https://www.alphaknockout.com Mouse Phldb2 Knockout Project (CRISPR/Cas9) Objective: To create a Phldb2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Phldb2 gene (NCBI Reference Sequence: NM_001252442 ; Ensembl: ENSMUSG00000033149 ) is located on Mouse chromosome 16. 19 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 19 (Transcript: ENSMUST00000076333). 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 conditional allele activated in neurons exhibit impaired LTP. Exon 2 starts from the coding region. Exon 2 covers 33.87% of the coding region. The size of effective KO region: ~1337 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 19 Legends Exon of mouse Phldb2 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(30.65% 613) | C(17.55% 351) | T(33.35% 667) | G(18.45% 369) 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(29.2% 584) | C(20.25% 405) | T(32.7% 654) | G(17.85% 357) 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% chr16 - 45826082 45828081 2000 browser details YourSeq 55 76 244 2000 92.2% chr3 + 60249907 60250710 804 browser details YourSeq 54 93 152 2000 96.7% chr17 - 85969726 85969791 66 browser details YourSeq 50 73 152 2000 98.1% chr11 - 68488759 68488876 118 browser details YourSeq 47 86 165 2000 87.5% chr2 - 43007845 43007922 78 browser details YourSeq 46 105 156 2000 98.0% chr1 - 180181024 180181087 64 browser details YourSeq 40 73 124 2000 97.7% chr3 + 30190123 30190194 72 browser details YourSeq 37 76 121 2000 93.4% chr12 + 7422777 7422834 58 browser details YourSeq 34 74 119 2000 81.1% chr18 + 43121623 43121664 42 browser details YourSeq 32 94 131 2000 94.8% chr4 + 100693959 100694008 50 browser details YourSeq 32 73 105 2000 100.0% chr13 + 88752862 88752896 35 browser details YourSeq 32 96 152 2000 79.0% chr1 + 99725138 99725201 64 browser details YourSeq 31 78 115 2000 94.5% chr14 + 83006092 83006137 46 browser details YourSeq 31 78 156 2000 97.0% chr1 + 167619074 167619290 217 browser details YourSeq 30 92 124 2000 87.1% chr12 - 7792326 7792356 31 browser details YourSeq 30 78 114 2000 85.3% chr1 - 109299423 109299457 35 browser details YourSeq 30 78 160 2000 59.4% chr7 + 80853790 80853822 33 browser details YourSeq 30 76 156 2000 59.4% chr16 + 55840816 55840848 33 browser details YourSeq 29 76 106 2000 96.8% chr2 - 136221670 136221700 31 browser details YourSeq 29 74 108 2000 91.5% chr5 + 65562512 65562546 35 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% chr16 - 45822759 45824758 2000 browser details YourSeq 27 1140 1169 2000 96.6% chr14 + 123205537 123205571 35 browser details YourSeq 26 668 693 2000 100.0% chr7 + 14343965 14343990 26 browser details YourSeq 23 367 397 2000 87.1% chr7 - 136094616 136094646 31 browser details YourSeq 21 1173 1193 2000 100.0% chr9 + 40940401 40940421 21 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: Phldb2 pleckstrin homology like domain, family B, member 2 [ Mus musculus (house mouse) ] Gene ID: 208177, updated on 12-Aug-2019 Gene summary Official Symbol Phldb2 provided by MGI Official Full Name pleckstrin homology like domain, family B, member 2 provided by MGI Primary source MGI:MGI:2444981 See related Ensembl:ENSMUSG00000033149 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 LL5b; LL5beta; AV253284; C820004H04Rik Expression Broad expression in bladder adult (RPKM 19.0), limb E14.5 (RPKM 13.1) and 20 other tissues See more Orthologs human all Genomic context Location: 16; 16 B5 See Phldb2 in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (45746230..45953598, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (45746346..45844491, complement) Chromosome 16 - NC_000082.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 11 transcripts Gene: Phldb2 ENSMUSG00000033149 Description pleckstrin homology like domain, family B, member 2 [Source:MGI Symbol;Acc:MGI:2444981] Gene Synonyms C820004H04Rik, LL5b, LL5beta Location Chromosome 16: 45,746,243-45,953,598 reverse strand. GRCm38:CM001009.2 About this gene This gene has 11 transcripts (splice variants), 317 orthologues, 6 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Phldb2-201 ENSMUST00000036355.12 5483 1249aa ENSMUSP00000046496.6 Protein coding CCDS28202 Q8K1N2 TSL:1 GENCODE basic APPRIS P3 Phldb2-202 ENSMUST00000076333.11 5347 1302aa ENSMUSP00000075672.5 Protein coding CCDS57031 Q8K1N2 TSL:1 GENCODE basic APPRIS ALT2 Phldb2-207 ENSMUST00000134802.7 3612 1131aa ENSMUSP00000123284.1 Protein coding - D3Z069 CDS 3' incomplete TSL:1 Phldb2-205 ENSMUST00000131003.7 2328 565aa ENSMUSP00000119718.1 Protein coding - F6QU68 CDS 5' incomplete TSL:1 Phldb2-209 ENSMUST00000136405.1 595 133aa ENSMUSP00000121800.1 Protein coding - D3Z231 CDS 3' incomplete TSL:3 Phldb2-206 ENSMUST00000133111.2 614 No protein - Retained intron - - TSL:5 Phldb2-204 ENSMUST00000128365.2 831 No protein - lncRNA - - TSL:5 Phldb2-211 ENSMUST00000151300.1 664 No protein - lncRNA - - TSL:3 Phldb2-208 ENSMUST00000136317.1 497 No protein - lncRNA - - TSL:2 Phldb2-210 ENSMUST00000142697.1 467 No protein - lncRNA - - TSL:5 Phldb2-203 ENSMUST00000125433.1 343 No protein - lncRNA - - TSL:3 Page 7 of 9 https://www.alphaknockout.com 227.36 kb Forward strand 45.75Mb 45.80Mb 45.85Mb 45.90Mb 45.95Mb Genes Gm15591-201 >lncRNA Gm15640-201 >lncRNA (Comprehensive set... Gm15638-203 >lncRNA Gm15638-201 >lncRNA Gm15638-202 >lncRNA Contigs < AC166572.1 AC164979.2 > Genes (Comprehensive set... < Abhd10-201protein coding < Phldb2-206retained intron < Phldb2-209protein coding < Abhd10-202nonsense mediated decay < Phldb2-203lncRNA < Phldb2-204lncRNA < Plcxd2-201protein coding < Abhd10-203lncRNA< Phldb2-211lncRNA < Phldb2-208lncRNA < Abhd10-204lncRNA < Phldb2-210lncRNA < Abhd10-205lncRNA < Phldb2-201protein coding < Phldb2-202protein coding < Phldb2-205protein coding < Phldb2-207protein coding Regulatory Build 45.75Mb 45.80Mb 45.85Mb 45.90Mb 45.95Mb Reverse strand 227.36 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 processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000076333 < Phldb2-202protein coding Reverse strand 97.84 kb ENSMUSP00000075... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF50729 SMART Pleckstrin homology domain Pfam Pleckstrin homology domain PROSITE profiles Pleckstrin homology domain PANTHER PTHR12156:SF21 PTHR12156 Gene3D PH-like domain superfamily CDD PHLDB1/2/3, PH domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 200 400 600 800 1000 1302 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.