https://www.alphaknockout.com

Mouse Ckap4 Knockout Project (CRISPR/Cas9)

Objective: To create a Ckap4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ckap4 (NCBI Reference Sequence: NM_175451 ; Ensembl: ENSMUSG00000046841 ) is located on Mouse 10. 2 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 2 (Transcript: ENSMUST00000053871). Exon 1~2 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:

Exon 1 starts from about 0.06% of the coding region. Exon 1~2 covers 100.0% of the coding region. The size of effective KO region: ~6392 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

Legends Exon of mouse Ckap4 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 start 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.

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(20.15% 403) | C(30.4% 608) | T(23.35% 467) | G(26.1% 522)

Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(25.0% 500) | C(22.35% 447) | T(29.55% 591) | G(23.1% 462)

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% chr10 - 84533867 84535866 2000 browser details YourSeq 31 409 442 2000 97.0% chr12 - 100181484 100181517 34 browser details YourSeq 25 397 425 2000 88.9% chr19 + 54728366 54728393 28 browser details YourSeq 21 1084 1104 2000 100.0% chr9 - 106182446 106182466 21 browser details YourSeq 21 1498 1518 2000 100.0% chr6 + 124947851 124947871 21 browser details YourSeq 20 1502 1523 2000 95.5% chr1 - 36274281 36274302 22

Note: The 2000 bp section upstream of start codon 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% chr10 - 84525473 84527472 2000 browser details YourSeq 108 1313 1446 2000 90.3% chr4 - 100973110 100973243 134 browser details YourSeq 105 1315 1439 2000 93.4% chr10 + 58795226 58795353 128 browser details YourSeq 70 1903 2000 2000 86.8% chr9 - 37274364 37274464 101 browser details YourSeq 69 1903 2000 2000 85.8% chr8 + 72452339 72452438 100 browser details YourSeq 66 1903 1993 2000 86.9% chr17 + 75090373 75090465 93 browser details YourSeq 62 1903 1993 2000 94.4% chr5 + 101294526 101294618 93 browser details YourSeq 61 1907 2000 2000 83.0% chr14 - 47640129 47640228 100 browser details YourSeq 61 1373 1437 2000 98.5% chr10 + 26758332 26758400 69 browser details YourSeq 60 1903 2000 2000 90.0% chr13 - 67044532 67044628 97 browser details YourSeq 60 1903 1989 2000 85.1% chr10 + 53103007 53103095 89 browser details YourSeq 60 1904 2000 2000 84.1% chr1 + 117517207 117517305 99 browser details YourSeq 59 1903 2000 2000 92.8% chr11 - 97593699 97593797 99 browser details YourSeq 59 1902 2000 2000 91.8% chr16 + 12895743 12895845 103 browser details YourSeq 58 1903 2000 2000 89.2% chr16 + 44341439 44341537 99 browser details YourSeq 57 1903 1990 2000 92.6% chr5 + 89407401 89407488 88 browser details YourSeq 57 1903 2000 2000 79.6% chr11 + 89203370 89203472 103 browser details YourSeq 55 1911 2000 2000 81.2% chr19 - 14920985 14921076 92 browser details YourSeq 55 1902 2000 2000 93.7% chr7 + 125440747 125440845 99 browser details YourSeq 55 1903 1984 2000 84.2% chr5 + 121052426 121052509 84

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: Ckap4 cytoskeleton-associated protein 4 [ Mus musculus (house mouse) ] Gene ID: 216197, updated on 24-Sep-2019

Gene summary

Official Symbol Ckap4 provided by MGI Official Full Name cytoskeleton-associated protein 4 provided by MGI Primary source MGI:MGI:2444926 See related Ensembl:ENSMUSG00000046841 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 P63; CLIMP-63; 5630400A09Rik Expression Broad expression in ovary adult (RPKM 52.9), limb E14.5 (RPKM 52.2) and 27 other tissues See more Orthologs human all

Genomic context

Location: 10; 10 C1 See Ckap4 in Genome Data Viewer Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (84526305..84533888, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (83989050..83996633, complement)

Chromosome 10 - NC_000076.6

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Transcript information: This gene has 2 transcripts

Gene: Ckap4 ENSMUSG00000046841

Description cytoskeleton-associated protein 4 [Source:MGI Symbol;Acc:MGI:2444926] Gene Synonyms 5630400A09Rik, CLIMP-63, P63 Location Chromosome 10: 84,526,305-84,534,062 reverse strand. GRCm38:CM001003.2 About this gene This gene has 2 transcripts (splice variants), 186 orthologues, 7 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

Ckap4-201 ENSMUST00000053871.4 3089 575aa ENSMUSP00000050336.3 Protein coding CCDS24080 Q8BMK4 TSL:1 GENCODE basic APPRIS P1

Ckap4-202 ENSMUST00000167671.1 2606 575aa ENSMUSP00000130304.1 Protein coding CCDS24080 Q8BMK4 TSL:1 GENCODE basic APPRIS P1

27.76 kb Forward strand

84.52Mb 84.53Mb 84.54Mb E230014E18Rik-201 >lncRNA (Comprehensive set...

Contigs AC150314.5 > < AC140333.2 Genes (Comprehensive set... < Ckap4-201protein coding

< Ckap4-202protein coding

Regulatory Build

84.52Mb 84.53Mb 84.54Mb Reverse strand 27.76 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000053871

< Ckap4-201protein coding

Reverse strand 7.76 kb

ENSMUSP00000050... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57997

PANTHER Cytoskeleton-associated protein 4

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

Variant Legend

missense variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 575

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

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