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Mouse Dnajc17 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dnajc17 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dnajc17 gene (NCBI Reference Sequence: NM_139139 ; Ensembl: ENSMUSG00000034278 ) is located on Mouse chromosome 2. 11 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 11 (Transcript: ENSMUST00000038439). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dnajc17 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-15I21 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 gene trapped allele die before implantation.

Exon 2 starts from about 8.69% of the coding region. The knockout of Exon 2~4 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 22272 bp, and the size of intron 4 for 3'-loxP site insertion: 1693 bp. The size of effective cKO region: ~1220 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 5 6 11 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Dnajc17 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(7720bp) | A(24.15% 1864) | C(24.42% 1885) | T(26.49% 2045) | G(24.95% 1926)

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% chr2 - 119186666 119189665 3000 browser details YourSeq 775 897 2172 3000 94.2% chr9 + 53689446 53690306 861 browser details YourSeq 769 1355 2172 3000 97.2% chr4 - 79327685 79328507 823 browser details YourSeq 763 1355 2172 3000 96.9% chr2 + 75191022 75191845 824 browser details YourSeq 759 1357 2172 3000 96.7% chr14 + 73796492 73797313 822 browser details YourSeq 754 1355 2169 3000 96.5% chr17 - 74487251 74488074 824 browser details YourSeq 750 1367 2172 3000 96.7% chrX + 69605458 69606268 811 browser details YourSeq 750 1367 2172 3000 96.9% chr4 + 86854664 86857349 2686 browser details YourSeq 739 1355 2164 3000 95.8% chr15 - 76337601 76338416 816 browser details YourSeq 731 1359 2172 3000 95.2% chr8 - 88806368 88807182 815 browser details YourSeq 729 1356 2172 3000 94.9% chr11 - 98185343 98186157 815 browser details YourSeq 727 1344 2164 3000 95.5% chr11 - 57449092 57450176 1085 browser details YourSeq 721 1355 2169 3000 94.6% chr10 - 65534634 65535454 821 browser details YourSeq 717 1359 2154 3000 95.6% chr5 + 12478828 12479633 806 browser details YourSeq 709 1358 2158 3000 94.6% chrX - 103037615 103038415 801 browser details YourSeq 669 1370 2160 3000 92.4% chr6 + 49542944 49543738 795 browser details YourSeq 665 1359 2160 3000 92.8% chr13 - 50950211 50951017 807 browser details YourSeq 611 1384 2153 3000 91.6% chr5 - 107241619 107242388 770 browser details YourSeq 581 1355 2156 3000 89.3% chr3 - 32260326 32261155 830 browser details YourSeq 574 1370 2176 3000 89.5% chrX - 85241275 85242763 1489

Note: The 3000 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 3000 1 3000 3000 100.0% chr2 - 119182446 119185445 3000 browser details YourSeq 200 604 1029 3000 98.1% chr11 - 5666383 5666881 499 browser details YourSeq 197 601 859 3000 97.6% chr12 - 91797416 91797838 423 browser details YourSeq 195 509 797 3000 97.2% chr11 - 104672442 104672995 554 browser details YourSeq 195 488 795 3000 91.3% chr3 + 116752659 116752894 236 browser details YourSeq 194 591 802 3000 96.7% chr5 + 51954775 51954986 212 browser details YourSeq 193 604 814 3000 98.6% chrX - 95068713 95069254 542 browser details YourSeq 193 603 803 3000 98.6% chr9 + 6624430 6624640 211 browser details YourSeq 193 597 797 3000 98.6% chr6 + 93552379 93552581 203 browser details YourSeq 193 591 797 3000 98.1% chr11 + 5707880 5708088 209 browser details YourSeq 192 604 831 3000 98.5% chr1 - 131912872 131913254 383 browser details YourSeq 192 604 850 3000 97.6% chr7 + 4816077 4816524 448 browser details YourSeq 191 581 789 3000 98.0% chr2 - 5923538 5923890 353 browser details YourSeq 191 603 797 3000 99.0% chr17 - 26109814 26110008 195 browser details YourSeq 191 591 799 3000 97.1% chr14 - 47680882 47681094 213 browser details YourSeq 191 604 803 3000 98.0% chr8 + 24911676 24911875 200 browser details YourSeq 190 604 806 3000 97.6% chr16 - 18698943 18699381 439 browser details YourSeq 190 604 797 3000 99.0% chr18 + 34514689 34514882 194 browser details YourSeq 189 604 799 3000 98.5% chr6 - 119141598 119141794 197 browser details YourSeq 189 602 797 3000 98.5% chr5 - 23820691 23820887 197

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: Dnajc17 DnaJ heat shock protein family (Hsp40) member C17 [ Mus musculus (house mouse) ] Gene ID: 69408, updated on 12-Aug-2019

Gene summary

Official Symbol Dnajc17 provided by MGI Official Full Name DnaJ heat shock protein family (Hsp40) member C17 provided by MGI Primary source MGI:MGI:1916658 See related Ensembl:ENSMUSG00000034278 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 C87112; D9Bwg1371e; 1700025B16Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 9.6), CNS E14 (RPKM 7.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2 E5; 2 59.97 cM See Dnajc17 in Genome Data Viewer

Exon count: 11

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (119172500..119208838, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (118998236..119034531, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 3 transcripts

Gene: Dnajc17 ENSMUSG00000034278

Description DnaJ heat shock protein family (Hsp40) member C17 [Source:MGI Symbol;Acc:MGI:1916658] Gene Synonyms 1700025B16Rik, D9Bwg1371e Location Chromosome 2: 119,172,500-119,208,795 reverse strand. GRCm38:CM000995.2 About this gene This gene has 3 transcripts (splice variants), 210 orthologues, 21 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dnajc17-201 ENSMUST00000038439.3 1008 303aa ENSMUSP00000041841.3 Protein coding CCDS16593 Q91WT4 TSL:1 GENCODE basic APPRIS P1

Dnajc17-202 ENSMUST00000125731.1 450 No protein - Retained intron - - TSL:2

Dnajc17-203 ENSMUST00000141009.1 616 No protein - lncRNA - - TSL:3

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56.30 kb Forward strand

119.17Mb 119.18Mb 119.19Mb 119.20Mb 119.21Mb Genes Gchfr-201 >protein coding Gm14138-201 >processed pseudogene Zfyve19-208 >lncRNA (Comprehensive set...

Gm14137-201 >protein coding Zfyve19-201 >protein coding

Gm14137-202 >protein coding Zfyve19-206 >lncRNA

Zfyve19-202 >protein coding

Zfyve19-205 >lncRNA

Zfyve19-204 >retained intron

Zfyve19-210 >retained intron

Zfyve19-211 >retained intron

Zfyve19-203 >retained intron

Zfyve19-207 >retained intron

Zfyve19-209 >lncRNA

Contigs < AL772264.8 Genes (Comprehensive set... < Dnajc17-201protein coding < Ppp1r14d-201protein coding

< Dnajc17-203lncRNA < Ppp1r14d-202protein coding

< Dnajc17-202retained intron

Regulatory Build

119.17Mb 119.18Mb 119.19Mb 119.20Mb 119.21Mb Reverse strand 56.30 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene pseudogene

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Transcript: ENSMUST00000038439

< Dnajc17-201protein coding

Reverse strand 36.30 kb

ENSMUSP00000041... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Chaperone J-domain superfamily RNA-binding domain superfamily

SMART DnaJ domain Prints DnaJ domain Pfam DnaJ domain RNA recognition motif domain

PROSITE profiles DnaJ domain PANTHER PTHR44313 Gene3D Chaperone J-domain superfamily Nucleotide-binding alpha-beta plait domain superfamily

CDD DnaJ domain DNAJC17, RNA recognition motif

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 303

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

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