https://www.alphaknockout.com
Mouse Cnot2 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Cnot2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cnot2 gene (NCBI Reference Sequence: NM_001037846 ; Ensembl: ENSMUSG00000020166 ) is located on Mouse chromosome 10. 16 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 16 (Transcript: ENSMUST00000105267). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cnot2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-133D24 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 4 starts from about 10.62% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 10443 bp, and the size of intron 4 for 3'-loxP site insertion: 10128 bp. The size of effective cKO region: ~567 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 4 16 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Cnot2 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(7067bp) | A(28.03% 1981) | C(15.49% 1095) | T(37.53% 2652) | G(18.95% 1339)
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% chr10 - 116517634 116520633 3000 browser details YourSeq 31 243 276 3000 97.1% chr14 + 78609746 78609947 202 browser details YourSeq 30 247 281 3000 84.9% chr1 + 190183098 190183130 33 browser details YourSeq 29 243 281 3000 96.8% chr3 + 9171147 9171191 45 browser details YourSeq 20 295 328 3000 79.5% chr1 - 139682490 139682523 34 browser details YourSeq 20 635 660 3000 88.5% chr11 + 8266646 8266671 26
Note: The 3000 bp section upstream of Exon 4 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% chr10 - 116514067 116517066 3000 browser details YourSeq 75 1700 1774 3000 100.0% chr10 - 116513470 116513544 75 browser details YourSeq 32 285 330 3000 84.8% chr11 + 50100633 50100678 46 browser details YourSeq 29 1832 1865 3000 94.0% chr1 - 180883523 180883557 35 browser details YourSeq 27 285 324 3000 96.6% chr13 - 38332266 38332307 42 browser details YourSeq 25 1930 1966 3000 83.8% chr18 + 87590831 87590867 37 browser details YourSeq 24 345 374 3000 96.2% chr3 - 47082570 47082601 32 browser details YourSeq 20 285 306 3000 95.5% chr13 - 93269370 93269391 22 browser details YourSeq 20 1641 1660 3000 100.0% chr11 - 42648672 42648691 20
Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Cnot2 CCR4-NOT transcription complex, subunit 2 [ Mus musculus (house mouse) ] Gene ID: 72068, updated on 24-Oct-2019
Gene summary
Official Symbol Cnot2 provided by MGI Official Full Name CCR4-NOT transcription complex, subunit 2 provided by MGI Primary source MGI:MGI:1919318 See related Ensembl:ENSMUSG00000020166 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 C79650; AA537049; AA959607; AW557563; 2600016M12Rik; 2810470K03Rik Expression Ubiquitous expression in limb E14.5 (RPKM 10.3), CNS E11.5 (RPKM 10.3) and 27 other tissues See more Orthologs human all
Genomic context
Location: 10; 10 D2 See Cnot2 in Genome Data Viewer
Exon count: 19
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (116485160..116581900, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (115922217..116018567, complement)
Chromosome 10 - NC_000076.6
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Transcript information: This gene has 19 transcripts
Gene: Cnot2 ENSMUSG00000020166
Description CCR4-NOT transcription complex, subunit 2 [Source:MGI Symbol;Acc:MGI:1919318] Gene Synonyms 2600016M12Rik, 2810470K03Rik Location Chromosome 10: 116,485,161-116,581,511 reverse strand. GRCm38:CM001003.2 About this gene This gene has 19 transcripts (splice variants), 252 orthologues, 1 paralogue and is a member of 2 Ensembl protein families. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cnot2- ENSMUST00000105267.7 2800 540aa ENSMUSP00000100902.1 Protein coding CCDS36064 Q8C5L3 TSL:1 203 GENCODE basic APPRIS P1
Cnot2- ENSMUST00000168036.7 2693 499aa ENSMUSP00000132315.1 Protein coding CCDS24185 E9Q027 TSL:5 210 GENCODE basic
Cnot2- ENSMUST00000164088.7 2618 499aa ENSMUSP00000127830.1 Protein coding CCDS24185 E9Q027 TSL:1 204 GENCODE basic
Cnot2- ENSMUST00000169921.7 2120 540aa ENSMUSP00000132152.1 Protein coding CCDS36064 Q8C5L3 TSL:1 213 GENCODE basic APPRIS P1
Cnot2- ENSMUST00000020374.5 1059 109aa ENSMUSP00000020374.5 Protein coding CCDS36065 H7BWX6 TSL:1 201 GENCODE basic
Cnot2- ENSMUST00000105265.7 2268 455aa ENSMUSP00000100900.1 Protein coding - Q8C5L3 TSL:1 202 GENCODE basic
Cnot2- ENSMUST00000167706.7 1621 490aa ENSMUSP00000128837.1 Protein coding - E9Q8D5 TSL:5 209 GENCODE basic
Cnot2- ENSMUST00000218744.1 358 78aa ENSMUSP00000151501.1 Protein coding - A0A1W2P771 CDS 3' 218 incomplete TSL:3
Cnot2- ENSMUST00000169576.7 2955 48aa ENSMUSP00000130192.1 Nonsense mediated - E9Q7M5 TSL:1 212 decay
Cnot2- ENSMUST00000169507.7 810 57aa ENSMUSP00000128720.1 Nonsense mediated - E9Q8R6 TSL:5 211 decay
Cnot2- ENSMUST00000218490.1 777 57aa ENSMUSP00000151847.1 Nonsense mediated - E9Q8R6 TSL:3 217 decay
Cnot2- ENSMUST00000219544.1 611 No - Retained intron - - TSL:2 219 protein
Cnot2- ENSMUST00000165527.7 1044 No - lncRNA - - TSL:1 206 protein
Cnot2- ENSMUST00000169937.7 891 No - lncRNA - - TSL:5 214 protein
Cnot2- ENSMUST00000164383.7 823 No - lncRNA - - TSL:5 205 protein
Cnot2- ENSMUST00000171214.7 623 No - lncRNA - - TSL:5 215 protein
Cnot2- ENSMUST00000167644.1 592 No - lncRNA - - TSL:3 208 protein
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Cnot2- ENSMUST00000166166.7 442 No - lncRNA - - TSL:3 207 protein
Cnot2- ENSMUST00000171944.7 341 No - lncRNA - - TSL:3 216 protein
116.35 kb Forward strand 116.48Mb 116.50Mb 116.52Mb 116.54Mb 116.56Mb 116.58Mb Genes Kcnmb4os2-203 >lncRNA 5330438D12Rik-203 >lncRNA Gm49344-201 >TEC (Comprehensive set...
5330438D12Rik-205 >lncRNA
5330438D12Rik-201 >pseudogene
5330438D12Rik-202 >lncRNA
5330438D12Rik-204 >lncRNA
Contigs AC139376.3 >
Genes < Cnot2-210protein coding (Comprehensive set...
< Cnot2-212nonsense mediated decay
< Cnot2-204protein coding
< Cnot2-203protein coding
< Cnot2-202protein coding
< Cnot2-213protein coding
< Cnot2-209protein coding
< Cnot2-219retained intron < Cnot2-218protein coding
< Cnot2-214lncRNA
< Cnot2-215lncRNA
< Cnot2-205lncRNA
< Cnot2-207lncRNA
< Cnot2-211nonsense mediated decay
< Cnot2-217nonsense mediated decay
< Cnot2-216lncRNA
< Cnot2-201protein coding
< Cnot2-206lncRNA
< Cnot2-208lncRNA
< Gm25190-201miRNA
Regulatory Build
116.48Mb 116.50Mb 116.52Mb 116.54Mb 116.56Mb 116.58Mb Reverse strand 116.35 kb
Regulation Legend
CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Page 7 of 9
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
pseudogene processed transcript RNA gene 116.35 kb Forward strand 116.48Mb 116.50Mb 116.52Mb 116.54Mb 116.56Mb 116.58Mb Genes Kcnmb4os2-203 >lncRNA 5330438D12Rik-203 >lncRNA Gm49344-201 >TEC (Comprehensive set...
5330438D12Rik-205 >lncRNA
5330438D12Rik-201 >pseudogene
5330438D12Rik-202 >lncRNA
5330438D12Rik-204 >lncRNA
Contigs AC139376.3 > Genes (Comprehensive set... < Cnot2-210protein coding
< Cnot2-212nonsense mediated decay
< Cnot2-204protein coding
< Cnot2-203protein coding
< Cnot2-202protein coding
< Cnot2-213protein coding
< Cnot2-209protein coding
< Cnot2-219retained intron < Cnot2-218protein coding
< Cnot2-214lncRNA
< Cnot2-215lncRNA
< Cnot2-205lncRNA
< Cnot2-207lncRNA
< Cnot2-211nonsense mediated decay
< Cnot2-217nonsense mediated decay
< Cnot2-216lncRNA
< Cnot2-201protein coding
< Cnot2-206lncRNA
< Cnot2-208lncRNA
< Gm25190-201miRNA
Regulatory Build
116.48Mb 116.50Mb 116.52Mb 116.54Mb 116.56Mb 116.58Mb Reverse strand 116.35 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter https://www.alphaknockout.com Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
pseudogene processed transcript RNA gene
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Transcript: ENSMUST00000105267
< Cnot2-203protein coding
Reverse strand 96.33 kb
ENSMUSP00000100... MobiDB lite Low complexity (Seg) Pfam NOT2/NOT3/NOT5, C-terminal PANTHER Not2/Not3/Not5
PTHR23326:SF3 Gene3D CCR4-NOT complex subunit 2/3/5, N-terminal domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 480 540
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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