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Mouse Rps6kc1 Knockout Project (CRISPR/Cas9)

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Mouse Rps6kc1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Rps6kc1 Knockout Project (CRISPR/Cas9) Objective: To create a Rps6kc1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rps6kc1 gene (NCBI Reference Sequence: NM_178775 ; Ensembl: ENSMUSG00000089872 ) is located on Mouse chromosome 1. 15 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 15 (Transcript: ENSMUST00000061611). Exon 2~3 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 2 starts from about 3.35% of the coding region. Exon 2~3 covers 4.96% of the coding region. The size of effective KO region: ~5668 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 3 15 Legends Exon of mouse Rps6kc1 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 3 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(22.2% 444) | C(20.75% 415) | T(31.75% 635) | G(25.3% 506) 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(21.05% 421) | C(29.85% 597) | T(29.8% 596) | G(19.3% 386) Note: The 2000 bp section downstream of Exon 3 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% chr1 - 190905119 190907118 2000 browser details YourSeq 166 316 780 2000 84.7% chr11 + 22965727 22966177 451 browser details YourSeq 157 456 644 2000 92.5% chr11 + 20668378 20668568 191 browser details YourSeq 155 472 651 2000 93.9% chr14 - 31449579 31449766 188 browser details YourSeq 155 465 651 2000 92.4% chr1 - 30841301 30841489 189 browser details YourSeq 155 465 652 2000 91.5% chr11 + 4934077 4934265 189 browser details YourSeq 154 460 651 2000 91.4% chr3 + 97926093 97926296 204 browser details YourSeq 153 465 643 2000 93.9% chr18 - 84621183 84621556 374 browser details YourSeq 152 402 644 2000 92.8% chr4 - 132161483 132161906 424 browser details YourSeq 152 460 651 2000 90.0% chr13 + 54476713 54476917 205 browser details YourSeq 151 463 652 2000 90.5% chr6 - 136730605 136730801 197 browser details YourSeq 151 460 650 2000 90.0% chr8 + 114334832 114335024 193 browser details YourSeq 150 459 652 2000 90.0% chr11 - 86746545 86746751 207 browser details YourSeq 148 470 652 2000 91.2% chr6 - 38325872 38326060 189 browser details YourSeq 148 472 651 2000 91.6% chr8 + 106156533 106156721 189 browser details YourSeq 148 469 644 2000 92.7% chr2 + 155323131 155323313 183 browser details YourSeq 148 465 651 2000 90.8% chr2 + 93575482 93575676 195 browser details YourSeq 147 465 651 2000 90.8% chrX - 41968968 41969167 200 browser details YourSeq 147 471 651 2000 91.1% chr2 - 69623966 69624150 185 browser details YourSeq 147 468 651 2000 91.1% chr7 + 79991274 79991470 197 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% chr1 - 190897451 190899450 2000 browser details YourSeq 78 1441 1583 2000 89.8% chr16 + 10020637 10020813 177 browser details YourSeq 75 1442 1561 2000 93.2% chr10 + 44395310 44395449 140 browser details YourSeq 67 1444 1583 2000 79.5% chr15 - 9278489 9278586 98 browser details YourSeq 66 1429 1531 2000 90.4% chr17 + 69024618 69024721 104 browser details YourSeq 64 1441 1525 2000 95.8% chr1 - 126498228 126498326 99 browser details YourSeq 64 1441 1522 2000 90.2% chr3 + 85437633 85437731 99 browser details YourSeq 58 1443 1523 2000 94.1% chr16 - 93680535 93680617 83 browser details YourSeq 57 1474 1606 2000 79.2% chr16 - 76003180 76003290 111 browser details YourSeq 55 1441 1515 2000 84.6% chr1 - 189961250 189961322 73 browser details YourSeq 54 1454 1519 2000 92.4% chr2 - 166301995 166302068 74 browser details YourSeq 53 1441 1516 2000 85.3% chr14 + 22603055 22603125 71 browser details YourSeq 52 1464 1523 2000 91.1% chrX - 136986048 136986105 58 browser details YourSeq 52 1441 1499 2000 94.9% chr14 + 103631164 103631226 63 browser details YourSeq 51 1463 1531 2000 94.8% chr5 + 133065354 133065428 75 browser details YourSeq 48 1454 1529 2000 81.9% chr11 + 114513777 114513842 66 browser details YourSeq 48 1452 1519 2000 87.5% chr1 + 11060661 11060726 66 browser details YourSeq 47 1429 1502 2000 71.7% chr2 - 171440572 171440624 53 browser details YourSeq 47 1461 1529 2000 94.9% chr12 - 12524563 12524631 69 browser details YourSeq 47 1460 1579 2000 87.1% chr10 + 23902883 23903000 118 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Rps6kc1 ribosomal protein S6 kinase polypeptide 1 [ Mus musculus (house mouse) ] Gene ID: 320119, updated on 12-Aug-2019 Gene summary Official Symbol Rps6kc1 provided by MGI Official Full Name ribosomal protein S6 kinase polypeptide 1 provided by MGI Primary source MGI:MGI:2443419 See related Ensembl:ENSMUSG00000089872 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 C80612; Rpk118; AA682037; B130003F20Rik Expression Ubiquitous expression in cortex adult (RPKM 3.4), CNS E14 (RPKM 3.1) and 28 other tissues See more Orthologs human all Genomic context Location: 1; 1 H6 See Rps6kc1 in Genome Data Viewer Exon count: 16 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (190772879..190913010, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (192596758..192735649, complement) Chromosome 1 - NC_000067.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Rps6kc1 ENSMUSG00000089872 Description ribosomal protein S6 kinase polypeptide 1 [Source:MGI Symbol;Acc:MGI:2443419] Gene Synonyms B130003F20Rik, RPK118 Location Chromosome 1: 190,700,202-190,911,770 reverse strand. GRCm38:CM000994.2 About this gene This gene has 9 transcripts (splice variants), 208 orthologues, 2 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 Rps6kc1- ENSMUST00000061611.14 3995 1056aa ENSMUSP00000061769.8 Protein coding - E9QMX4 TSL:5 201 GENCODE basic APPRIS P1 Rps6kc1- ENSMUST00000159066.1 503 131aa ENSMUSP00000124558.1 Protein coding - E0CY13 CDS 3' 202 incomplete TSL:3 Rps6kc1- ENSMUST00000159367.7 4103 60aa ENSMUSP00000124383.1 Nonsense mediated - E0CYA1 TSL:5 203 decay Rps6kc1- ENSMUST00000159624.7 3276 336aa ENSMUSP00000125010.1 Nonsense mediated - Q8BLK9 TSL:2 204 decay Rps6kc1- ENSMUST00000160889.7 2694 22aa ENSMUSP00000123733.1 Nonsense mediated - F6VA16 CDS 5' 206 decay incomplete TSL:1 Rps6kc1- ENSMUST00000159823.7 4792 No - Retained intron - - TSL:2 205 protein Rps6kc1- ENSMUST00000162500.7 2913 No - Retained intron - - TSL:1 208 protein Rps6kc1- ENSMUST00000160891.1 518 No - Retained intron - - TSL:3 207 protein Rps6kc1- ENSMUST00000162692.1 406 No - lncRNA - - TSL:3 209 protein Page 7 of 9 https://www.alphaknockout.com 231.57 kb Forward strand 190.7Mb 190.8Mb 190.9Mb Contigs AC137146.9 > AC112675.12 > Genes (Comprehensive set... < Rps6kc1-204nonsense mediated decay < Rps6kc1-201protein coding < Rps6kc1-203nonsense mediated decay < Rps6kc1-206nonsense mediated decay < Rps6kc1-209lncRNA < Rps6kc1-205retained intron < Rps6kc1-202protein coding < Rps6kc1-208retained intron < Rps6kc1-207retained intron Regulatory Build 190.7Mb 190.8Mb 190.9Mb Reverse strand 231.57 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 RNA gene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000061611 < Rps6kc1-201protein coding Reverse strand 138.89 kb ENSMUSP00000061... MobiDB lite Low complexity (Seg) Superfamily PX domain superfamily Protein kinase-like domain superfamily MIT domain superfamily SMART Phox homologous domain MIT Protein kinase domain Pfam Phox homologous domain MIT Protein kinase domain PROSITE profiles Phox homologous domain Protein kinase domain PANTHER PTHR15508:SF2 PTHR15508 Gene3D PX domain superfamily 1.20.58.280 1.10.510.10 CDD cd02677 RPK118-like, kinase domain Ribosomal protein S6 kinase delta-1, PX domain All sequence SNPs/i..
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