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Mouse Ppp2r5b Knockout Project (CRISPR/Cas9)

Objective: To create a Ppp2r5b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ppp2r5b gene (NCBI Reference Sequence: NM_198168 ; Ensembl: ENSMUSG00000024777 ) is located on Mouse chromosome 19. 14 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 14 (Transcript: ENSMUST00000025695). Exon 2~11 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 2 starts from the coding region. Exon 2~11 covers 74.85% of the coding region. The size of effective KO region: ~4944 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' 10

1 2 3 4 5 6 7 8 9 11 14

Legends Exon of mouse Ppp2r5b Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 412 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 806 bp section downstream of Exon 11 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(412bp) | A(19.9% 82) | C(30.1% 124) | T(21.84% 90) | G(28.16% 116)

Note: The 412 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(806bp) | A(23.7% 191) | C(27.17% 219) | T(24.19% 195) | G(24.94% 201)

Note: The 806 bp section downstream of Exon 11 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 412 1 412 412 100.0% chr19 - 6234832 6235243 412 browser details YourSeq 25 113 144 412 96.3% chr14 - 78577730 78577763 34 browser details YourSeq 23 359 383 412 87.5% chr5 - 8364365 8364388 24 browser details YourSeq 21 101 121 412 100.0% chr19 - 15753905 15753925 21 browser details YourSeq 21 31 51 412 100.0% chr16 + 13923634 13923654 21 browser details YourSeq 20 31 50 412 100.0% chr14 - 35486923 35486942 20

Note: The 412 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 806 1 806 806 100.0% chr19 - 6229350 6230155 806 browser details YourSeq 30 656 805 806 65.7% chr11 + 109379423 109379547 125 browser details YourSeq 23 264 287 806 100.0% chr10 + 15231316 15231343 28 browser details YourSeq 22 784 805 806 100.0% chr10 - 125674781 125674802 22 browser details YourSeq 21 186 206 806 100.0% chr1 + 126925227 126925247 21 browser details YourSeq 20 82 101 806 100.0% chr10 - 36960978 36960997 20 browser details YourSeq 20 604 623 806 100.0% chr12 + 108359086 108359105 20

Note: The 806 bp section downstream of Exon 11 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ppp2r5b protein phosphatase 2, regulatory subunit B', beta [ Mus musculus (house mouse) ] Gene ID: 225849, updated on 12-Aug-2019

Gene summary

Official Symbol Ppp2r5b provided by MGI Official Full Name protein phosphatase 2, regulatory subunit B', beta provided by MGI Primary source MGI:MGI:2388480 See related Ensembl:ENSMUSG00000024777 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 B'beta; BC026670 Expression Ubiquitous expression in adrenal adult (RPKM 89.9), cerebellum adult (RPKM 51.2) and 23 other tissues See more Orthologs human all

Genomic context

Location: 19; 19 A See Ppp2r5b in Genome Data Viewer Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (6227765..6236264, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (6227767..6235840, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 4 transcripts

Gene: Ppp2r5b ENSMUSG00000024777

Description protein phosphatase 2, regulatory subunit B', beta [Source:MGI Symbol;Acc:MGI:2388480] Gene Synonyms B'beta Location Chromosome 19: 6,227,765-6,235,872 reverse strand. GRCm38:CM001012.2 View alleles of this gene on alternative sequences About this gene This gene has 4 transcripts (splice variants), 1 gene allele, 175 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ppp2r5b-201 ENSMUST00000025695.9 2742 497aa ENSMUSP00000025695.9 Protein coding CCDS29499 Q6PD28 TSL:1 GENCODE basic APPRIS P1

Ppp2r5b-204 ENSMUST00000236699.1 740 No protein - Retained intron - - -

Ppp2r5b-203 ENSMUST00000153155.1 389 No protein - Retained intron - - TSL:3

Ppp2r5b-202 ENSMUST00000148693.1 467 No protein - lncRNA - - TSL:5

28.11 kb Forward strand 6.22Mb 6.23Mb 6.24Mb Genes Majin-201 >protein coding Gpha2-201 >protein coding Atg2a-201 >protein coding (Comprehensive set...

Majin-202 >protein coding Gpha2-202 >protein coding Atg2a-204 >protein coding

Contigs AC127556.4 > Genes (Comprehensive set... < Ppp2r5b-201protein coding

< Ppp2r5b-204retained intron < Ppp2r5b-202lncRNA

< Ppp2r5b-203retained intron

Regulatory Build

6.22Mb 6.23Mb 6.24Mb Reverse strand 28.11 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000025695

< Ppp2r5b-201protein coding

Reverse strand 8.11 kb

ENSMUSP00000025... MobiDB lite Low complexity (Seg) Superfamily Armadillo-type fold

Pfam Protein phosphatase 2A, regulatory B subunit, B56

PIRSF Protein phosphatase 2A, regulatory B subunit, B56

PANTHER PTHR10257:SF4

Protein phosphatase 2A, regulatory B subunit, B56 Gene3D Armadillo-like helical

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 497

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

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