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Mouse Nmd3 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Nmd3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Nmd3 gene (NCBI Reference Sequence: NM_133787 ; Ensembl: ENSMUSG00000027787 ) is located on Mouse chromosome 3. 16 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000029358). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Nmd3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-12P9 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 3 starts from about 2.98% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1790 bp, and the size of intron 4 for 3'-loxP site insertion: 2880 bp. The size of effective cKO region: ~2701 bp. The cKO 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 16 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Nmd3 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(9201bp) | A(25.38% 2335) | C(20.16% 1855) | T(33.53% 3085) | G(20.93% 1926)

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% chr3 + 69721008 69724007 3000 browser details YourSeq 68 312 804 3000 71.8% chr4 - 8791871 8792064 194 browser details YourSeq 67 470 738 3000 93.6% chr10 - 41581896 41582356 461 browser details YourSeq 65 582 775 3000 82.4% chr9 + 73226639 73226828 190 browser details YourSeq 55 709 780 3000 91.1% chr4 - 116498783 116498857 75 browser details YourSeq 54 709 775 3000 93.6% chr6 - 88460547 88460616 70 browser details YourSeq 53 582 749 3000 82.1% chr2 - 32426595 32426758 164 browser details YourSeq 53 698 770 3000 90.7% chr11 - 118044551 118044624 74 browser details YourSeq 52 709 775 3000 89.6% chr11 - 78602577 78602646 70 browser details YourSeq 51 703 775 3000 85.0% chr1 - 135399361 135399433 73 browser details YourSeq 51 2423 2518 3000 94.8% chr5 + 130041146 130041246 101 browser details YourSeq 50 583 749 3000 96.3% chr9 - 44755635 44756131 497 browser details YourSeq 50 585 749 3000 89.1% chr10 - 45317459 45317623 165 browser details YourSeq 48 693 746 3000 96.3% chr6 - 86781054 86781392 339 browser details YourSeq 48 709 780 3000 94.5% chr11 - 87037402 87037476 75 browser details YourSeq 48 709 776 3000 94.6% chr10 + 127684072 127684142 71 browser details YourSeq 45 562 777 3000 74.6% chr14 - 16182026 16182216 191 browser details YourSeq 45 714 768 3000 91.0% chr2 + 79653233 79653287 55 browser details YourSeq 45 711 761 3000 94.2% chr17 + 15407227 15407277 51 browser details YourSeq 44 580 737 3000 93.9% chr6 - 39517955 39518112 158

Note: The 3000 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 3000 1 3000 3000 100.0% chr3 + 69726709 69729708 3000 browser details YourSeq 136 2056 2383 3000 89.2% chr5 - 38826670 38827012 343 browser details YourSeq 135 2051 2397 3000 86.1% chr11 - 21709313 21709664 352 browser details YourSeq 112 2218 2492 3000 92.5% chr2 + 154995885 154996166 282 browser details YourSeq 111 2155 2403 3000 89.6% chr8 - 74839878 74840132 255 browser details YourSeq 111 2105 2379 3000 87.1% chr12 + 27043011 27043286 276 browser details YourSeq 110 1433 1602 3000 90.5% chr4 - 124864905 124865074 170 browser details YourSeq 110 2155 2504 3000 88.9% chr15 + 27649684 27650050 367 browser details YourSeq 107 1448 1602 3000 84.6% chr1 - 63152541 63152695 155 browser details YourSeq 106 1469 1967 3000 74.2% chr11 + 4899165 4899323 159 browser details YourSeq 105 1413 1602 3000 82.7% chr7 - 107045312 107045611 300 browser details YourSeq 104 1433 1597 3000 89.4% chr15 + 12353146 12353324 179 browser details YourSeq 104 1447 1602 3000 80.8% chr1 + 131586613 131586763 151 browser details YourSeq 102 1461 1602 3000 86.0% chr4 + 44021116 44021257 142 browser details YourSeq 102 2055 2377 3000 91.0% chr2 + 111757197 111757522 326 browser details YourSeq 100 1461 1604 3000 83.7% chr10 - 99694779 99694921 143 browser details YourSeq 100 1467 1602 3000 86.8% chr7 + 101045409 101045544 136 browser details YourSeq 99 1447 1602 3000 81.9% chr9 - 123781600 123781737 138 browser details YourSeq 98 1433 1608 3000 93.0% chr9 - 72277476 72277674 199 browser details YourSeq 98 1437 1567 3000 88.9% chr2 - 31552958 31553092 135

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: Nmd3 NMD3 ribosome export adaptor [ Mus musculus (house mouse) ] Gene ID: 97112, updated on 12-Aug-2019

Gene summary

Official Symbol Nmd3 provided by MGI Official Full Name NMD3 ribosome export adaptor provided by MGI Primary source MGI:MGI:2140103 See related Ensembl:ENSMUSG00000027787 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 C87860 Expression Ubiquitous expression in placenta adult (RPKM 13.4), CNS E11.5 (RPKM 10.0) and 28 other tissues See more Orthologs human all

Genomic context

Location: 3; 3 E1 See Nmd3 in Genome Data Viewer

Exon count: 16

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (69722055..69749047)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (69525977..69552969)

Chromosome 3 - NC_000069.6

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Transcript information: This gene has 8 transcripts

Gene: Nmd3 ENSMUSG00000027787

Description NMD3 ribosome export adaptor [Source:MGI Symbol;Acc:MGI:2140103] Gene Synonyms C87860 Location Chromosome 3: 69,721,985-69,756,373 forward strand. GRCm38:CM000996.2 About this gene This gene has 8 transcripts (splice variants), 209 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nmd3- ENSMUST00000029358.14 9135 503aa ENSMUSP00000029358.8 Protein coding CCDS17407 Q99L48 TSL:1 201 GENCODE basic APPRIS P1

Nmd3- ENSMUST00000143249.1 396 76aa ENSMUSP00000115736.1 Protein coding - D3YYX2 CDS 3' 205 incomplete TSL:3

Nmd3- ENSMUST00000135266.7 697 144aa ENSMUSP00000142290.1 Nonsense mediated - A0A0A6YY59 TSL:3 203 decay

Nmd3- ENSMUST00000143041.7 494 36aa ENSMUSP00000116113.1 Nonsense mediated - D6RHT4 TSL:2 204 decay

Nmd3- ENSMUST00000127211.6 945 No - Retained intron - - TSL:1 202 protein

Nmd3- ENSMUST00000194168.1 848 No - Retained intron - - TSL:3 208 protein

Nmd3- ENSMUST00000150210.1 746 No - Retained intron - - TSL:2 207 protein

Nmd3- ENSMUST00000149680.7 401 No - Retained intron - - TSL:2 206 protein

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54.39 kb Forward strand

69.72Mb 69.73Mb 69.74Mb 69.75Mb 69.76Mb Genes (Comprehensive set... Nmd3-201 >protein coding

Nmd3-206 >retained intron Nmd3-207 >retained intron

Nmd3-204 >nonsense mediated decay

Nmd3-203 >nonsense mediated decay

Nmd3-202 >retained intron

Nmd3-205 >protein coding

Nmd3-208 >retained intron

Contigs < AC162790.2 AC115035.5 > Genes < Rpl32-ps-201processed pseudogene (Comprehensive set...

Regulatory Build

69.72Mb 69.73Mb 69.74Mb 69.75Mb 69.76Mb Reverse strand 54.39 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000029358

34.39 kb Forward strand

Nmd3-201 >protein coding

ENSMUSP00000029... Pfam Nmd3, N-terminal PANTHER Ribosomal export protein Nmd3

PTHR12746:SF2

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 503

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

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