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Mouse Stra6l Conditional Knockout Project (CRISPR/Cas9)

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Mouse Stra6l Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Stra6l Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Stra6l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stra6l gene (NCBI Reference Sequence: NM_028788 ; Ensembl: ENSMUSG00000028327 ) is located on Mouse chromosome 4. 18 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000030011). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Stra6l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-386D19 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 5 starts from about 17.66% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 1171 bp, and the size of intron 5 for 3'-loxP site insertion: 908 bp. The size of effective cKO region: ~621 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 6 18 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Stra6l Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com 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(7121bp) | A(22.82% 1625) | C(26.64% 1897) | T(27.0% 1923) | G(23.54% 1676) 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. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr4 + 45862926 45865925 3000 browser details YourSeq 325 890 1506 3000 93.9% chr2 + 75673095 75791647 118553 browser details YourSeq 302 928 1499 3000 94.7% chr12 - 84420857 84421443 587 browser details YourSeq 300 928 1507 3000 88.7% chr13 + 100801701 100802068 368 browser details YourSeq 285 947 1497 3000 91.3% chr9 - 113754779 113755155 377 browser details YourSeq 277 931 1486 3000 92.5% chr1 - 60160629 60161162 534 browser details YourSeq 276 937 1499 3000 93.4% chr18 + 34534699 34575666 40968 browser details YourSeq 266 937 1475 3000 87.4% chr4 - 32976534 32976860 327 browser details YourSeq 260 924 1436 3000 90.2% chr5 - 139470233 139470534 302 browser details YourSeq 255 937 1458 3000 93.6% chr7 - 45480073 45480739 667 browser details YourSeq 247 923 1466 3000 92.6% chr12 + 55338726 55339251 526 browser details YourSeq 244 869 1439 3000 89.1% chr2 - 75690301 75690595 295 browser details YourSeq 242 923 1397 3000 96.9% chr2 - 172967916 172968605 690 browser details YourSeq 239 926 1436 3000 93.8% chr4 + 41142499 41143170 672 browser details YourSeq 228 950 1405 3000 96.8% chr9 + 44938731 44939380 650 browser details YourSeq 222 942 1457 3000 90.2% chr6 + 125103418 125103804 387 browser details YourSeq 220 925 1456 3000 91.4% chr18 + 77710296 77710686 391 browser details YourSeq 212 931 1479 3000 90.4% chr11 + 84417673 84418099 427 browser details YourSeq 211 504 1135 3000 91.7% chr8 - 71501382 71501793 412 browser details YourSeq 207 907 1133 3000 94.9% chr5 - 86138593 86138808 216 Note: The 3000 bp section upstream of Exon 5 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% chr4 + 45866547 45869546 3000 browser details YourSeq 62 1997 2114 3000 80.3% chr1 - 178549077 178549182 106 browser details YourSeq 45 2185 2237 3000 98.1% chr18 - 59449937 59449994 58 browser details YourSeq 45 2026 2205 3000 66.2% chr2 + 6402098 6402189 92 browser details YourSeq 40 2000 2060 3000 72.4% chr1 - 178549070 178549118 49 browser details YourSeq 38 1997 2059 3000 91.0% chr18 + 66795014 66795098 85 browser details YourSeq 36 1156 1260 3000 89.4% chr19 + 21022775 21022892 118 browser details YourSeq 35 2000 2054 3000 71.8% chr10 + 127384513 127384551 39 browser details YourSeq 34 2190 2227 3000 97.3% chr13 + 23826508 23826551 44 browser details YourSeq 33 2002 2060 3000 70.3% chr10 + 119837042 119837080 39 browser details YourSeq 29 1999 2055 3000 96.8% chr2 + 173367926 173368059 134 browser details YourSeq 25 2036 2061 3000 100.0% chr5 - 112818427 112818457 31 browser details YourSeq 25 2032 2058 3000 96.3% chr12 - 95690040 95690066 27 browser details YourSeq 25 2036 2060 3000 100.0% chr10 - 90720461 90720485 25 browser details YourSeq 24 2036 2062 3000 96.3% chr2 - 75776028 75776058 31 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Stra6l STRA6-like [ Mus musculus (house mouse) ] Gene ID: 74152, updated on 14-Aug-2019 Gene summary Official Symbol Stra6l provided by MGI Official Full Name STRA6-like provided by MGI Primary source MGI:MGI:1921402 See related Ensembl:ENSMUSG00000028327 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 Rbpr2; 1300002K09Rik Expression Biased expression in liver adult (RPKM 35.2), liver E18 (RPKM 4.8) and 2 other tissues See more Genomic context Location: 4; 4 B1 See Stra6l in Genome Data Viewer Exon count: 23 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (45824293..45887010) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (45861819..45899880) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Stra6l ENSMUSG00000028327 Description STRA6-like [Source:MGI Symbol;Acc:MGI:1921402] Gene Synonyms 1300002K09Rik, Rbpr2 Location Chromosome 4: 45,848,664-45,887,008 forward strand. GRCm38:CM000997.2 About this gene This gene has 5 transcripts (splice variants), 161 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 2 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stra6l-202 ENSMUST00000107782.7 4009 530aa ENSMUSP00000103411.1 Protein coding CCDS80094 Q9DBN1 TSL:1 GENCODE basic APPRIS ALT2 Stra6l-203 ENSMUST00000107783.7 3669 621aa ENSMUSP00000103412.1 Protein coding CCDS18141 Q9DBN1 TSL:5 GENCODE basic APPRIS P3 Stra6l-201 ENSMUST00000030011.5 3606 621aa ENSMUSP00000030011.5 Protein coding CCDS18141 Q9DBN1 TSL:1 GENCODE basic APPRIS P3 Stra6l-205 ENSMUST00000165478.1 7254 No protein - Retained intron - - TSL:2 Stra6l-204 ENSMUST00000128947.1 734 No protein - lncRNA - - TSL:5 58.34 kb Forward strand 45.84Mb 45.85Mb 45.86Mb 45.87Mb 45.88Mb 45.89Mb Genes (Comprehensive set... Stra6l-203 >protein coding Ccdc180-204 >protein coding Stra6l-202 >protein coding Ccdc180-202 >protein coding Stra6l-201 >protein coding Ccdc180-201 >lncRNA Stra6l-204 >lncRNA Stra6l-205 >retained intron Contigs AL772381.5 > Regulatory Build 45.84Mb 45.85Mb 45.86Mb 45.87Mb 45.88Mb 45.89Mb Reverse strand 58.34 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000030011 38.06 kb Forward strand Stra6l-201 >protein coding ENSMUSP00000030... Transmembrane heli... Low complexity (Seg) Pfam PF14752 PANTHER PTHR21444:SF17 PTHR21444 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 621 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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