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Mouse Tmem117 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem117 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem117 gene (NCBI Reference Sequence: NM_178789 ; Ensembl: ENSMUSG00000063296 ) is located on Mouse chromosome 15. 8 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000080141). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Tmem117 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-72C12 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:

Exon 4 starts from about 26.65% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 164386 bp, and the size of intron 4 for 3'-loxP site insertion: 52315 bp. The size of effective cKO region: ~600 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 4 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tmem117 Homology arm 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(7100bp) | A(28.39% 2016) | C(20.63% 1465) | T(29.69% 2108) | G(21.28% 1511)

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.

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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% chr15 + 94876132 94879131 3000 browser details YourSeq 309 2048 2666 3000 84.5% chr11 - 79479490 79480084 595 browser details YourSeq 277 2048 2533 3000 86.4% chr9 - 61450646 61451132 487 browser details YourSeq 261 2048 2536 3000 82.4% chr11 - 54680146 54680647 502 browser details YourSeq 248 2059 2534 3000 87.9% chr11 + 29817567 29818047 481 browser details YourSeq 239 2054 2649 3000 82.6% chr7 - 68450758 68451342 585 browser details YourSeq 236 2059 2489 3000 87.2% chr17 + 52002911 52003342 432 browser details YourSeq 204 2048 2653 3000 83.5% chrX - 9227736 9228325 590 browser details YourSeq 195 2092 2487 3000 75.4% chr4 + 84709140 84709540 401 browser details YourSeq 194 2048 2405 3000 82.2% chr13 - 101131940 101132316 377 browser details YourSeq 194 2053 2506 3000 87.4% chr8 + 78926352 78926815 464 browser details YourSeq 186 2133 2534 3000 87.5% chr12 - 116518857 116519261 405 browser details YourSeq 182 2048 2452 3000 87.2% chr4 - 88293647 88294053 407 browser details YourSeq 179 2058 2533 3000 86.0% chr10 - 85288796 85289270 475 browser details YourSeq 176 2324 2592 3000 88.2% chr3 + 28542571 28542847 277 browser details YourSeq 174 1116 1513 3000 87.8% chr9 + 14259749 14260220 472 browser details YourSeq 171 2048 2448 3000 88.0% chr10 + 67839336 67839738 403 browser details YourSeq 168 2058 2458 3000 85.3% chr7 + 108584245 108584663 419 browser details YourSeq 166 2048 2592 3000 83.7% chr8 - 69304908 69305436 529 browser details YourSeq 153 2059 2451 3000 85.2% chr9 - 33743760 33744168 409

Note: The 3000 bp section upstream of Exon 4 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% chr15 + 94879732 94882731 3000 browser details YourSeq 33 1315 1358 3000 97.3% chr14 - 5124420 5124463 44 browser details YourSeq 33 1315 1358 3000 97.3% chr14 + 7051954 7051997 44 browser details YourSeq 24 1426 1450 3000 100.0% chr2 - 131318896 131318922 27 browser details YourSeq 23 1507 1530 3000 100.0% chr1 - 8778116 8778141 26 browser details YourSeq 22 393 417 3000 95.9% chr11 - 97361191 97361217 27 browser details YourSeq 21 1430 1453 3000 95.9% chr1 - 113850252 113850276 25

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Tmem117 transmembrane protein 117 [ Mus musculus (house mouse) ] Gene ID: 320709, updated on 12-Aug-2019

Gene summary

Official Symbol Tmem117 provided by MGI Official Full Name transmembrane protein 117 provided by MGI Primary source MGI:MGI:2444580 See related Ensembl:ENSMUSG00000063296 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 B930062P21Rik Expression Biased expression in large intestine adult (RPKM 7.6), cerebellum adult (RPKM 2.2) and 14 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 E3 See Tmem117 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (94629185..95096097)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (94459616..94926528)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 2 transcripts

Gene: Tmem117 ENSMUSG00000063296

Description transmembrane protein 117 [Source:MGI Symbol;Acc:MGI:2444580] Gene Synonyms B930062P21Rik Location Chromosome 15: 94,629,232-95,096,098 forward strand. GRCm38:CM001008.2 About this gene This gene has 2 transcripts (splice variants), 197 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmem117-201 ENSMUST00000080141.5 2717 514aa ENSMUSP00000079038.4 Protein coding CCDS27774 Q8BH18 TSL:1 GENCODE basic APPRIS P1

Tmem117-202 ENSMUST00000229677.1 2662 No protein - Retained intron - - -

486.87 kb Forward strand 94.7Mb 94.8Mb 94.9Mb 95.0Mb 95.1Mb Genes (Comprehensive set... Tmem117-202 >retained intron

Tmem117-201 >protein coding

Contigs AC147160.2 > AC158918.7 > < AC118683.10 AC102905.9 > Genes < Gm25546-201snoRNA < Gm23129-201snoRNA (Comprehensive set...

< 1700129L04Rik-202lncRNA

< 1700129L04Rik-201lncRNA

< Nell2-203protein coding

Regulatory Build

94.7Mb 94.8Mb 94.9Mb 95.0Mb 95.1Mb Reverse strand 486.87 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

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Transcript: ENSMUST00000080141

466.85 kb Forward strand

Tmem117-201 >protein coding

ENSMUSP00000079... Transmembrane heli... MobiDB lite Low complexity (Seg) Pfam TMEM117 protein PANTHER TMEM117 protein

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

Variant Legend missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 514

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

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