https://www.alphaknockout.com

Mouse Tmem43 Knockout Project (CRISPR/Cas9)

Objective: To create a Tmem43 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tmem43 (NCBI Reference Sequence: NM_028766 ; Ensembl: ENSMUSG00000030095 ) is located on Mouse 6. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000032183). Exon 2~12 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: In a high-throughput screen, female homozygous mutant mice exhibited an increased anxiety-like response during open field activity testing when compared with their gender-matched wild-type littermates and the historical mean. Homozygous KO or certain codon substitution mutants don't affect heart function.

Exon 2 starts from about 1.08% of the coding region. Exon 2~12 covers 99.0% of the coding region. The size of effective KO region: ~9700 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 2 3 4 5 6 7 11 12

Legends Exon of mouse Tmem43 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 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 2000 bp section downstream of stop codon 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(2000bp) | A(24.6% 492) | C(24.95% 499) | T(27.4% 548) | G(23.05% 461)

Note: The 2000 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(2000bp) | A(23.3% 466) | C(25.15% 503) | T(28.65% 573) | G(22.9% 458)

Note: The 2000 bp section downstream of stop codon 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% chr6 + 91475246 91477245 2000 browser details YourSeq 22 429 450 2000 100.0% chr3 + 36694660 36694681 22 browser details YourSeq 21 1536 1556 2000 100.0% chr8 + 89011976 89011996 21 browser details YourSeq 20 319 338 2000 100.0% chr1 - 135637676 135637695 20 browser details YourSeq 20 879 898 2000 100.0% chr1 + 58952629 58952648 20

Note: The 2000 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 2000 1 2000 2000 100.0% chr6 + 91486946 91488945 2000 browser details YourSeq 29 735 765 2000 90.0% chr3 - 37137579 37137608 30 browser details YourSeq 24 1370 1393 2000 100.0% chr9 - 70172938 70172961 24 browser details YourSeq 23 55 77 2000 100.0% chr14 - 22766559 22766581 23 browser details YourSeq 21 354 374 2000 100.0% chr2 - 162092386 162092406 21 browser details YourSeq 21 736 756 2000 100.0% chr13 - 103262361 103262381 21 browser details YourSeq 21 1787 1807 2000 100.0% chr2 + 35435366 35435386 21 browser details YourSeq 20 25 44 2000 100.0% chr1 - 51995833 51995852 20 browser details YourSeq 20 946 967 2000 95.5% chr1 + 123734231 123734252 22

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

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Gene and information: Tmem43 transmembrane protein 43 [ Mus musculus (house mouse) ] Gene ID: 74122, updated on 10-Sep-2019

Gene summary

Official Symbol Tmem43 provided by MGI Official Full Name transmembrane protein 43 provided by MGI Primary source MGI:MGI:1921372 See related Ensembl:ENSMUSG00000030095 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 LUMA; 1200015A22Rik Expression Ubiquitous expression in subcutaneous fat pad adult (RPKM 68.5), mammary gland adult (RPKM 49.0) and 26 other Orthologs tissues See more human all

Genomic context

Location: 6; 6 D1 See Tmem43 in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (91473707..91488463)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (91423745..91438452)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 5 transcripts

Gene: Tmem43 ENSMUSG00000030095

Description transmembrane protein 43 [Source:MGI Symbol;Acc:MGI:1921372] Gene Synonyms 1200015A22Rik, LUMA Location Chromosome 6: 91,473,703-91,488,463 forward strand. GRCm38:CM000999.2 About this gene This gene has 5 transcripts (splice variants), 195 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tmem43-201 ENSMUST00000032183.5 2903 400aa ENSMUSP00000032183.4 Protein coding CCDS20368 Q9DBS1 TSL:1 GENCODE basic APPRIS P1

Tmem43-202 ENSMUST00000140246.7 860 No protein - Retained intron - - TSL:2

Tmem43-204 ENSMUST00000153179.1 561 No protein - Retained intron - - TSL:2

Tmem43-203 ENSMUST00000144246.1 465 No protein - lncRNA - - TSL:3

Tmem43-205 ENSMUST00000205954.1 376 No protein - lncRNA - - TSL:5

34.76 kb Forward strand

91.47Mb 91.48Mb 91.49Mb (Comprehensive set... Tmem43-201 >protein coding

Tmem43-204 >retained intron

Tmem43-205 >lncRNA

Tmem43-202 >retained intron

Tmem43-203 >lncRNA

Contigs < AC161456.3 Genes < Chchd4-201protein coding < Xpc-201protein coding (Comprehensive set...

< Chchd4-202retained intron < Xpc-202nonsense mediated decay

Regulatory Build

91.47Mb 91.48Mb 91.49Mb Reverse strand 34.76 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000032183

14.76 kb Forward strand

Tmem43-201 >protein coding

ENSMUSP00000032... Transmembrane heli... Pfam Transmembrane protein 43 family

PANTHER PTHR13416:SF2

Transmembrane protein 43 family

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

Variant Legend

synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400

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

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