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Mouse Erc2 Knockout Project (CRISPR/Cas9)

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Mouse Erc2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Erc2 Knockout Project (CRISPR/Cas9) Objective: To create a Erc2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Erc2 gene (NCBI Reference Sequence: NM_177814 ; Ensembl: ENSMUSG00000040640 ) is located on Mouse chromosome 14. 16 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000090302). Exon 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: Mice homozygous for targeted disruptions of this gene are viable and fertile. However, homozygotes for one allele display abnormal CNS synaptic transmission. Homozygotes for a second allele display retinal abnormalities and impaired vision. Exon 2 starts from the coding region. Exon 2 covers 21.86% of the coding region. The size of effective KO region: ~797 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 16 Legends Exon of mouse Erc2 Knockout region Page 2 of 9 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 Exon 2 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 2 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(27.85% 557) | C(18.2% 364) | T(31.4% 628) | G(22.55% 451) Note: The 2000 bp section upstream of Exon 2 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(23.45% 469) | C(23.9% 478) | T(32.9% 658) | G(19.75% 395) Note: The 2000 bp section downstream of Exon 2 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 9 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% chr14 + 27650828 27652827 2000 browser details YourSeq 36 1541 1651 2000 65.9% chr5 - 98976976 98977026 51 browser details YourSeq 35 1610 1742 2000 80.0% chr1 - 93503776 93503904 129 browser details YourSeq 30 47 94 2000 81.3% chr4 - 8172523 8172570 48 browser details YourSeq 30 55 92 2000 96.9% chr10 + 129760237 129760281 45 browser details YourSeq 28 53 85 2000 96.8% chr12 + 69950349 69950381 33 browser details YourSeq 27 1723 1749 2000 100.0% chr7 - 99006485 99006511 27 browser details YourSeq 26 60 91 2000 96.6% chr10 + 60957204 60957236 33 browser details YourSeq 25 1725 1749 2000 100.0% chr2 - 92461441 92461465 25 browser details YourSeq 25 1722 1746 2000 100.0% chr11 - 6152042 6152066 25 browser details YourSeq 24 1722 1747 2000 96.2% chr8 - 4307686 4307711 26 browser details YourSeq 24 1454 1477 2000 100.0% chr3 - 108976449 108976472 24 browser details YourSeq 24 1722 1747 2000 96.2% chr16 - 29040836 29040861 26 browser details YourSeq 24 1450 1473 2000 100.0% chr12 - 112356389 112356412 24 browser details YourSeq 24 51 74 2000 100.0% chr10 + 123898078 123898101 24 browser details YourSeq 23 1540 1562 2000 100.0% chr1 - 172394681 172394703 23 browser details YourSeq 23 663 689 2000 88.0% chr5 + 40798956 40798981 26 browser details YourSeq 23 1455 1477 2000 100.0% chr10 + 118621281 118621303 23 browser details YourSeq 22 1711 1732 2000 100.0% chr17 - 46175931 46175952 22 browser details YourSeq 22 1066 1087 2000 100.0% chr14 - 116813741 116813762 22 Note: The 2000 bp section upstream of Exon 2 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% chr14 + 27653485 27655484 2000 browser details YourSeq 45 1714 1760 2000 97.9% chr12 - 93551518 93551564 47 browser details YourSeq 29 1564 1609 2000 74.2% chr15 + 49335568 49335604 37 browser details YourSeq 24 605 643 2000 82.1% chr15 - 80003876 80003915 40 browser details YourSeq 24 268 298 2000 84.7% chr17 + 62503784 62503812 29 browser details YourSeq 21 1963 1983 2000 100.0% chr13 - 101540361 101540381 21 browser details YourSeq 21 929 949 2000 100.0% chr16 + 96561781 96561801 21 browser details YourSeq 20 1189 1214 2000 88.5% chr9 - 103687946 103687971 26 browser details YourSeq 20 1686 1705 2000 100.0% chr1 - 24281467 24281486 20 Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Erc2 ELKS/RAB6-interacting/CAST family member 2 [ Mus musculus (house mouse) ] Gene ID: 238988, updated on 12-Aug-2019 Gene summary Official Symbol Erc2 provided by MGI Official Full Name ELKS/RAB6-interacting/CAST family member 2 provided by MGI Primary source MGI:MGI:1098749 See related Ensembl:ENSMUSG00000040640 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 CAST; 6430531D06; CAST1/ERC2; ELKS2alpha; D14Ertd171e Expression Biased expression in CNS E18 (RPKM 6.8), frontal lobe adult (RPKM 6.0) and 6 other tissues See more Orthologs human all Genomic context Location: 14 A3; 14 16.51 cM See Erc2 in Genome Data Viewer Exon count: 29 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (27622275..28478537) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (28435628..29291723) Chromosome 14 - NC_000080.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 14 transcripts Gene: Erc2 ENSMUSG00000040640 Description ELKS/RAB6-interacting/CAST family member 2 [Source:MGI Symbol;Acc:MGI:1098749] Gene Synonyms CAST, D14Ertd171e, ELKS2alpha Location Chromosome 14: 27,622,428-28,478,537 forward strand. GRCm38:CM001007.2 About this gene This gene has 14 transcripts (splice variants), 202 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 16 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Erc2-205 ENSMUST00000210135.1 6233 977aa ENSMUSP00000147981.1 Protein coding CCDS84102 Q3UHT7 TSL:1 GENCODE basic APPRIS P2 Erc2-201 ENSMUST00000090302.5 6083 1002aa ENSMUSP00000087773.5 Protein coding CCDS26887 Q6PH08 TSL:1 GENCODE basic Erc2-208 ENSMUST00000210924.1 4871 623aa ENSMUSP00000147744.1 Protein coding - Q6PH08 TSL:1 GENCODE basic Erc2-210 ENSMUST00000211145.1 2874 957aa ENSMUSP00000147886.1 Protein coding - Q5J8K6 Q6PH08 TSL:1 GENCODE basic APPRIS ALT2 Erc2-209 ENSMUST00000211087.1 1356 347aa ENSMUSP00000147814.1 Protein coding - A0A1B0GS69 CDS 3' incomplete TSL:5 Erc2-213 ENSMUST00000211684.1 1074 51aa ENSMUSP00000148033.1 Protein coding - A0A1B0GSQ8 TSL:5 GENCODE basic Erc2-206 ENSMUST00000210327.1 1099 251aa ENSMUSP00000148076.1 Non stop decay - A0A1B0GSU7 TSL:5 Erc2-204 ENSMUST00000209800.1 3361 No protein - Retained intron - - TSL:1 Erc2-203 ENSMUST00000209752.1 3339 No protein - Retained intron - - TSL:1 Erc2-207 ENSMUST00000210524.1 638 No protein - Retained intron - - TSL:3 Erc2-211 ENSMUST00000211627.1 1216 No protein - lncRNA - - TSL:1 Erc2-202 ENSMUST00000209476.1 1098 No protein - lncRNA - - TSL:1 Erc2-212 ENSMUST00000211636.1 486 No protein - lncRNA - - TSL:5 Erc2-214 ENSMUST00000224045.1 429 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 876.11 kb Forward strand 27.8Mb 28.0Mb 28.2Mb 28.4Mb Genes (Comprehensive set... Erc2-205 >protein coding Erc2-201 >protein coding Erc2-209 >protein coding Erc2-212 >lncRNA Erc2-204 >retained intron Erc2-203 >retained intron Gm20242-201 >TEC Erc2-206 >non stop decay Erc2-202 >lncRNA Erc2-214 >lncRNA Erc2-210 >protein coding Gm18398-201 >processed pseudogene Erc2-211 >lncRNA Erc2-213 >protein coding Erc2-208 >protein coding Erc2-207 >retained intron Contigs < AC154729.2 < AC138300.8 < CT010523.15 < CT025681.8 Genes < Gm45645-201lncRNA (Comprehensive set... Regulatory Build 27.8Mb 28.0Mb 28.2Mb 28.4Mb Reverse strand 876.11 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000090302 856.10 kb Forward strand Erc2-201 >protein coding ENSMUSP00000087... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57997 Pfam Active zone protein ELKS PANTHER ERC protein 2 PTHR18861 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant splice region variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1002 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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