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Mouse Phlda1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Phlda1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Phlda1 (NCBI Reference Sequence: NM_009344 ; Ensembl: ENSMUSG00000020205 ) is located on Mouse 10. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000164773). Exon 1 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Phlda1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-69B10 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 targeted null mutation are viable, fertile and morphologically normal. Relative to wild-type littermates, homozygous null mice display no obvious defects in immune function, Fas expression or T-cell apoptosis.

Exon 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~1254 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

1 2

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Phlda1 cKO region loxP site

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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(7215bp) | A(25.6% 1847) | C(23.24% 1677) | T(25.6% 1847) | G(25.56% 1844)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr10 + 111503406 111506405 3000 browser details YourSeq 40 2910 2973 3000 93.5% chr14 - 33446873 33446942 70 browser details YourSeq 32 442 479 3000 94.5% chr8 - 40289634 40289696 63 browser details YourSeq 32 2917 2961 3000 71.5% chr11 + 52351019 52351053 35 browser details YourSeq 29 2528 2560 3000 87.5% chr14 + 46285441 46285472 32 browser details YourSeq 27 1686 1712 3000 100.0% chr6 - 22577325 22577351 27 browser details YourSeq 27 2237 2283 3000 89.7% chr12 - 59310705 59310750 46 browser details YourSeq 27 2911 2941 3000 96.8% chr18 + 13972311 13972343 33 browser details YourSeq 25 1685 1713 3000 96.3% chr4 + 130067306 130067334 29 browser details YourSeq 24 1686 1712 3000 84.0% chr9 - 55924056 55924080 25 browser details YourSeq 24 1686 1712 3000 84.0% chr3 + 151812622 151812646 25 browser details YourSeq 24 1675 1701 3000 96.3% chr12 + 99844525 99844553 29 browser details YourSeq 23 1686 1711 3000 83.4% chr2 - 71744215 71744238 24 browser details YourSeq 23 1685 1709 3000 87.5% chr1 - 163429341 163429364 24 browser details YourSeq 22 1682 1703 3000 100.0% chr5 + 125029558 125029579 22

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% chr10 + 111507621 111510620 3000 browser details YourSeq 94 1480 1883 3000 99.0% chr10 - 111509100 111509503 404 browser details YourSeq 94 1694 1923 3000 83.9% chr10 - 60121960 60122174 215 browser details YourSeq 90 1697 1927 3000 85.1% chr19 + 24911607 24911826 220 browser details YourSeq 89 1691 1927 3000 84.5% chr10 - 69934166 69934384 219 browser details YourSeq 86 1692 1933 3000 83.2% chr12 - 92017911 92018147 237 browser details YourSeq 86 1692 1927 3000 85.0% chr1 - 75020544 75020766 223 browser details YourSeq 84 1693 1956 3000 91.2% chr8 + 95184686 95185091 406 browser details YourSeq 82 1693 1923 3000 82.4% chr5 - 53936925 53937143 219 browser details YourSeq 81 1695 1927 3000 79.6% chr11 - 82550077 82550236 160 browser details YourSeq 81 1672 1927 3000 81.2% chr5 + 132780381 132780616 236 browser details YourSeq 81 1697 1935 3000 83.1% chr17 + 6116965 6117197 233 browser details YourSeq 79 1692 1927 3000 78.3% chr7 + 132366636 132366831 196 browser details YourSeq 79 1694 1927 3000 79.6% chr10 + 69012656 69012865 210 browser details YourSeq 77 1692 1926 3000 80.7% chr2 - 153537299 153537503 205 browser details YourSeq 77 1697 1932 3000 79.6% chr17 + 78207018 78207220 203 browser details YourSeq 76 1695 1927 3000 83.6% chr11 - 31923608 31923819 212 browser details YourSeq 75 1720 1924 3000 81.2% chr11 + 86966830 86967011 182 browser details YourSeq 74 1691 1924 3000 91.1% chr6 + 137657121 137657356 236 browser details YourSeq 73 1691 1942 3000 82.4% chrX + 163099108 163099342 235

Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Phlda1 pleckstrin homology like domain, family A, member 1 [ Mus musculus (house mouse) ] Gene ID: 21664, updated on 10-Oct-2019

Gene summary

Official Symbol Phlda1 provided by MGI Official Full Name pleckstrin homology like domain, family A, member 1 provided by MGI Primary source MGI:MGI:1096880 See related Ensembl:ENSMUSG00000020205 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 Tdag; TDAG51; DT1P1B11 Expression Broad expression in adrenal adult (RPKM 67.6), stomach adult (RPKM 50.4) and 23 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 D1 See Phlda1 in Genome Data Viewer

Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (111506286..111508649)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (110943342..110945705)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 2 transcripts

Gene: Phlda1 ENSMUSG00000020205

Description pleckstrin homology like domain, family A, member 1 [Source:MGI Symbol;Acc:MGI:1096880] Gene Synonyms DT1P1B11, TDAG51, Tdag Location Chromosome 10: 111,506,286-111,508,645 forward strand. GRCm38:CM001003.2 About this gene This gene has 2 transcripts (splice variants), 156 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Phlda1-201 ENSMUST00000164773.1 1949 405aa ENSMUSP00000132815.1 Protein coding CCDS24167 Q62392 TSL:1 GENCODE basic APPRIS P1

Phlda1-202 ENSMUST00000186359.1 362 No protein - lncRNA - - TSL:3

22.36 kb Forward strand

111.500Mb 111.505Mb 111.510Mb 111.515Mb (Comprehensive set... Nap1l1-201 >protein coding Gm5176-201 >lncRNA Phlda1-201 >protein coding

Nap1l1-203 >protein coding Gm5176-202 >transcribed processed pseudogene Phlda1-202 >lncRNA

Contigs AC166253.18 >

Genes < Gm28592-201lncRNA (Comprehensive set...

Regulatory Build

111.500Mb 111.505Mb 111.510Mb 111.515Mb Reverse strand 22.36 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene pseudogene

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Transcript: ENSMUST00000164773

2.36 kb Forward strand

Phlda1-201 >protein coding

ENSMUSP00000132... MobiDB lite Low complexity (Seg) Superfamily SSF50729 SMART Pleckstrin homology domain PANTHER PTHR15478

PTHR15478:SF4

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

Variant Legend inframe insertion inframe deletion missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 405

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

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