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Mouse Dok6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Dok6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Dok6 gene (NCBI Reference Sequence: NM_001039173 ; Ensembl: ENSMUSG00000073514 ) is located on Mouse chromosome 18. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000097495). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Dok6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-356C16 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 17.62% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 38566 bp, and the size of intron 3 for 3'-loxP site insertion: 63053 bp. The size of effective cKO region: ~615 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 3 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Dok6 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(7115bp) | A(34.27% 2438) | C(16.8% 1195) | T(30.3% 2156) | G(18.64% 1326)

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% chr18 - 89560368 89563367 3000 browser details YourSeq 703 2 1046 3000 87.1% chr14 + 75092088 75093136 1049 browser details YourSeq 692 13 1061 3000 86.8% chr14 - 103383935 103384997 1063 browser details YourSeq 684 2 1035 3000 87.1% chr6 - 7762018 7763053 1036 browser details YourSeq 681 2 1060 3000 86.6% chr6 + 126992583 126993630 1048 browser details YourSeq 662 1 1065 3000 87.5% chr7 + 120302796 120411963 109168 browser details YourSeq 662 1 1061 3000 84.7% chr12 + 44505860 44506909 1050 browser details YourSeq 661 1 984 3000 87.0% chr4 + 87358314 87359299 986 browser details YourSeq 660 1 1046 3000 86.8% chr16 - 33642909 33643952 1044 browser details YourSeq 659 1 984 3000 86.3% chr18 - 32299499 32300495 997 browser details YourSeq 659 1 1046 3000 86.5% chr12 - 90028339 90067808 39470 browser details YourSeq 658 2 1059 3000 87.5% chr9 - 77861653 77862706 1054 browser details YourSeq 658 1 1046 3000 86.2% chr8 - 74159675 74160720 1046 browser details YourSeq 656 1 987 3000 85.8% chr5 + 101047151 101048126 976 browser details YourSeq 652 1 997 3000 87.4% chr1 + 176555694 176556694 1001 browser details YourSeq 650 2 1035 3000 86.8% chr6 - 24737415 24738442 1028 browser details YourSeq 650 8 1046 3000 88.3% chr16 + 37895804 38025233 129430 browser details YourSeq 650 2 1046 3000 84.7% chr10 + 123428099 123429143 1045 browser details YourSeq 649 54 1062 3000 87.1% chr16 + 15922305 15923630 1326 browser details YourSeq 649 1 1061 3000 86.8% chr11 + 63821286 63822338 1053

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% chr18 - 89556753 89559752 3000 browser details YourSeq 43 1452 1567 3000 90.4% chr4 - 53432673 53432788 116 browser details YourSeq 39 1506 1568 3000 71.2% chr3 + 123655392 123655436 45 browser details YourSeq 38 1236 1557 3000 72.8% chr2 + 147349886 147350185 300 browser details YourSeq 33 1528 1567 3000 84.3% chr5 - 36455780 36455817 38 browser details YourSeq 33 1522 1562 3000 82.1% chr3 + 34120991 34121029 39 browser details YourSeq 32 1522 1557 3000 88.6% chr10 - 80599526 80599560 35 browser details YourSeq 29 1541 1573 3000 90.4% chr3 - 154034645 154034676 32 browser details YourSeq 29 1541 1576 3000 87.5% chr13 - 114470197 114470231 35 browser details YourSeq 27 655 689 3000 86.3% chr1 - 43800979 43801011 33 browser details YourSeq 27 657 690 3000 86.3% chr2 + 135510540 135510571 32 browser details YourSeq 23 1543 1568 3000 83.4% chr2 - 127310029 127310052 24

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Dok6 docking protein 6 [ Mus musculus (house mouse) ] Gene ID: 623279, updated on 12-Aug-2019

Gene summary

Official Symbol Dok6 provided by MGI Official Full Name docking protein 6 provided by MGI Primary source MGI:MGI:3639495 See related Ensembl:ENSMUSG00000073514 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 Dok-6 Expression Biased expression in frontal lobe adult (RPKM 4.5), cortex adult (RPKM 4.0) and 5 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 E4 See Dok6 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (89292423..89769540, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (89470527..89938528, complement)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 2 transcripts

Gene: Dok6 ENSMUSG00000073514

Description docking protein 6 [Source:MGI Symbol;Acc:MGI:3639495] Gene Synonyms Dok-6 Location Chromosome 18: 89,292,424-89,769,528 reverse strand. GRCm38:CM001011.2 About this gene This gene has 2 transcripts (splice variants), 197 orthologues, 7 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Dok6-201 ENSMUST00000097495.4 10099 331aa ENSMUSP00000095103.3 Protein coding CCDS37878 Q2MHE5 TSL:1 GENCODE basic APPRIS P1

Dok6-202 ENSMUST00000160328.1 4082 No protein - Retained intron - - TSL:1

497.11 kb Forward strand

89.3Mb 89.4Mb 89.5Mb 89.6Mb 89.7Mb Genes Gm16297-201 >processed pseudogene Gm22694-201 >miRNA (Comprehensive set...

Contigs < AC116387.7 AC110375.4 > AC164619.5 > AC151406.2 >

Genes (Comprehensive set... < Dok6-201protein coding

< Gm50204-201processed pseudogene < Gm16296-201processed pseudogene

< Dok6-202retained intron

Regulatory Build

89.3Mb 89.4Mb 89.5Mb 89.6Mb 89.7Mb Reverse strand 497.11 kb

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

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

pseudogene RNA gene processed transcript

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Transcript: ENSMUST00000097495

< Dok6-201protein coding

Reverse strand 477.11 kb

ENSMUSP00000095... MobiDB lite Low complexity (Seg) Superfamily SSF50729 SMART Pleckstrin homology domain IRS-type PTB domain

SM01244 Pfam IRS-type PTB domain PROSITE profiles IRS-type PTB domain PANTHER PTHR21258:SF43

PTHR21258 Gene3D PH-like domain superfamily CDD DOK4/5/6, PH domain cd13164

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

Variant Legend synonymous variant

Scale bar 0 40 80 120 160 200 240 280 331

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

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