https://www.alphaknockout.com Mouse Sec14l2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Sec14l2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Sec14l2 gene (NCBI Reference Sequence: NM_144520 ; Ensembl: ENSMUSG00000003585 ) is located on Mouse chromosome 11. 12 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 12 (Transcript: ENSMUST00000003681). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Sec14l2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-81P12 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: Mice homozygous for a null allele exhibit decreased cholesterol synthesis and plasma levels under fasting conditions compared to wild-type mice. Exon 2 starts from about 4.55% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1868 bp, and the size of intron 2 for 3'-loxP site insertion: 4743 bp. The size of effective cKO region: ~576 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 2 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Homology arm Exon of mouse Sec14l2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7076bp) | A(22.67% 1604) | C(24.46% 1731) | T(25.27% 1788) | G(27.6% 1953) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. 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% chr11 - 4117007 4120006 3000 browser details YourSeq 24 991 1017 3000 96.3% chr1 + 48980552 48980591 40 browser details YourSeq 23 818 844 3000 88.0% chr15 - 12682061 12682086 26 browser details YourSeq 23 2857 2879 3000 100.0% chr15 + 8303929 8303951 23 browser details YourSeq 22 2859 2880 3000 100.0% chr12 - 80875225 80875246 22 browser details YourSeq 22 1675 1696 3000 100.0% chr12 + 80258605 80258626 22 browser details YourSeq 22 717 739 3000 100.0% chr1 + 13615358 13615381 24 browser details YourSeq 21 1590 1618 3000 86.3% chr6 - 13975481 13975509 29 browser details YourSeq 21 2861 2881 3000 100.0% chr15 + 78194771 78194791 21 browser details YourSeq 21 2856 2876 3000 100.0% chr10 + 61045651 61045671 21 Note: The 3000 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 3000 1 3000 3000 100.0% chr11 - 4113431 4116430 3000 browser details YourSeq 193 2357 2579 3000 97.1% chr1 + 131115798 131116427 630 browser details YourSeq 191 2379 2582 3000 97.6% chr2 + 173349081 173349293 213 browser details YourSeq 189 2388 2602 3000 93.6% chr17 - 32261378 32261581 204 browser details YourSeq 187 2384 2598 3000 95.7% chr9 - 40814629 40814843 215 browser details YourSeq 186 2384 2580 3000 97.5% chr11 - 102744501 102744707 207 browser details YourSeq 185 2388 2582 3000 97.5% chr9 - 37591750 37591944 195 browser details YourSeq 185 2388 2581 3000 98.0% chr11 - 85562886 85563083 198 browser details YourSeq 185 2388 2583 3000 97.5% chr1 - 58471251 58471447 197 browser details YourSeq 184 2388 2583 3000 97.0% chr12 - 108230729 108230924 196 browser details YourSeq 184 2388 2582 3000 97.5% chr11 + 87413073 87413271 199 browser details YourSeq 183 2388 2580 3000 97.5% chr2 - 170448735 170448927 193 browser details YourSeq 183 2388 2579 3000 98.0% chr14 - 56772446 56772638 193 browser details YourSeq 183 2388 2579 3000 98.0% chr11 - 54798765 54798958 194 browser details YourSeq 183 2388 2582 3000 97.0% chr10 - 80661940 80662134 195 browser details YourSeq 183 2385 2579 3000 97.5% chr1 - 84919703 84919910 208 browser details YourSeq 183 2388 2580 3000 97.5% chr7 + 49463829 49464021 193 browser details YourSeq 183 2388 2580 3000 97.5% chr4 + 132769978 132770170 193 browser details YourSeq 183 2385 2579 3000 97.0% chr3 + 68486903 68487097 195 browser details YourSeq 183 2388 2580 3000 97.5% chr19 + 40345399 40345591 193 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Sec14l2 SEC14-like lipid binding 2 [ Mus musculus (house mouse) ] Gene ID: 67815, updated on 10-Oct-2019 Gene summary Official Symbol Sec14l2 provided by MGI Official Full Name SEC14-like lipid binding 2 provided by MGI Primary source MGI:MGI:1915065 See related Ensembl:ENSMUSG00000003585 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Spf; TAP; 1300013M05Rik Expression Biased expression in liver adult (RPKM 140.8), liver E18 (RPKM 27.5) and 6 other tissues See more Orthologs human all Genomic context Location: 11; 11 A1 See Sec14l2 in Genome Data Viewer Exon count: 12 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (4097039..4118729, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (3997042..4018732, complement) Chromosome 11 - NC_000077.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Sec14l2 ENSMUSG00000003585 Description SEC14-like lipid binding 2 [Source:MGI Symbol;Acc:MGI:1915065] Gene Synonyms 1300013M05Rik, Spf, tap Location Chromosome 11: 4,097,039-4,123,415 reverse strand. GRCm38:CM001004.2 About this gene This gene has 6 transcripts (splice variants), 197 orthologues, 5 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 Sec14l2-201 ENSMUST00000003681.7 2631 403aa ENSMUSP00000003681.7 Protein coding CCDS24377 Q99J08 TSL:1 GENCODE basic APPRIS P1 Sec14l2-202 ENSMUST00000123901.7 1624 No protein - lncRNA - - TSL:5 Sec14l2-203 ENSMUST00000132421.7 1229 No protein - lncRNA - - TSL:5 Sec14l2-204 ENSMUST00000133631.1 880 No protein - lncRNA - - TSL:3 Sec14l2-205 ENSMUST00000136420.1 429 No protein - lncRNA - - TSL:2 Sec14l2-206 ENSMUST00000145173.1 423 No protein - lncRNA - - TSL:5 46.38 kb Forward strand 4.09Mb 4.10Mb 4.11Mb 4.12Mb 4.13Mb Genes Gm11955-201 >processed pseudogene (Comprehensive set... Contigs AL807395.8 > Genes (Comprehensive set... < Mtfp1-201protein codin<g Sec14l2-202lncRNA < Gm11957-201processed pseudogene < Mtfp1-202lncRNA < Sec14l2-204lncRNA < Sec14l2-206lncRNA < Sec14l2-201protein coding < Sec14l2-203lncRNA < Sec14l2-205lncRNA Regulatory Build 4.09Mb 4.10Mb 4.11Mb 4.12Mb 4.13Mb Reverse strand 46.38 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000003681 < Sec14l2-201protein coding Reverse strand 21.79 kb ENSMUSP00000003... Superfamily CRAL-TRIO lipid binding domain superfamily GOLD domain superfamily CRAL/TRIO, N-terminal domain superfamily SMART CRAL/TRIO, N-terminal domain CRAL-TRIO lipid binding domain Prints PR00180 Pfam CRAL-TRIO lipid binding domain PROSITE profiles CRAL-TRIO lipid binding domain GOLD domain PANTHER PTHR23324:SF64 PTHR23324 Gene3D CRAL-TRIO lipid binding domain superfamily 2.60.120.680 CDD CRAL-TRIO lipid binding domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 403 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.