https://www.alphaknockout.com

Mouse Calml3 Knockout Project (CRISPR/Cas9)

Objective: To create a Calml3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Calml3 (NCBI Reference Sequence: NM_027416 ; Ensembl: ENSMUSG00000063130 ) is located on Mouse 13. 1 exon is identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 1 (Transcript: ENSMUST00000077698). Exon 1 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 0.22% of the coding region. Exon 1 covers 100.0% of the coding region. The size of effective KO region: ~445 bp. The KO region does not have any other known gene.

Page 1 of 8 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1

Legends Exon of mouse Calml3 Knockout region

Page 2 of 8 https://www.alphaknockout.com

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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

Page 3 of 8 https://www.alphaknockout.com

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

Sequence 12

Summary: Full Length(2000bp) | A(26.15% 523) | C(26.25% 525) | T(26.95% 539) | G(20.65% 413)

Note: The 2000 bp section upstream of start 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(22.55% 451) | C(22.85% 457) | T(31.6% 632) | G(23.0% 460)

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.

Page 4 of 8 https://www.alphaknockout.com

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 - 3804204 3806203 2000 browser details YourSeq 35 1553 1591 2000 97.4% chr16 + 23035910 23035953 44 browser details YourSeq 35 1552 1594 2000 90.0% chr15 + 66502290 66502331 42 browser details YourSeq 34 1552 1594 2000 88.9% chr14 - 11183083 11183123 41 browser details YourSeq 33 1552 1592 2000 84.3% chr6 + 30878141 30878179 39 browser details YourSeq 33 1551 1591 2000 94.6% chr11 + 113116054 113116103 50 browser details YourSeq 31 1558 1594 2000 81.3% chr2 + 26111496 26111527 32 browser details YourSeq 31 1530 1576 2000 75.8% chr12 + 100874841 100874878 38 browser details YourSeq 30 1557 1587 2000 100.0% chr16 - 33024438 33024469 32 browser details YourSeq 30 1551 1580 2000 100.0% chr10 - 44872050 44872079 30 browser details YourSeq 30 1551 1594 2000 75.0% chr2 + 127047328 127047362 35 browser details YourSeq 29 1553 1581 2000 100.0% chr11 - 67575133 67575161 29 browser details YourSeq 29 1553 1582 2000 100.0% chr1 - 27231966 27231997 32 browser details YourSeq 29 1553 1581 2000 100.0% chr11 + 50004285 50004313 29 browser details YourSeq 28 1553 1581 2000 100.0% chr13 - 113944546 113944577 32 browser details YourSeq 28 1553 1580 2000 100.0% chr11 - 80319009 80319036 28 browser details YourSeq 28 1553 1580 2000 100.0% chr1 - 89283020 89283047 28 browser details YourSeq 28 1553 1581 2000 100.0% chr19 + 49506955 49506986 32 browser details YourSeq 28 1554 1582 2000 100.0% chr15 + 18440866 18440897 32 browser details YourSeq 28 1553 1580 2000 100.0% chr11 + 113798555 113798582 28

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% chr13 - 3801757 3803756 2000 browser details YourSeq 63 1399 1493 2000 88.9% chr16 + 48592792 48592889 98 browser details YourSeq 59 972 1493 2000 66.7% chr11 + 35719455 35719771 317 browser details YourSeq 57 1420 1534 2000 86.1% chr7 + 100564257 100564589 333 browser details YourSeq 56 1445 1592 2000 87.9% chr3 - 95599503 95600030 528 browser details YourSeq 55 1409 1493 2000 88.9% chr7 - 122062042 122062580 539 browser details YourSeq 55 955 1493 2000 71.1% chr1 + 18385085 18385582 498 browser details YourSeq 54 951 1493 2000 63.2% chr10 + 12929438 12929676 239 browser details YourSeq 52 1399 1493 2000 80.3% chr11 - 119097976 119098068 93 browser details YourSeq 51 1400 1493 2000 71.6% chr1 - 63161494 63161581 88 browser details YourSeq 47 1445 1539 2000 86.2% chr11 - 96090538 96091004 467 browser details YourSeq 47 1406 1493 2000 85.1% chr18 + 4614600 4614688 89 browser details YourSeq 46 1424 1495 2000 83.9% chr9 - 72853125 72853203 79 browser details YourSeq 46 1406 1493 2000 83.9% chr9 - 32620961 32621048 88 browser details YourSeq 46 1422 1493 2000 82.0% chr10 - 81163174 81163245 72 browser details YourSeq 46 948 1493 2000 63.0% chrX + 149047874 149048233 360 browser details YourSeq 45 1416 1492 2000 79.3% chr18 - 36482542 36482618 77 browser details YourSeq 45 971 1493 2000 57.0% chr10 + 56330674 56330760 87 browser details YourSeq 44 1446 1528 2000 82.7% chrX - 56344029 56344263 235 browser details YourSeq 43 1420 1493 2000 85.3% chr12 - 8763867 8763952 86

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

Page 5 of 8 https://www.alphaknockout.com

Gene and information: Calml3 -like 3 [ Mus musculus (house mouse) ] Gene ID: 70405, updated on 12-Aug-2019

Gene summary

Official Symbol Calml3 provided by MGI Official Full Name calmodulin-like 3 provided by MGI Primary source MGI:MGI:1917655 See related Ensembl:ENSMUSG00000063130 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 AI326174; 2310068O22Rik Orthologs human all

Genomic context

Location: 13; 13 A1 See Calml3 in Genome Data Viewer

Exon count: 1

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 13 NC_000079.6 (3802893..3804318, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 13 NC_000079.5 (3802139..3803564, complement)

Chromosome 13 - NC_000079.6

Page 6 of 8 https://www.alphaknockout.com

Transcript information: This gene has 2 transcripts

Gene: Calml3 ENSMUSG00000063130

Description calmodulin-like 3 [Source:MGI Symbol;Acc:MGI:1917655] Gene Synonyms 2310068O22Rik Location Chromosome 13: 3,802,896-3,804,316 reverse strand. GRCm38:CM001006.2 About this gene This gene has 2 transcripts (splice variants), 204 orthologues, 22 paralogues and is a member of 2 Ensembl protein families. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Calml3-201 ENSMUST00000077698.4 1421 149aa ENSMUSP00000076880.3 Protein coding CCDS26214 Q9D6P8 TSL:NA GENCODE basic APPRIS P1

Calml3-202 ENSMUST00000222976.1 509 115aa ENSMUSP00000152735.1 Protein coding - A0A1Y7VK66 CDS 5' incomplete TSL:5

21.42 kb Forward strand 3.795Mb 3.800Mb 3.805Mb 3.810Mb Contigs < AC132239.4

Genes (Comprehensive set... < Calml3-201protein coding

< Calml3-202protein coding

Regulatory Build

3.795Mb 3.800Mb 3.805Mb 3.810Mb Reverse strand 21.42 kb

Regulation Legend

Enhancer Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000077698

< Calml3-201protein coding

Reverse strand 1.42 kb

ENSMUSP00000076... Superfamily EF-hand domain pair SMART EF-hand domain Pfam EF-hand domain PROSITE profiles EF-hand domain PROSITE patterns EF-Hand 1, calcium-binding site PANTHER PTHR23050

Calmodulin Gene3D 1.10.238.10 CDD EF-hand domain

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 149

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

Page 8 of 8