https://www.alphaknockout.com Mouse Epb41l3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Epb41l3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Epb41l3 gene (NCBI Reference Sequence: NM_013813 ; Ensembl: ENSMUSG00000024044 ) is located on Mouse chromosome 17. 22 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 21 (Transcript: ENSMUST00000080208). 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 Epb41l3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-391O3 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 one knock-out allele display a normal phenotype. Mice homozygous for a different knock-out allele exhibit ataxia, gait abnormalities, clasping, hypermyelination, abnormal axon morphology, and decreased internode length. Exon 2 starts from about 100% 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: 50948 bp, and the size of intron 2 for 3'-loxP site insertion: 2109 bp. The size of effective cKO region: ~707 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 22 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Epb41l3 Homology arm cKO region loxP site Page 2 of 8 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(7207bp) | A(26.14% 1884) | C(23.16% 1669) | T(28.15% 2029) | G(22.55% 1625) 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 8 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% chr17 + 69204621 69207620 3000 browser details YourSeq 381 1581 2970 3000 89.0% chr4 + 33979851 34363567 383717 browser details YourSeq 333 1442 1964 3000 88.3% chr13 + 70013624 70014162 539 browser details YourSeq 322 1458 1975 3000 87.1% chr4 + 12666430 12666958 529 browser details YourSeq 315 1485 1966 3000 86.3% chr2 - 27277730 27278131 402 browser details YourSeq 313 1492 1957 3000 85.7% chr1 - 194103493 194103945 453 browser details YourSeq 313 1492 1964 3000 88.3% chr7 + 110761865 110762310 446 browser details YourSeq 311 1428 1954 3000 87.2% chr16 - 93663084 93666954 3871 browser details YourSeq 311 1437 1964 3000 85.7% chr3 + 27380225 27380771 547 browser details YourSeq 305 1493 1962 3000 87.1% chr14 + 77125051 77125510 460 browser details YourSeq 302 1463 1957 3000 85.8% chr18 - 52568935 52569372 438 browser details YourSeq 302 1416 1948 3000 86.8% chr12 - 35480113 35480661 549 browser details YourSeq 297 1581 1964 3000 88.2% chr2 + 136438490 136438871 382 browser details YourSeq 296 1492 1962 3000 89.3% chr13 - 59865891 59866428 538 browser details YourSeq 295 1492 1964 3000 86.5% chr5 - 26272704 26273200 497 browser details YourSeq 291 1460 1964 3000 88.6% chr11 - 110141851 110142364 514 browser details YourSeq 289 1582 1964 3000 88.6% chr16 - 57294806 57295170 365 browser details YourSeq 287 1492 1964 3000 85.7% chrX - 136042482 136042911 430 browser details YourSeq 287 1421 1964 3000 86.9% chr9 - 60499074 60499625 552 browser details YourSeq 286 1490 1957 3000 85.9% chr11 + 45182026 45182454 429 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% chr17 + 69208328 69211327 3000 browser details YourSeq 365 1106 1505 3000 95.3% chr5 + 127729413 127729811 399 browser details YourSeq 362 1106 1503 3000 95.3% chr14 - 64773391 64773787 397 browser details YourSeq 360 1105 1503 3000 94.2% chr12 + 54569920 54570315 396 browser details YourSeq 359 1106 1636 3000 93.9% chr17 + 79810961 79811771 811 browser details YourSeq 358 1106 1503 3000 94.2% chr1 - 189905009 189905403 395 browser details YourSeq 357 1106 1504 3000 94.2% chr2 - 19167389 19167785 397 browser details YourSeq 357 1105 1506 3000 94.7% chr5 + 31355270 31355670 401 browser details YourSeq 357 1106 1503 3000 95.9% chr17 + 69164058 69164458 401 browser details YourSeq 357 1099 1505 3000 93.5% chr1 + 181322242 181322644 403 browser details YourSeq 356 1108 1522 3000 92.3% chr19 - 38393335 38393735 401 browser details YourSeq 356 1100 1505 3000 93.3% chr19 - 24178401 24178803 403 browser details YourSeq 356 1100 1505 3000 93.0% chr1 - 191308771 191309172 402 browser details YourSeq 356 1105 1504 3000 94.0% chr13 + 82748269 82748667 399 browser details YourSeq 355 1099 1505 3000 93.4% chr3 - 84354284 84354689 406 browser details YourSeq 355 1105 1511 3000 95.2% chr14 - 74954512 74954927 416 browser details YourSeq 354 1110 1504 3000 93.9% chr4 - 137203382 137203771 390 browser details YourSeq 354 1105 1505 3000 94.0% chr8 + 95216939 95217338 400 browser details YourSeq 354 1094 1504 3000 92.9% chr4 + 111303642 111304051 410 browser details YourSeq 353 1106 1503 3000 93.5% chr3 - 57632758 57633151 394 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Epb41l3 erythrocyte membrane protein band 4.1 like 3 [ Mus musculus (house mouse) ] Gene ID: 13823, updated on 24-Oct-2019 Gene summary Official Symbol Epb41l3 provided by MGI Official Full Name erythrocyte membrane protein band 4.1 like 3 provided by MGI Primary source MGI:MGI:103008 See related Ensembl:ENSMUSG00000024044 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 4.1B; Dal1; NBL3; DAL1P; Epb4.1l3 Expression Broad expression in cerebellum adult (RPKM 14.3), frontal lobe adult (RPKM 11.1) and 15 other tissues See more Orthologs human all Genomic context Location: 17 E1.3; 17 40.15 cM See Epb41l3 in Genome Data Viewer Exon count: 33 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (69075647..69289991) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (69506150..69639329) Chromosome 17 - NC_000083.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 20 transcripts Gene: Epb41l3 ENSMUSG00000024044 Description erythrocyte membrane protein band 4.1 like 3 [Source:MGI Symbol;Acc:MGI:103008] Gene Synonyms 4.1B, DAL1P, Epb4.1l3, NBL3 Location Chromosome 17: 69,075,683-69,289,989 forward strand. GRCm38:CM001010.2 About this gene This gene has 20 transcripts (splice variants), 199 orthologues, 11 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Epb41l3- ENSMUST00000080208.6 4047 929aa ENSMUSP00000079098.5 Protein coding CCDS28952 Q9WV92 TSL:1 201 GENCODE basic APPRIS P2 Epb41l3- ENSMUST00000239167.1 4692 951aa ENSMUSP00000158911.1 Protein coding - - GENCODE 220 basic APPRIS ALT2 Epb41l3- ENSMUST00000238898.1 4368 1052aa ENSMUSP00000158929.1 Protein coding - - GENCODE 219 basic Epb41l3- ENSMUST00000112680.7 4073 939aa ENSMUSP00000108300.1 Protein coding - Q9WV92 TSL:5 202 GENCODE basic APPRIS ALT2 Epb41l3- ENSMUST00000223703.1 3687 852aa ENSMUSP00000153611.1 Protein coding - A0A286YDY4 CDS 5' 205 incomplete Epb41l3- ENSMUST00000225977.1 3188 876aa ENSMUSP00000153228.1 Protein coding - A7YY80 GENCODE 215 basic APPRIS ALT2 Epb41l3- ENSMUST00000233509.1 2736 911aa ENSMUSP00000156566.1 Protein coding - D0VYV6 GENCODE 217 basic APPRIS ALT2 Epb41l3- ENSMUST00000233964.1 2703 819aa ENSMUSP00000156827.1 Protein coding - A0A3B2WBE1 CDS 5' 218 incomplete Epb41l3- ENSMUST00000232706.1 2439 812aa ENSMUSP00000156921.1 Protein coding - A0A3B2WD96 GENCODE 216 basic APPRIS ALT2 Epb41l3- ENSMUST00000224523.1 1817 605aa ENSMUSP00000153133.1 Protein coding - A0A286YD84 CDS 5' 207 incomplete Epb41l3- ENSMUST00000224951.1 786 262aa ENSMUSP00000153190.1 Protein coding - A0A286YCY1 CDS 5' and 3' 208 incomplete Epb41l3- ENSMUST00000225695.1 670 223aa ENSMUSP00000153138.1 Protein coding - A0A286YCT6 CDS 5' and 3' 213 incomplete Epb41l3- ENSMUST00000225062.1 666 29aa ENSMUSP00000153146.1 Protein coding - A0A286YD94