https://www.alphaknockout.com Mouse Ipo7 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ipo7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ipo7 gene (NCBI Reference Sequence: NM_181517 ; Ensembl: ENSMUSG00000066232 ) is located on Mouse chromosome 7. 25 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000084731). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ipo7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-15F24 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 3 starts from about 5.36% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 2538 bp, and the size of intron 3 for 3'-loxP site insertion: 1763 bp. The size of effective cKO region: ~654 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 3 4 25 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ipo7 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(7154bp) | A(26.74% 1913) | C(18.02% 1289) | T(34.4% 2461) | G(20.84% 1491) 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% chr7 + 110026442 110029441 3000 browser details YourSeq 330 1844 2399 3000 89.8% chr16 - 20669198 20669893 696 browser details YourSeq 293 1756 2413 3000 89.7% chr1 - 134452354 134453008 655 browser details YourSeq 291 1883 2400 3000 88.6% chr16 + 20667783 20668355 573 browser details YourSeq 286 1750 2397 3000 85.1% chr19 - 4373142 4373714 573 browser details YourSeq 286 1927 2412 3000 88.7% chr16 + 32223514 32224095 582 browser details YourSeq 275 1760 2400 3000 87.2% chr5 + 115376506 115377218 713 browser details YourSeq 266 1747 2414 3000 84.5% chr3 - 116935956 116936562 607 browser details YourSeq 257 1748 2414 3000 89.4% chr13 - 55216990 55217706 717 browser details YourSeq 255 1759 2415 3000 91.3% chr1 + 181875512 181876503 992 browser details YourSeq 251 1847 2172 3000 89.7% chr15 + 99046033 99046472 440 browser details YourSeq 249 1927 2416 3000 83.8% chr4 - 45042426 45042903 478 browser details YourSeq 248 1847 2176 3000 88.9% chr1 + 135335592 135336032 441 browser details YourSeq 241 1852 2175 3000 88.3% chr2 - 170188020 170188698 679 browser details YourSeq 238 1928 2396 3000 87.7% chr6 + 48115227 48115894 668 browser details YourSeq 234 1844 2176 3000 89.3% chr17 - 46761856 46762323 468 browser details YourSeq 228 1759 2400 3000 83.2% chr7 + 126718338 126718692 355 browser details YourSeq 226 1743 2186 3000 86.0% chr4 + 133248979 133249310 332 browser details YourSeq 225 1756 2190 3000 85.9% chrX + 74463180 74463500 321 browser details YourSeq 221 1759 2176 3000 82.3% chr2 + 180106357 180106658 302 Note: The 3000 bp section upstream of Exon 3 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% chr7 + 110030096 110033095 3000 browser details YourSeq 178 629 844 3000 89.5% chr10 - 42442482 42442690 209 browser details YourSeq 176 637 1144 3000 84.2% chr13 - 100525567 100525801 235 browser details YourSeq 174 626 829 3000 91.5% chr10 - 53514745 53514945 201 browser details YourSeq 173 629 829 3000 93.5% chr6 - 108560871 108561073 203 browser details YourSeq 173 368 829 3000 86.7% chr2 - 121523341 121523752 412 browser details YourSeq 172 626 831 3000 93.1% chr10 + 86737592 86737801 210 browser details YourSeq 171 636 828 3000 94.8% chr1 - 58390731 58390932 202 browser details YourSeq 169 633 831 3000 93.0% chr9 - 57206146 57206764 619 browser details YourSeq 169 636 831 3000 93.4% chr4 - 129792125 129792331 207 browser details YourSeq 169 636 828 3000 94.4% chr13 - 66785679 66785877 199 browser details YourSeq 169 634 830 3000 93.4% chr11 - 69816171 69816369 199 browser details YourSeq 169 634 843 3000 92.1% chr10 - 62696202 62696427 226 browser details YourSeq 169 626 833 3000 90.2% chr19 + 4430803 4431008 206 browser details YourSeq 168 639 834 3000 93.4% chr4 - 117936136 117936334 199 browser details YourSeq 168 637 834 3000 92.9% chr8 + 37967472 37967679 208 browser details YourSeq 167 636 834 3000 91.8% chr9 - 72680321 72680517 197 browser details YourSeq 167 626 829 3000 90.5% chr17 - 88588641 88588842 202 browser details YourSeq 167 636 829 3000 93.8% chr17 - 23614893 23615087 195 browser details YourSeq 167 636 851 3000 91.6% chr13 + 5363076 5363295 220 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Ipo7 importin 7 [ Mus musculus (house mouse) ] Gene ID: 233726, updated on 12-Aug-2019 Gene summary Official Symbol Ipo7 provided by MGI Official Full Name importin 7 provided by MGI Primary source MGI:MGI:2152414 See related Ensembl:ENSMUSG00000066232 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 Imp7; Ranbp7; C330016G14; A330055O14Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 27.1), placenta adult (RPKM 21.3) and 28 other tissues See more Orthologs human all Genomic context Location: 7; 7 E3 See Ipo7 in Genome Data Viewer Exon count: 25 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (110018315..110056609) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (117161939..117198628) Chromosome 7 - NC_000073.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Ipo7 ENSMUSG00000066232 Description importin 7 [Source:MGI Symbol;Acc:MGI:2152414] Gene Synonyms A330055O14Rik, Imp7, RanBP7 Location Chromosome 7: 110,018,274-110,056,609 forward strand. GRCm38:CM001000.2 About this gene This gene has 5 transcripts (splice variants), 224 orthologues, 4 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ipo7- ENSMUST00000084731.4 4763 1038aa ENSMUSP00000081782.3 Protein coding CCDS40085 Q9EPL8 TSL:1 201 GENCODE basic APPRIS P1 Ipo7- ENSMUST00000208951.1 1883 70aa ENSMUSP00000146367.1 Nonsense mediated - A0A140LHD0 TSL:1 205 decay Ipo7- ENSMUST00000208408.1 4159 No - Retained intron - - TSL:NA 203 protein Ipo7- ENSMUST00000208821.1 3867 No - Retained intron - - TSL:1 204 protein Ipo7- ENSMUST00000207277.1 3784 No - Retained intron - - TSL:1 202 protein Page 6 of 8 https://www.alphaknockout.com 58.34 kb Forward strand 110.01Mb 110.02Mb 110.03Mb 110.04Mb 110.05Mb 110.06Mb Genes (Comprehensive set... Ipo7-201 >protein coding Zfp143-204 >protein coding Ipo7-205 >nonsense mediated decay Zfp143-201 >protein coding Ipo7-202 >retained intron Zfp143-205 >protein coding Ipo7-204 >retained intron Zfp143-202 >protein coding Gm25636-201 >snoRNA Snora23-201 >snoRNA Ipo7-203 >retained intron Contigs < AC140235.3 AC121995.2 > Genes < 1600010M07Rik-201lncRNA (Comprehensive set... < AA474408-201TEC Regulatory Build 110.01Mb 110.02Mb 110.03Mb 110.04Mb 110.05Mb 110.06Mb Reverse strand 58.34 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000084731 38.34 kb Forward strand Ipo7-201 >protein coding ENSMUSP00000081... MobiDB lite Low complexity (Seg) Superfamily Armadillo-type fold SMART Importin-beta, N-terminal domain Pfam Exportin-2, central domain Importin-beta, N-terminal domain PROSITE profiles Importin-beta, N-terminal domain PANTHER PTHR10997:SF27 PTHR10997 Gene3D Armadillo-like helical All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1038 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.