https://www.alphaknockout.com Mouse Ptges3 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ptges3 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ptges3 gene (NCBI Reference Sequence: NM_019766 ; Ensembl: ENSMUSG00000071072 ) is located on Mouse chromosome 10. 8 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 8 (Transcript: ENSMUST00000052798). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ptges3 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-362F3 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: Disruption of gene function results in neonatal lethality, respiratory system abnormalities, as well as skin morphological and physiological defects. Exon 2 starts from about 0.63% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 9385 bp, and the size of intron 3 for 3'-loxP site insertion: 848 bp. The size of effective cKO region: ~1114 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 3 4 8 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ptges3 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(7614bp) | A(25.98% 1978) | C(19.81% 1508) | T(32.28% 2458) | G(21.93% 1670) 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% chr10 + 128065423 128068422 3000 browser details YourSeq 294 861 1447 3000 89.1% chr1 + 17234590 17235041 452 browser details YourSeq 281 868 1447 3000 90.6% chr3 + 95090236 95493352 403117 browser details YourSeq 273 876 1447 3000 88.1% chr5 + 144353827 144354311 485 browser details YourSeq 258 871 1446 3000 91.2% chr2 - 119206080 119206653 574 browser details YourSeq 258 861 1432 3000 88.8% chrX + 38567659 38568157 499 browser details YourSeq 253 872 1455 3000 84.9% chr13 + 100801712 100802067 356 browser details YourSeq 251 877 1450 3000 88.4% chr4 + 48558181 48558493 313 browser details YourSeq 247 878 1436 3000 92.8% chr1 - 85661167 85661738 572 browser details YourSeq 240 874 1506 3000 84.7% chr18 - 77700207 77700535 329 browser details YourSeq 236 878 1446 3000 83.0% chr3 - 103001284 103001606 323 browser details YourSeq 236 887 1447 3000 85.5% chr10 + 95732840 95733182 343 browser details YourSeq 234 378 1051 3000 87.6% chr4 + 83515885 83516324 440 browser details YourSeq 220 873 1447 3000 86.4% chr17 + 15614044 15614480 437 browser details YourSeq 211 765 1375 3000 86.0% chr15 + 103465849 103466179 331 browser details YourSeq 209 766 1435 3000 84.4% chr15 + 34148657 34149032 376 browser details YourSeq 203 403 1050 3000 87.3% chr16 + 22041923 22042522 600 browser details YourSeq 201 865 1433 3000 84.3% chr10 - 63376315 63376729 415 browser details YourSeq 198 385 1051 3000 85.3% chr1 - 63184386 63184829 444 browser details YourSeq 187 873 1363 3000 85.8% chr15 - 102219894 102220149 256 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% chr10 + 128069537 128072536 3000 browser details YourSeq 230 1159 1777 3000 84.2% chr3 + 151764334 151764804 471 browser details YourSeq 218 773 1275 3000 89.3% chr1 - 64505611 64506129 519 browser details YourSeq 197 1016 1274 3000 92.7% chr10 - 127314790 127315270 481 browser details YourSeq 188 1143 1758 3000 81.4% chr1 - 181830163 181830662 500 browser details YourSeq 188 598 2621 3000 87.4% chr6 + 85844166 85844354 189 browser details YourSeq 187 833 1294 3000 91.0% chr14 + 30580319 30580827 509 browser details YourSeq 182 1000 1281 3000 91.5% chr12 - 101063081 101063540 460 browser details YourSeq 170 598 2621 3000 83.6% chr4 - 25541899 25542096 198 browser details YourSeq 164 837 1213 3000 89.7% chr10 + 81796675 81797293 619 browser details YourSeq 144 1000 1256 3000 91.4% chr11 - 69731357 69731702 346 browser details YourSeq 134 1147 1626 3000 93.7% chr3 - 102154586 102155074 489 browser details YourSeq 126 2267 2403 3000 97.1% chr12 - 102347468 102347902 435 browser details YourSeq 125 2269 2405 3000 96.4% chr12 - 54732378 54732526 149 browser details YourSeq 125 2269 2405 3000 96.4% chr12 + 54716410 54716558 149 browser details YourSeq 124 2269 2405 3000 96.3% chr11 - 121023395 121023534 140 browser details YourSeq 122 963 1245 3000 89.7% chr11 - 106800741 106801613 873 browser details YourSeq 120 2277 2404 3000 96.9% chr11 + 78293678 78293805 128 browser details YourSeq 118 1510 1823 3000 84.0% chr19 - 56639666 56639979 314 browser details YourSeq 118 1443 1758 3000 81.6% chr14 - 13623448 13623741 294 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ptges3 prostaglandin E synthase 3 [ Mus musculus (house mouse) ] Gene ID: 56351, updated on 10-Oct-2019 Gene summary Official Symbol Ptges3 provided by MGI Official Full Name prostaglandin E synthase 3 provided by MGI Primary source MGI:MGI:1929282 See related Ensembl:ENSMUSG00000071072 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 p23; Tebp; Ptges; cPGES; sid3177; 5730442A20Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 32.9), adrenal adult (RPKM 31.2) and 28 other tissues See more Orthologs human all Genomic context Location: 10; 10 D3 See Ptges3 in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (128058982..128077254) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (127496038..127514310) Chromosome 10 - NC_000076.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Ptges3 ENSMUSG00000071072 Description prostaglandin E synthase 3 [Source:MGI Symbol;Acc:MGI:1929282] Gene Synonyms 5730442A20Rik, Ptges, Tebp, cPGES, p23 cochaperone, sid3177 Location Chromosome 10: 128,058,954-128,077,272 forward strand. GRCm38:CM001003.2 About this gene This gene has 3 transcripts (splice variants), 285 orthologues, 1 paralogue, is a member of 1 Ensembl protein family and is associated with 22 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ptges3-201 ENSMUST00000052798.13 2000 160aa ENSMUSP00000050292.7 Protein coding CCDS36087 Q9R0Q7 TSL:1 GENCODE basic APPRIS P1 Ptges3-202 ENSMUST00000084771.2 1559 130aa ENSMUSP00000081827.2 Protein coding - D3Z7C6 TSL:5 GENCODE basic Ptges3-203 ENSMUST00000148943.1 420 No protein - Retained intron - - TSL:5 38.32 kb Forward strand 128.05Mb 128.06Mb 128.07Mb 128.08Mb Genes (Comprehensive set... Ptges3-201 >protein coding Atp5b-208 >retained intron Ptges3-202 >protein coding Atp5b-205 >retained intron Ptges3-203 >retained intron Snord59a-201 >snoRNA Atp5b-201 >protein coding Gm50449-201 >snoRNA Mir677-201 >miRNA Atp5b-207 >lncRNA Atp5b-203 >lncRNA Contigs AC131120.4 > Genes < Gm47438-201lncRNA (Comprehensive set... Regulatory Build 128.05Mb 128.06Mb 128.07Mb 128.08Mb Reverse strand 38.32 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000052798 18.32 kb Forward strand Ptges3-201 >protein coding ENSMUSP00000050... MobiDB lite Low complexity (Seg) Superfamily HSP20-like chaperone Pfam CS domain PROSITE profiles CS domain PANTHER PTHR22932 PTHR22932:SF3 Gene3D HSP20-like chaperone CDD cd00237 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.