 36.38Mb 36.40Mb 36.42Mb 36.44Mb 36.46Mb Genes (Comprehensive set... Pcgf5-211 >protein coding Pcgf5-205 >lncRNA Pcgf5-202 >protein coding Gm47773-201 >processed pseudogene Pcgf5-207 >protein coding Pcgf5-203 >protein coding Pcgf5-204 >protein coding Gm9042-201 >processed pseudogene Pcgf5-208 >retained intron Pcgf5-201 >protein coding Pcgf5-210 >protein coding Pcgf5-206 >protein coding Pcgf5-209 >retained intron Contigs < AC117769.9 Genes < Gm47735-201lncRNA < Gm23170-201snRNA < F530104D19Rik-201lncRNA (Comprehensive set... Regulatory Build 36.34Mb 36.36Mb 36.38Mb 36.40Mb 36.42Mb 36.44Mb 36.46Mb Reverse strand 132.66 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 pseudogene processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000071267 77.14 kb Forward strand Pcgf5-202 >protein coding ENSMUSP00000071..
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