https://www.alphaknockout.com

Mouse Shc3 Knockout Project (CRISPR/Cas9)

Objective: To create a Shc3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Shc3 (NCBI Reference Sequence: NM_009167 ; Ensembl: ENSMUSG00000021448 ) is located on Mouse 13. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000021898). Exon 3~4 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: Mice homozygous for disruptions in this gene display a normal phenotype.

Exon 3 starts from about 13.08% of the coding region. Exon 3~4 covers 12.94% of the coding region. The size of effective KO region: ~2847 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 4 12

Legends Exon of mouse Shc3 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. 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 Exon 4 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(23.2% 464) | C(22.15% 443) | T(31.6% 632) | G(23.05% 461)

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(22.4% 448) | C(22.75% 455) | T(33.75% 675) | G(21.1% 422)

Note: The 2000 bp section downstream of Exon 4 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% chr13 - 51482946 51484945 2000 browser details YourSeq 793 1 799 2000 99.3% chr1 + 122421134 122421929 796 browser details YourSeq 791 1 799 2000 99.2% chr7 - 106837843 106838638 796 browser details YourSeq 791 1 799 2000 99.2% chr4 + 123885525 123886320 796 browser details YourSeq 791 1 799 2000 99.2% chr14 + 22933688 22934483 796 browser details YourSeq 789 1 799 2000 99.0% chr2 + 132479289 132480084 796 browser details YourSeq 789 1 799 2000 99.0% chr18 + 68026281 68027076 796 browser details YourSeq 789 1 799 2000 99.0% chr10 + 115277554 115278349 796 browser details YourSeq 788 1 799 2000 99.3% chr5 + 128674919 128675716 798 browser details YourSeq 788 1 799 2000 99.2% chr2 + 40152910 40153706 797 browser details YourSeq 787 1 799 2000 98.9% chrX - 169442030 169442825 796 browser details YourSeq 787 1 799 2000 98.9% chr5 - 102331717 102332512 796 browser details YourSeq 787 1 799 2000 98.9% chr2 - 105542972 105543767 796 browser details YourSeq 787 1 799 2000 98.9% chr10 - 19697167 19697962 796 browser details YourSeq 787 1 799 2000 98.9% chrX + 58582711 58583506 796 browser details YourSeq 787 1 799 2000 98.9% chr5 + 67584955 67585750 796 browser details YourSeq 787 1 799 2000 98.9% chr10 + 88628783 88629578 796 browser details YourSeq 786 1 799 2000 99.0% chr8 + 116491992 116492788 797 browser details YourSeq 785 1 806 2000 98.9% chrX - 96310547 96311352 806 browser details YourSeq 785 1 799 2000 98.8% chr3 - 61100810 61101605 796

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% chr13 - 51478099 51480098 2000 browser details YourSeq 267 476 1013 2000 91.2% chr6 - 80549113 80549455 343 browser details YourSeq 166 797 1011 2000 96.7% chr19 - 60294681 60294986 306 browser details YourSeq 139 824 1013 2000 95.5% chr8 + 95515555 95515847 293 browser details YourSeq 133 713 1007 2000 97.9% chr11 - 111352737 111719352 366616 browser details YourSeq 129 711 1013 2000 86.9% chr8 + 95515565 95515815 251 browser details YourSeq 118 475 1013 2000 83.5% chrY + 1059330 1059450 121 browser details YourSeq 112 474 1013 2000 83.9% chr1 + 22327575 22327892 318 browser details YourSeq 108 824 995 2000 87.4% chrY - 6627792 6627916 125 browser details YourSeq 101 898 1013 2000 97.2% chr5 + 85018665 85018909 245 browser details YourSeq 99 817 1013 2000 85.6% chr6 + 136736419 136736583 165 browser details YourSeq 97 700 995 2000 83.2% chr4 - 80345424 80345563 140 browser details YourSeq 96 913 1018 2000 92.3% chr11 + 62724365 62724467 103 browser details YourSeq 94 913 1013 2000 98.0% chr9 - 48217646 48217831 186 browser details YourSeq 94 913 1013 2000 98.0% chr19 - 52835430 53096440 261011 browser details YourSeq 94 913 1013 2000 98.0% chr17 + 51968181 51968346 166 browser details YourSeq 94 699 1013 2000 82.2% chr16 + 49314953 49315047 95 browser details YourSeq 93 913 1013 2000 92.9% chr9 - 48217614 48217711 98 browser details YourSeq 93 913 1013 2000 92.9% chr9 - 44350828 44350925 98 browser details YourSeq 93 913 1013 2000 92.9% chr8 - 71960793 71960890 98

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Shc3 src homology 2 domain-containing transforming protein C3 [ Mus musculus (house mouse) ] Gene ID: 20418, updated on 24-Oct-2019

Gene summary

Official Symbol Shc3 provided by MGI Official Full Name src homology 2 domain-containing transforming protein C3 provided by MGI Primary source MGI:MGI:106179 See related Ensembl:ENSMUSG00000021448 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 Rai; ShcC; N-Shc Expression Biased expression in cortex adult (RPKM 15.6), frontal lobe adult (RPKM 13.9) and 6 other tissues See more Orthologs human all

Genomic context

Location: 13 A5; 13 26.12 cM See Shc3 in Genome Data Viewer Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (51423389..51569451, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (51526410..51662453, complement)

Chromosome 13 - NC_000079.6

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Transcript information: This gene has 4 transcripts

Gene: Shc3 ENSMUSG00000021448

Description src homology 2 domain-containing transforming protein C3 [Source:MGI Symbol;Acc:MGI:106179] Gene Synonyms N-Shc, Rai, ShcC Location Chromosome 13: 51,431,041-51,569,487 reverse strand. GRCm38:CM001006.2 About this gene This gene has 4 transcripts (splice variants), 200 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Shc3-201 ENSMUST00000021898.5 1537 474aa ENSMUSP00000021898.5 Protein coding CCDS49265 Q61120 TSL:1 GENCODE basic APPRIS P2

Shc3-204 ENSMUST00000239056.1 3940 594aa ENSMUSP00000158865.1 Protein coding - - GENCODE basic APPRIS ALT2

Shc3-203 ENSMUST00000223543.2 3644 461aa ENSMUSP00000152080.2 Protein coding - Q3ZAX3 TSL:1 GENCODE basic

Shc3-202 ENSMUST00000221850.1 3163 No protein - Retained intron - - TSL:NA

158.45 kb Forward strand 51.44Mb 51.46Mb 51.48Mb 51.50Mb 51.52Mb 51.54Mb 51.56Mb S1pr3-201 >protein coding (Comprehensive set...

Contigs < AC159104.2 Genes (Comprehensive set... < Shc3-201protein coding

< Shc3-203protein coding

< Shc3-204protein coding

< Shc3-202retained intron

Regulatory Build

51.44Mb 51.46Mb 51.48Mb 51.50Mb 51.52Mb 51.54Mb 51.56Mb Reverse strand 158.45 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000021898

< Shc3-201protein coding

Reverse strand 136.04 kb

ENSMUSP00000021... MobiDB lite Superfamily SSF50729 SH2 domain superfamily

SMART PTB/PI domain SH2 domain

Prints Phosphotyrosine interaction domain, Shc-like SH2 domain Pfam PTB/PI domain SH2 domain

PROSITE profiles PTB/PI domain SH2 domain

PANTHER PTHR10337

SHC-transforming protein 3 Gene3D PH-like domain superfamily SH2 domain superfamily

CDD Phosphotyrosine interaction domain, Shc-like SH2 adaptor protein C, SH2 domain

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 400 474

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

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