https://www.alphaknockout.com Mouse Ifit3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ifit3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ifit3 gene (NCBI Reference Sequence: NM_010501 ; Ensembl: ENSMUSG00000074896 ) is located on Mouse chromosome 19. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000102825). 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 Ifit3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-111N1 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 2 is not frameshift exon, and covers 99.59% of the coding region. The size of intron 1 for 5'-loxP site insertion: 3456 bp. The size of effective cKO region: ~2421 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 5' gRNA region gRNA region 3' 1 2 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ifit3 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. 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(8171bp) | A(28.66% 2342) | C(19.35% 1581) | T(29.68% 2425) | G(22.31% 1823) 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% chr19 + 34583812 34586811 3000 browser details YourSeq 2808 1 3000 3000 97.1% chr19 + 34608207 34611181 2975 browser details YourSeq 139 861 1032 3000 90.7% chr2 + 39076761 39076935 175 browser details YourSeq 135 523 1032 3000 80.7% chr10 + 8347999 8348253 255 browser details YourSeq 134 852 1032 3000 88.5% chr1 + 154441033 154441211 179 browser details YourSeq 131 524 1031 3000 80.2% chr5 - 92565092 92565274 183 browser details YourSeq 131 852 1032 3000 84.0% chr10 - 21180112 21180285 174 browser details YourSeq 131 850 1032 3000 85.7% chr3 + 90203322 90203502 181 browser details YourSeq 130 852 1032 3000 86.1% chr5 - 146902886 146903066 181 browser details YourSeq 129 523 1032 3000 81.5% chr11 - 3178375 3178562 188 browser details YourSeq 127 863 1029 3000 88.5% chr1 - 30774846 30775012 167 browser details YourSeq 126 862 1032 3000 92.6% chr10 - 77225149 77225322 174 browser details YourSeq 124 852 1024 3000 87.3% chr1 - 60904982 60905134 153 browser details YourSeq 124 852 1029 3000 87.6% chr17 + 59614260 59614434 175 browser details YourSeq 123 864 1032 3000 88.2% chr12 + 54801756 54801924 169 browser details YourSeq 122 852 1032 3000 90.7% chr7 + 27959888 27960084 197 browser details YourSeq 122 523 1032 3000 79.4% chr18 + 38107629 38107822 194 browser details YourSeq 120 852 1020 3000 94.2% chrX - 100272816 100272993 178 browser details YourSeq 120 898 1032 3000 94.9% chr19 - 24237880 24238015 136 browser details YourSeq 120 871 1032 3000 92.9% chr15 - 93515970 93516520 551 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% chr19 + 34588733 34591732 3000 browser details YourSeq 2653 9 3000 3000 96.1% chr19 + 34613108 34616041 2934 browser details YourSeq 160 2580 2785 3000 90.2% chr10 - 45007820 45008184 365 browser details YourSeq 160 2565 2776 3000 95.0% chr12 + 94408773 94409017 245 browser details YourSeq 158 2486 2776 3000 92.9% chr17 + 75663686 75664310 625 browser details YourSeq 152 2580 2783 3000 94.3% chr10 - 45007897 45008200 304 browser details YourSeq 152 2579 2785 3000 91.7% chr3 + 149745644 149745937 294 browser details YourSeq 150 2579 2776 3000 93.7% chr8 + 65558306 65558673 368 browser details YourSeq 149 2556 2776 3000 95.8% chr2 - 9773347 9774153 807 browser details YourSeq 146 2598 2786 3000 92.1% chr15 - 87206035 87206238 204 browser details YourSeq 144 2580 2776 3000 93.5% chr10 - 50432015 50432401 387 browser details YourSeq 143 2582 2785 3000 92.9% chr7 - 79930902 79931260 359 browser details YourSeq 138 2580 2785 3000 92.5% chr2 - 90416485 90416758 274 browser details YourSeq 137 2588 2776 3000 94.8% chr16 - 55705795 55706182 388 browser details YourSeq 137 2588 2785 3000 93.2% chrX + 152390496 152390752 257 browser details YourSeq 137 2580 2776 3000 90.4% chr6 + 31820921 31821110 190 browser details YourSeq 135 2589 2776 3000 95.4% chr3 - 137745531 137745867 337 browser details YourSeq 135 2579 2776 3000 91.4% chr17 - 44576402 44576593 192 browser details YourSeq 132 2579 2785 3000 93.3% chrX + 152390587 152390816 230 browser details YourSeq 132 2579 2782 3000 86.4% chr3 + 126159060 126159248 189 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: Ifit3 interferon-induced protein with tetratricopeptide repeats 3 [ Mus musculus (house mouse) ] Gene ID: 15959, updated on 12-Aug-2019 Gene summary Official Symbol Ifit3 provided by MGI Official Full Name interferon-induced protein with tetratricopeptide repeats 3 provided by MGI Primary source MGI:MGI:1101055 See related Ensembl:ENSMUSG00000074896 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 P49; Ifi49 Expression Broad expression in bladder adult (RPKM 9.6), liver E18 (RPKM 8.0) and 17 other tissues See more Genomic context Location: 19; 19 C1 See Ifit3 in Genome Data Viewer Exon count: 4 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (34583119..34588982) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (34658019..34663472) Chromosome 19 - NC_000085.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Ifit3 ENSMUSG00000074896 Description interferon-induced protein with tetratricopeptide repeats 3 [Source:MGI Symbol;Acc:MGI:1101055] Gene Synonyms Ifi49 Location Chromosome 19: 34,583,531-34,588,731 forward strand. GRCm38:CM001012.2 About this gene This gene has 1 transcript (splice variant), 451 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ifit3-201 ENSMUST00000102825.3 1745 403aa ENSMUSP00000099889.3 Protein coding CCDS29762 Q5FW82 Q64345 TSL:1 GENCODE basic APPRIS P1 25.20 kb Forward strand 34.575Mb 34.580Mb 34.585Mb 34.590Mb 34.595Mb Genes (Comprehensive set... Ifit2-201 >protein coding Ifit3-201 >protein coding Contigs AC102249.16 > AC016791.23 > Genes < Ifit1bl1-202protein coding (Comprehensive set... < Ifit1bl1-201protein coding Regulatory Build 34.575Mb 34.580Mb 34.585Mb 34.590Mb 34.595Mb Reverse strand 25.20 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000102825 5.20 kb Forward strand Ifit3-201 >protein coding ENSMUSP00000099... Low complexity (Seg) Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam Tetratricopeptide repeat PF14559 PROSITE profiles Tetratricopeptide repeat-containing domain PANTHER Interferon-induced protein with tetratricopeptide repeats 3 PTHR10271 Gene3D Tetratricopeptide-like helical domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend stop gained 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.