https://www.alphaknockout.com

Mouse Ppp3r2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp3r2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp3r2 gene (NCBI Reference Sequence: NM_001004025 ; Ensembl: ENSMUSG00000028310 ) is located on Mouse chromosome 4. 2 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000029991). Exon 1~2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp3r2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-185H19 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit male infertility due to reduced hyperactivated sperm motility and midpiece rigidity.

Exon 1~2 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 2. The size of effective cKO region: ~822 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

gRNA region gRNA region

Wildtype allele A T A T

5' G A 3'

1 2

Targeting vector A T A T G A

Targeted allele A T A T G A

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ppp3r2 cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(6789bp) | A(28.18% 1913) | C(22.42% 1522) | T(28.33% 1923) | G(21.08% 1431)

Note: The sequence of homologous arms and cKO region 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 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr4 - 49681949 49684948 3000 browser details YourSeq 77 1300 1451 3000 82.8% chr11 + 72701244 72701410 167 browser details YourSeq 71 1305 1451 3000 82.6% chr2 - 163978757 163978904 148 browser details YourSeq 65 1304 1452 3000 85.2% chr5 - 36496688 36496839 152 browser details YourSeq 64 1484 1681 3000 82.9% chr12 - 116120366 116120554 189 browser details YourSeq 64 1315 1451 3000 82.7% chr3 + 131618012 131618162 151 browser details YourSeq 60 1291 1379 3000 80.5% chr7 + 81061206 81061292 87 browser details YourSeq 57 1423 1583 3000 95.4% chr18 + 32030158 32159144 128987 browser details YourSeq 56 1310 1383 3000 87.9% chr11 - 97306644 97306717 74 browser details YourSeq 56 1304 1389 3000 82.6% chr11 - 74550033 74550118 86 browser details YourSeq 56 1310 1451 3000 71.0% chr14 + 20221439 20221568 130 browser details YourSeq 55 1303 1379 3000 85.8% chrX - 159669573 159669649 77 browser details YourSeq 54 1310 1389 3000 81.1% chr15 - 80738747 80738825 79 browser details YourSeq 53 1301 1383 3000 82.0% chr7 - 120872935 120873017 83 browser details YourSeq 52 1310 1445 3000 92.0% chr3 + 55240953 55241107 155 browser details YourSeq 51 1311 1378 3000 87.9% chr2 - 32765496 32765563 68 browser details YourSeq 51 1301 1383 3000 80.8% chr18 + 65415634 65415716 83 browser details YourSeq 51 1301 1379 3000 82.3% chr12 + 103990440 103990518 79 browser details YourSeq 51 1305 1373 3000 87.0% chr11 + 68757605 68757673 69 browser details YourSeq 50 1301 1382 3000 85.5% chr6 + 127052977 127053056 80

Note: The 3000 bp section upstream of Exon 1 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 3000 1 3000 3000 100.0% chr4 - 49678160 49681159 3000 browser details YourSeq 99 2594 2757 3000 85.0% chr1 + 94607581 94607731 151 browser details YourSeq 98 2602 2752 3000 92.4% chr10 + 69432227 69432749 523 browser details YourSeq 94 2602 2757 3000 92.1% chr10 + 32046781 32047043 263 browser details YourSeq 93 2583 2733 3000 89.8% chr1 - 192270418 192270679 262 browser details YourSeq 85 2582 2767 3000 87.7% chr3 + 31365056 31365373 318 browser details YourSeq 84 2583 2753 3000 85.5% chr11 - 51536706 51536865 160 browser details YourSeq 83 2628 2776 3000 93.0% chr10 - 126278040 126278240 201 browser details YourSeq 81 2587 2748 3000 80.0% chr6 - 122547738 122547884 147 browser details YourSeq 81 2591 2757 3000 89.3% chr10 - 42001043 42001247 205 browser details YourSeq 80 2609 2741 3000 91.0% chr10 - 61307521 61307995 475 browser details YourSeq 78 2582 2724 3000 92.4% chr7 - 34899466 34899676 211 browser details YourSeq 76 2583 2753 3000 93.2% chr10 - 42100359 42100809 451 browser details YourSeq 76 2603 2728 3000 94.5% chr1 + 64981228 64981711 484 browser details YourSeq 73 2510 2736 3000 95.2% chr1 - 158173249 158173927 679 browser details YourSeq 72 2619 2753 3000 91.9% chr10 - 119080239 119080424 186 browser details YourSeq 72 2652 2757 3000 95.2% chr1 + 125658781 125659020 240 browser details YourSeq 71 2603 2750 3000 94.0% chr10 - 67957080 67957251 172 browser details YourSeq 70 2658 2753 3000 95.0% chr12 - 108072524 108072741 218 browser details YourSeq 69 2587 2754 3000 94.0% chr6 - 17950148 17950361 214

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Ppp3r2 protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type II) [ Mus musculus (house mouse) ] Gene ID: 19059, updated on 12-Aug-2019

Gene summary

Official Symbol Ppp3r2 provided by MGI Official Full Name protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type II) provided by MGI Primary source MGI:MGI:107171 See related Ensembl:ENSMUSG00000028310 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 CnB2; CaNB2 Expression Restricted expression toward testis adult (RPKM 63.5) See more Orthologs human all

Genomic context

Location: 4; 4 B1 See Ppp3r2 in Genome Data Viewer

Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (49678747..49681983, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (49691619..49694855, complement)

Chromosome 4 - NC_000070.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 1 transcript

Gene: Ppp3r2 ENSMUSG00000028310

Description protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type II) [Source:MGI Symbol;Acc:MGI:107171] Gene Synonyms CaNB2, CnB2, PP2B beta 2 Location Chromosome 4: 49,678,747-49,682,024 reverse strand. GRCm38:CM000997.2 About this gene This gene has 1 transcript (splice variant), 33 orthologues, 9 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp3r2-201 ENSMUST00000029991.2 3026 179aa ENSMUSP00000029991.2 Protein coding CCDS18179 Q497S1 Q63811 TSL:1 GENCODE basic APPRIS P1

23.28 kb Forward strand 49.67Mb 49.68Mb 49.69Mb Contigs AL732521.18 > Genes < Grin3a-201protein coding (Comprehensive set...

< Grin3a-202protein coding

< Ppp3r2-201protein coding

Regulatory Build

49.67Mb 49.68Mb 49.69Mb Reverse strand 23.28 kb

Regulation Legend CTCF Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000029991

< Ppp3r2-201protein coding

Reverse strand 3.28 kb

ENSMUSP00000029... Superfamily EF-hand domain pair SMART EF-hand domain Pfam EF-hand domain PROSITE profiles EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR45942

PTHR45942:SF3 Gene3D 1.10.238.10 CDD EF-hand domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 179

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

Page 7 of 7