https://www.alphaknockout.com

Mouse Tmem117 Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Tmem117 (NCBI Reference Sequence: NM_178789 ; Ensembl: ENSMUSG00000063296 ) is located on Mouse 15. 8 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000080141). Exon 3 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 3 starts from about 18.03% of the coding region. Exon 3 covers 8.63% of the coding region. The size of effective KO region: ~133 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'

1 3 8

Legends Exon of mouse Tmem117 Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 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 3 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(29.2% 584) | C(20.15% 403) | T(26.45% 529) | G(24.2% 484)

Note: The 2000 bp section upstream of Exon 3 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(26.95% 539) | C(18.85% 377) | T(33.45% 669) | G(20.75% 415)

Note: The 2000 bp section downstream of Exon 3 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 2000 1 2000 2000 100.0% chr15 + 94712863 94714862 2000 browser details YourSeq 134 396 655 2000 93.6% chr11 + 121016746 121017026 281 browser details YourSeq 130 478 673 2000 95.8% chr11 - 78802835 78803047 213 browser details YourSeq 121 396 634 2000 95.6% chr12 - 19385696 19671392 285697 browser details YourSeq 120 465 610 2000 96.3% chr5 + 9381116 9381607 492 browser details YourSeq 116 467 628 2000 92.0% chr11 + 53625978 53626201 224 browser details YourSeq 109 411 639 2000 95.9% chr11 - 83838359 83918058 79700 browser details YourSeq 106 451 598 2000 94.4% chr10 + 75590389 75590575 187 browser details YourSeq 106 469 691 2000 81.9% chr1 + 64143609 64143736 128 browser details YourSeq 105 467 637 2000 93.5% chr10 - 84740047 84740275 229 browser details YourSeq 104 530 691 2000 96.5% chr1 - 171024771 171025144 374 browser details YourSeq 104 467 669 2000 93.4% chr5 + 120098919 120099126 208 browser details YourSeq 103 467 596 2000 92.7% chr10 + 113929555 113929728 174 browser details YourSeq 99 440 602 2000 93.0% chr18 - 5092358 5092532 175 browser details YourSeq 99 470 600 2000 94.8% chr12 - 7890048 7890416 369 browser details YourSeq 98 430 558 2000 94.7% chr12 + 26468408 26468559 152 browser details YourSeq 97 482 604 2000 93.0% chr11 + 91577917 91578110 194 browser details YourSeq 95 461 596 2000 92.9% chr1 - 121149699 121149904 206 browser details YourSeq 92 467 574 2000 93.6% chr1 - 36484621 36484788 168 browser details YourSeq 92 396 596 2000 80.4% chr10 + 80510747 80510897 151

Note: The 2000 bp section upstream of Exon 3 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% chr15 + 94714996 94716995 2000 browser details YourSeq 27 439 466 2000 100.0% chr10 + 119257438 119257468 31 browser details YourSeq 24 683 716 2000 85.3% chr5 + 70672041 70672074 34 browser details YourSeq 23 285 310 2000 96.0% chrY - 79116481 79116510 30 browser details YourSeq 23 285 310 2000 96.0% chrY - 63543117 63543146 30 browser details YourSeq 22 1780 1804 2000 95.9% chr10 - 104374190 104374216 27

Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and 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 (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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