https://www.alphaknockout.com Mouse Stk39 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Stk39 conditional knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stk39 gene (NCBI Reference Sequence: NM_016866 ; Ensembl: ENSMUSG00000027030 ) is located on Mouse chromosome 2. 18 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 18 (Transcript: ENSMUST00000102715). 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 Stk39 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-153H9 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 a null allele exhibit reduced bumetanide-sensitive thallium, a potassium tracer, uptake in dorsal root ganglion neurons and reduced fertility. Mice with an ENU mutation in intron 8 exhibit elevated albumin- creatinine (ACR) ratios. Exon 2 starts from about 14.69% 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: 61492 bp, and the size of intron 2 for 3'-loxP site insertion: 11496 bp. The size of effective cKO region: ~613 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 18 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Stk39 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. 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(7113bp) | A(24.45% 1739) | C(21.69% 1543) | T(31.38% 2232) | G(22.48% 1599) 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% chr2 - 68410392 68413391 3000 browser details YourSeq 76 1201 1283 3000 93.8% chr7 + 64988801 64988881 81 browser details YourSeq 74 1206 1279 3000 100.0% chr14 - 10634115 10634188 74 browser details YourSeq 74 1198 1271 3000 100.0% chr18 + 40689269 40689342 74 browser details YourSeq 61 1224 1287 3000 98.5% chr2 + 58591923 58591992 70 browser details YourSeq 51 1203 1253 3000 100.0% chr14 - 95916193 95916243 51 browser details YourSeq 50 1151 1200 3000 100.0% chr8 + 58177099 58177148 50 browser details YourSeq 46 1229 1274 3000 100.0% chr13 - 17959588 17959633 46 browser details YourSeq 45 1152 1198 3000 93.5% chr6 - 94978171 94978216 46 browser details YourSeq 41 1160 1203 3000 90.5% chr16 + 72975145 72975186 42 browser details YourSeq 40 1154 1197 3000 95.5% chr19 + 47522910 47522953 44 browser details YourSeq 34 1766 1861 3000 91.9% chr13 - 49895386 49895480 95 browser details YourSeq 32 1768 1861 3000 91.5% chr13 + 50847494 50847586 93 browser details YourSeq 32 959 1087 3000 97.1% chr12 + 106557731 106558038 308 browser details YourSeq 31 1061 1201 3000 97.0% chr9 - 39007506 39007646 141 browser details YourSeq 31 1171 1203 3000 97.0% chr2 + 45895234 45895266 33 browser details YourSeq 28 1152 1183 3000 93.8% chr6 + 91870700 91870731 32 browser details YourSeq 26 937 965 3000 96.5% chr10 - 9686245 9686276 32 browser details YourSeq 23 2845 2871 3000 88.0% chr12 - 70473746 70473771 26 browser details YourSeq 21 2387 2407 3000 100.0% chr16 - 71959241 71959261 21 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% chr2 - 68406779 68409778 3000 browser details YourSeq 43 2525 2574 3000 93.9% chr10 - 21325057 21325106 50 browser details YourSeq 24 166 189 3000 100.0% chr11 - 77727640 77727663 24 browser details YourSeq 23 1003 1026 3000 100.0% chr17 - 72000706 72000730 25 browser details YourSeq 22 1056 1080 3000 95.9% chr8 - 79679671 79679696 26 browser details YourSeq 22 390 413 3000 87.0% chr5 - 17571069 17571091 23 browser details YourSeq 22 1783 1804 3000 100.0% chr3 - 70634881 70634902 22 browser details YourSeq 22 457 478 3000 100.0% chr11 - 35105505 35105526 22 browser details YourSeq 21 889 909 3000 100.0% chr6 - 59362931 59362951 21 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: Stk39 serine/threonine kinase 39 [ Mus musculus (house mouse) ] Gene ID: 53416, updated on 10-Oct-2019 Gene summary Official Symbol Stk39 provided by MGI Official Full Name serine/threonine kinase 39 provided by MGI Primary source MGI:MGI:1858416 See related Ensembl:ENSMUSG00000027030 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 DCHT; Rnl5; SPAK; RF005; AW227544; AW556857 Expression Broad expression in testis adult (RPKM 29.2), cerebellum adult (RPKM 12.7) and 19 other tissues See more Orthologs human all Genomic context Location: 2; 2 C1.3 See Stk39 in Genome Data Viewer Exon count: 22 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (68210445..68472108, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (68048504..68310038, complement) Chromosome 2 - NC_000068.7 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 8 transcripts Gene: Stk39 ENSMUSG00000027030 Description serine/threonine kinase 39 [Source:MGI Symbol;Acc:MGI:1858416] Gene Synonyms DCHT, RF005, Rnl5, SPAK Location Chromosome 2: 68,210,445-68,472,268 reverse strand. GRCm38:CM000995.2 About this gene This gene has 8 transcripts (splice variants), 205 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 25 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stk39-201 ENSMUST00000102715.3 3536 556aa ENSMUSP00000099776.3 Protein coding CCDS16084 A2AQL0 Q9Z1W9 TSL:1 GENCODE basic APPRIS P1 Stk39-203 ENSMUST00000126663.7 2578 No protein - Retained intron - - TSL:1 Stk39-202 ENSMUST00000123781.7 2038 No protein - Retained intron - - TSL:1 Stk39-208 ENSMUST00000149581.1 737 No protein - lncRNA - - TSL:5 Stk39-205 ENSMUST00000134442.7 492 No protein - lncRNA - - TSL:3 Stk39-206 ENSMUST00000139407.1 473 No protein - lncRNA - - TSL:3 Stk39-204 ENSMUST00000130380.7 452 No protein - lncRNA - - TSL:3 Stk39-207 ENSMUST00000144457.1 414 No protein - lncRNA - - TSL:3 Page 6 of 8 https://www.alphaknockout.com 281.82 kb Forward strand 68.25Mb 68.30Mb 68.35Mb 68.40Mb 68.45Mb Genes Gm13596-201 >lncRNA (Comprehensive set... Contigs BX936296.17 > AL845486.6 > AL844841.10 > Genes (Comprehensive set... < Stk39-201protein coding < Stk39-203retained intron < Stk39-207lncRNA < Stk39-202retained intron < Stk39-205lncRNA < Stk39-208lncRNA < Stk39-204lncRNA < Stk39-206lncRNA Regulatory Build 68.25Mb 68.30Mb 68.35Mb 68.40Mb 68.45Mb Reverse strand 281.82 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000102715 < Stk39-201protein coding Reverse strand 261.82 kb ENSMUSP00000099... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Superfamily Protein kinase-like domain superfamily SMART Protein kinase domain Pfam Protein kinase domain Serine/threonine-protein kinase OSR1/WNK, CCT domain PROSITE profiles Protein kinase domain PROSITE patterns Protein kinase, ATP binding site PANTHER PTHR24361 PTHR24361:SF671 Gene3D 3.30.200.20 1.10.510.10 3.10.20.90 CDD cd06610 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 556 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.