https://www.alphaknockout.com Mouse Errfi1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Errfi1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Errfi1 gene (NCBI Reference Sequence: NM_133753 ; Ensembl: ENSMUSG00000028967 ) is located on Mouse chromosome 4. 4 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000030811). Exon 2~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Errfi1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-110K6 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: Homozygous inactivation of this gene may lead to premature death, delayed eyelid opening, epidermal hyperplasia, degenerative joint disease, skin or lung cancer, gastrointestinal tract tumors, increased susceptibility to chemically- induced tumors, and impaired lung development and vascularization. Exon 2~4 covers 100.0% of the coding region. Start codon is in exon 2, and stop codon is in exon 4. The size of intron 1 for 5'-loxP site insertion: 9803 bp. The size of effective cKO region: ~2625 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy gRNA region Wildtype allele T A 5' gRNA region A 3' 1 2 3 4 Targeting vector T A A Targeted allele T A A Constitutive KO allele (After Cre recombination) Legends Exon of mouse Errfi1 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(8852bp) | A(27.42% 2427) | C(20.63% 1826) | T(28.59% 2531) | G(23.36% 2068) 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 + 150861899 150864898 3000 browser details YourSeq 35 2710 2753 3000 90.7% chr11 - 113905184 113905234 51 browser details YourSeq 34 2707 2754 3000 88.7% chr11 - 24379581 24379634 54 browser details YourSeq 32 2719 2761 3000 88.1% chr11 + 57424157 57424211 55 browser details YourSeq 30 2710 2754 3000 87.5% chr1 - 84658206 84658255 50 browser details YourSeq 30 2717 2754 3000 83.8% chr1 + 59319545 59319581 37 browser details YourSeq 29 2712 2752 3000 89.2% chr11 - 97931155 97931201 47 browser details YourSeq 28 2724 2754 3000 96.7% chr15 - 73575756 73575786 31 browser details YourSeq 27 2724 2756 3000 91.0% chr2 - 164379387 164379419 33 browser details YourSeq 27 2724 2754 3000 93.6% chr16 - 11247256 11247286 31 browser details YourSeq 27 2724 2754 3000 93.6% chr14 - 100205958 100205988 31 browser details YourSeq 27 2724 2754 3000 93.6% chr1 - 189623956 189623986 31 browser details YourSeq 27 2718 2754 3000 93.4% chr14 + 47534956 47534992 37 browser details YourSeq 27 2721 2753 3000 91.0% chr11 + 5232746 5232778 33 browser details YourSeq 26 2724 2755 3000 90.7% chr11 - 59792020 59792051 32 browser details YourSeq 26 2724 2753 3000 93.4% chr1 + 68564223 68564252 30 browser details YourSeq 25 2724 2754 3000 90.4% chr14 - 79472606 79472636 31 browser details YourSeq 25 2725 2753 3000 93.2% chr11 - 95266803 95266831 29 browser details YourSeq 25 2724 2754 3000 90.4% chr17 + 84970866 84970896 31 browser details YourSeq 25 2724 2754 3000 90.4% chr15 + 102650921 102650951 31 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% chr4 + 150867501 150870500 3000 browser details YourSeq 75 1564 1716 3000 74.6% chr5 + 119541169 119541321 153 browser details YourSeq 74 1562 1723 3000 91.2% chr9 + 60986876 61089069 102194 browser details YourSeq 71 1564 1710 3000 81.0% chr5 + 64965769 64965913 145 browser details YourSeq 67 1564 1786 3000 71.7% chrX + 36830676 36831283 608 browser details YourSeq 64 1562 1707 3000 72.0% chr13 - 113347056 113347201 146 browser details YourSeq 63 1577 1703 3000 86.3% chr10 - 42024139 42024263 125 browser details YourSeq 54 1574 1716 3000 69.3% chr11 - 103784439 104086956 302518 browser details YourSeq 54 1574 1701 3000 71.1% chr1 - 58208296 58208423 128 browser details YourSeq 53 1577 1708 3000 82.2% chr11 + 53552614 53552743 130 browser details YourSeq 51 1565 1676 3000 89.3% chr10 + 61444146 61444261 116 browser details YourSeq 50 1577 1813 3000 93.3% chr11 - 102529381 102529879 499 browser details YourSeq 50 1577 1705 3000 81.5% chr11 - 52206641 52206767 127 browser details YourSeq 50 1577 1702 3000 90.4% chr16 + 10727381 10727508 128 browser details YourSeq 49 1658 1786 3000 86.6% chr11 - 117984530 117984692 163 browser details YourSeq 49 1576 1706 3000 68.8% chr14 + 67617874 67618004 131 browser details YourSeq 47 1587 1706 3000 81.4% chr1 - 120680561 120680681 121 browser details YourSeq 47 1577 1703 3000 82.9% chr10 + 66906866 66906991 126 browser details YourSeq 46 1631 1688 3000 89.7% chr8 + 53340091 53340148 58 browser details YourSeq 45 1631 1701 3000 85.8% chr11 + 106722311 106722382 72 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Errfi1 ERBB receptor feedback inhibitor 1 [ Mus musculus (house mouse) ] Gene ID: 74155, updated on 24-Oct-2019 Gene summary Official Symbol Errfi1 provided by MGI Official Full Name ERBB receptor feedback inhibitor 1 provided by MGI Primary source MGI:MGI:1921405 See related Ensembl:ENSMUSG00000028967 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 Mig6; RALT; Mig-6; AI788755; 1300002F13Rik Expression Broad expression in liver adult (RPKM 96.9), kidney adult (RPKM 81.7) and 23 other tissuesS ee more Orthologs human all Genomic context Location: 4; 4 E2 See Errfi1 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (150853796..150868889) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (150229200..150242989) Chromosome 4 - NC_000070.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Errfi1 ENSMUSG00000028967 Description ERBB receptor feedback inhibitor 1 [Source:MGI Symbol;Acc:MGI:1921405] Gene Synonyms 1300002F13Rik, Mig-6, RALT Location Chromosome 4: 150,853,919-150,868,892 forward strand. GRCm38:CM000997.2 About this gene This gene has 3 transcripts (splice variants), 241 orthologues, is a member of 1 Ensembl protein family and is associated with 44 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Errfi1-201 ENSMUST00000030811.1 3043 461aa ENSMUSP00000030811.1 Protein coding CCDS18974 Q3TK48 Q99JZ7 TSL:1 GENCODE basic APPRIS P1 Errfi1-202 ENSMUST00000073600.8 3036 461aa ENSMUSP00000073285.2 Protein coding CCDS18974 Q3TK48 Q99JZ7 TSL:1 GENCODE basic APPRIS P1 Errfi1-203 ENSMUST00000128640.7 546 No protein - lncRNA - - TSL:1 34.97 kb Forward strand 150.85Mb 150.86Mb 150.87Mb Genes (Comprehensive set... Rps15a-ps3-201 >processed pseudEorgrefin1e-202 >protein coding Errfi1-201 >protein coding Errfi1-203 >lncRNA Contigs AL607084.14 > Genes < 1700045H11Rik-201lncRNA (Comprehensive set... Regulatory Build 150.85Mb 150.86Mb 150.87Mb Reverse strand 34.97 kb Regulation Legend CTCF Enhancer Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000030811 13.81 kb Forward strand Errfi1-201 >protein coding ENSMUSP00000030... MobiDB lite Low complexity (Seg) Pfam Cdc42 binding domain-like Mig-6 domain PANTHER PTHR14254:SF5 PTHR14254 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 461 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.