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Mouse Nfkbib Knockout Project (CRISPR/Cas9)

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Mouse Nfkbib Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Nfkbib Knockout Project (CRISPR/Cas9) Objective: To create a Nfkbib knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Nfkbib gene (NCBI Reference Sequence: NM_010908 ; Ensembl: ENSMUSG00000030595 ) is located on Mouse chromosome 7. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000032815). 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: Mice homozygous for one knock-out allele exhibit decreased susceptibility to endotoxin shock and induced arthritis. Exon 2 starts from about 16.71% of the coding region. Exon 2~3 covers 40.85% of the coding region. The size of effective KO region: ~530 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 7 Legends Exon of mouse Nfkbib 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1336 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(21.6% 432) | C(23.25% 465) | T(30.25% 605) | G(24.9% 498) 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(1336bp) | A(23.95% 320) | C(27.02% 361) | T(25.22% 337) | G(23.8% 318) Note: The 1336 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% chr7 - 28762132 28764131 2000 browser details YourSeq 163 38 280 2000 97.7% chr11 - 53330154 53708066 377913 browser details YourSeq 163 43 276 2000 94.6% chr1 - 89011948 89177826 165879 browser details YourSeq 150 107 280 2000 93.7% chrX - 71511145 71511547 403 browser details YourSeq 142 132 288 2000 93.6% chr13 - 67609865 67610019 155 browser details YourSeq 142 132 286 2000 96.2% chr5 + 122507240 122507397 158 browser details YourSeq 141 113 280 2000 93.9% chr11 + 88328316 88328734 419 browser details YourSeq 141 132 299 2000 93.3% chr1 + 72818255 72818422 168 browser details YourSeq 137 132 280 2000 94.6% chr11 - 59710172 59710319 148 browser details YourSeq 137 133 283 2000 95.4% chr5 + 125507585 125507735 151 browser details YourSeq 136 135 282 2000 96.0% chr15 - 76670266 76670413 148 browser details YourSeq 135 134 280 2000 96.6% chr10 + 94157443 94157592 150 browser details YourSeq 134 132 286 2000 93.6% chr14 + 119485578 119485748 171 browser details YourSeq 133 135 281 2000 93.9% chr8 - 111702685 111702830 146 browser details YourSeq 133 134 299 2000 90.2% chr16 - 11169336 11169497 162 browser details YourSeq 133 48 280 2000 92.9% chr1 + 86560415 86560990 576 browser details YourSeq 132 134 281 2000 93.2% chr6 - 146645485 146645631 147 browser details YourSeq 132 134 282 2000 94.6% chr18 - 44682836 44682984 149 browser details YourSeq 131 138 280 2000 95.9% chr11 - 59918759 59918901 143 browser details YourSeq 131 130 272 2000 96.6% chr10 - 63242373 63242518 146 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 1336 1 1336 1336 100.0% chr7 - 28760266 28761601 1336 browser details YourSeq 161 1062 1279 1336 88.2% chr9 - 7732387 7732580 194 browser details YourSeq 161 1091 1288 1336 88.9% chr1 + 78558629 78558818 190 browser details YourSeq 160 1097 1295 1336 91.7% chr11 + 4903487 4903690 204 browser details YourSeq 160 1093 1285 1336 91.0% chr1 + 180575536 180575726 191 browser details YourSeq 156 1093 1290 1336 88.6% chr5 - 142297043 142297237 195 browser details YourSeq 156 1097 1286 1336 92.5% chr4 - 132361110 132361515 406 browser details YourSeq 156 1091 1285 1336 89.5% chr2 - 180475382 180475573 192 browser details YourSeq 155 1092 1285 1336 91.1% chr2 + 130483714 130483918 205 browser details YourSeq 153 1103 1285 1336 90.6% chr15 + 44308631 44308811 181 browser details YourSeq 152 1095 1285 1336 89.7% chr4 - 139294980 139295167 188 browser details YourSeq 152 1098 1285 1336 89.2% chr3 - 30917619 30917804 186 browser details YourSeq 152 1091 1285 1336 91.0% chr10 - 117797502 117797702 201 browser details YourSeq 151 1091 1278 1336 90.9% chr12 - 102709069 102709260 192 browser details YourSeq 151 1096 1280 1336 91.5% chr2 + 127878120 127878302 183 browser details YourSeq 151 1091 1285 1336 88.4% chr2 + 92499181 92499373 193 browser details YourSeq 150 1092 1278 1336 92.7% chr5 - 64000524 64000717 194 browser details YourSeq 149 1098 1285 1336 90.4% chr14 - 56743329 56743516 188 browser details YourSeq 149 1095 1285 1336 89.8% chr14 + 18362580 18362783 204 browser details YourSeq 149 1093 1285 1336 87.3% chr13 + 107673489 107673678 190 Note: The 1336 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: Nfkbib nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, beta [ Mus musculus (house mouse) ] Gene ID: 18036, updated on 12-Aug-2019 Gene summary Official Symbol Nfkbib provided by MGI Official Full Name nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, beta provided by MGI Primary source MGI:MGI:104752 See related Ensembl:ENSMUSG00000030595 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as IkB; IkBb; ikB-B; IKB-beta; IKappaBbeta; NF-kappa-BIB; I-kappa-B-beta; I(Kappa)B(beta) Summary This gene encodes an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB). The encoded Expression protein prevents NF-kappaB-mediated transcription activation by sequestering it in the cytosol. In response to signals that induce NF-kappaB, such as cytokines and growth factors, the encoded protein undergoes phosphorylation, triggering its rapid ubiquitination and proteasomal degradation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2015] Orthologs Ubiquitous expression in testis adult (RPKM 35.6), duodenum adult (RPKM 28.6) and 28 other tissues See more human all Genomic context Location: 7; 7 B1 See Nfkbib in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (28758251..28766644, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (29543270..29551543, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Nfkbib ENSMUSG00000030595 Description nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, beta [Source:MGI Symbol;Acc:MGI:104752] Gene Synonyms IKappaBbeta, IkB Location Chromosome 7: 28,758,251-28,767,512 reverse strand. GRCm38:CM001000.2 About this gene This gene has 3 transcripts (splice variants), 117 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 18 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Nfkbib-201 ENSMUST00000032815.10 2061 359aa ENSMUSP00000032815.4 Protein coding CCDS21054 Q60778 TSL:1 GENCODE basic APPRIS P1 Nfkbib-202 ENSMUST00000085851.11 1243 359aa ENSMUSP00000083012.5 Protein coding CCDS21054 Q60778 TSL:1 GENCODE basic APPRIS P1 Nfkbib-203 ENSMUST00000137121.1 572 150aa ENSMUSP00000117769.1 Protein coding - D3Z358 CDS 3' incomplete TSL:3 Page 7 of 9 https://www.alphaknockout.com 29.26 kb Forward strand 28.75Mb 28.76Mb 28.77Mb Genes Sars2-201 >protein coding Ccer2-201 >protein coding Sirt2-202 >protein coding (Comprehensive set... Sirt2-201 >protein coding Sirt2-209 >protein coding Sirt2-208 >protein coding Sirt2-210 >retained intron Sirt2-206 >retained intron Sirt2-207 >retained intron Contigs < AC171210.1 Genes < Nfkbib-201protein coding (Comprehensive set... < Nfkbib-202protein
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