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Mouse Calml3 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Calml3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Calml3 gene (NCBI Reference Sequence: NM_027416.3 ; Ensembl: ENSMUSG00000063130 ) is located on Mouse chromosome 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 conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Calml3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-175A12 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 1 covers 100.0% of the coding region. Start codon is in exon 1, and stop codon is in exon 1. The size of effective cKO region: ~1818 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
gRNA region
Wildtype allele A T
5' G gRNA region 3'
1
Targeting vector A T G
Targeted allele A T G
Constitutive KO allele (After Cre recombination)
Legends Homology arm Exon of mouse Calml3 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(6447bp) | A(26.17% 1687) | C(23.5% 1515) | T(27.8% 1792) | G(22.54% 1453)
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% chr13 - 3804204 3807203 3000 browser details YourSeq 89 682 780 3000 97.0% chr17 - 6075615 6075778 164 browser details YourSeq 80 681 864 3000 80.7% chr10 + 14447033 14447179 147 browser details YourSeq 74 682 780 3000 94.0% chr9 - 63476090 63476251 162 browser details YourSeq 74 683 778 3000 91.6% chr2 - 47107380 47107474 95 browser details YourSeq 68 700 778 3000 96.2% chr8 - 107398231 107398809 579 browser details YourSeq 67 682 777 3000 96.1% chr12 + 116708087 116708325 239 browser details YourSeq 66 685 779 3000 93.5% chr10 + 127242994 127243225 232 browser details YourSeq 62 727 843 3000 95.6% chr11 + 83280246 83280475 230 browser details YourSeq 60 685 778 3000 81.3% chr5 - 144424607 144424677 71 browser details YourSeq 53 221 358 3000 84.7% chr1 + 74166276 74166433 158 browser details YourSeq 52 709 776 3000 93.6% chr10 - 125750915 125751111 197 browser details YourSeq 51 247 399 3000 89.1% chr11 - 53302470 53302800 331 browser details YourSeq 51 363 583 3000 73.2% chr10 + 7803395 7803586 192 browser details YourSeq 50 567 780 3000 94.8% chr3 - 143169000 143169307 308 browser details YourSeq 50 727 778 3000 98.1% chr6 + 31966820 31966871 52 browser details YourSeq 49 229 521 3000 93.0% chr14 + 54414736 54415184 449 browser details YourSeq 49 680 769 3000 72.8% chr12 + 37795051 37795107 57 browser details YourSeq 47 363 533 3000 66.2% chr14 + 79542251 79542357 107 browser details YourSeq 46 728 778 3000 89.8% chr10 + 83026980 83027028 49
Note: The 3000 bp section upstream of Exon 1 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% chr13 - 3800757 3803756 3000 browser details YourSeq 63 1399 1493 3000 88.9% chr16 + 48592792 48592889 98 browser details YourSeq 59 972 1493 3000 66.7% chr11 + 35719455 35719771 317 browser details YourSeq 57 1420 1534 3000 86.1% chr7 + 100564257 100564589 333 browser details YourSeq 56 1445 1592 3000 87.9% chr3 - 95599503 95600030 528 browser details YourSeq 55 1409 1493 3000 88.9% chr7 - 122062042 122062580 539 browser details YourSeq 55 955 1493 3000 71.1% chr1 + 18385085 18385582 498 browser details YourSeq 54 951 1493 3000 63.2% chr10 + 12929438 12929676 239 browser details YourSeq 52 1399 1493 3000 80.3% chr11 - 119097976 119098068 93 browser details YourSeq 51 1400 1493 3000 71.6% chr1 - 63161494 63161581 88 browser details YourSeq 47 1445 1539 3000 86.2% chr11 - 96090538 96091004 467 browser details YourSeq 47 1406 1493 3000 85.1% chr18 + 4614600 4614688 89 browser details YourSeq 46 1424 1495 3000 83.9% chr9 - 72853125 72853203 79 browser details YourSeq 46 1406 1493 3000 83.9% chr9 - 32620961 32621048 88 browser details YourSeq 46 1422 1493 3000 82.0% chr10 - 81163174 81163245 72 browser details YourSeq 46 948 1493 3000 63.0% chrX + 149047874 149048233 360 browser details YourSeq 45 1416 1492 3000 79.3% chr18 - 36482542 36482618 77 browser details YourSeq 45 1590 1689 3000 79.5% chr1 - 136760681 136760778 98 browser details YourSeq 45 971 1493 3000 57.0% chr10 + 56330674 56330760 87 browser details YourSeq 44 1446 1528 3000 82.7% chrX - 56344029 56344263 235
Note: The 3000 bp section downstream of Exon 1 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Calml3 calmodulin-like 3 [ Mus musculus (house mouse) ] Gene ID: 70405, updated on 26-Jun-2020
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
108.20200622 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
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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), 227 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
merged Ensembl/Havana Ensembl protein coding
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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 Calmodulin
PTHR23050 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.
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