https://www.alphaknockout.com Mouse Kif1c Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Kif1c conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Kif1c gene (NCBI Reference Sequence: NM_153103 ; Ensembl: ENSMUSG00000020821 ) is located on Mouse chromosome 11. 23 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 23 (Transcript: ENSMUST00000094499). Exon 6~11 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Kif1c gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-72D6 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 reporter allele are viable, fertile and overtly normal and display normal motor-dependent retrograde Golgi apparatus-to-endoplasmic reticulum transport. Exon 6 starts from about 11.03% of the coding region. The knockout of Exon 6~11 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 579 bp, and the size of intron 11 for 3'-loxP site insertion: 552 bp. The size of effective cKO region: ~2171 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 5' gRNA region gRNA region 3' 11 1 2 3 4 5 6 7 8 9 10 12 13 23 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Kif1c 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(8671bp) | A(21.96% 1904) | C(25.1% 2176) | T(26.25% 2276) | G(26.7% 2315) 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% chr11 + 70701209 70704208 3000 browser details YourSeq 84 2542 2643 3000 91.2% chr1 - 93077715 93077816 102 browser details YourSeq 27 169 198 3000 85.8% chr16 - 15934215 15934242 28 browser details YourSeq 22 1773 1796 3000 87.0% chr16 + 69985264 69985286 23 Note: The 3000 bp section upstream of Exon 6 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% chr11 + 70706380 70709379 3000 browser details YourSeq 144 1115 1312 3000 88.8% chr18 + 74735080 74735279 200 browser details YourSeq 138 1115 1312 3000 85.8% chr4 + 31818964 31819161 198 browser details YourSeq 136 1114 1309 3000 87.8% chr11 - 19672464 19672662 199 browser details YourSeq 136 1115 1312 3000 85.9% chr12 + 7686100 7686298 199 browser details YourSeq 136 1115 1310 3000 88.4% chr10 + 7597033 7597989 957 browser details YourSeq 135 1115 1309 3000 89.6% chr2 - 109744994 109745212 219 browser details YourSeq 134 1115 1308 3000 93.0% chr5 - 38018872 38019092 221 browser details YourSeq 133 1115 1278 3000 90.8% chr4 + 131695663 131695827 165 browser details YourSeq 132 1138 1312 3000 90.8% chr7 - 102495890 102496072 183 browser details YourSeq 132 1117 1309 3000 88.6% chr17 - 49132134 49132328 195 browser details YourSeq 131 1117 1319 3000 88.3% chr12 - 73509484 73509686 203 browser details YourSeq 131 1115 1321 3000 89.7% chr16 + 33712811 33713032 222 browser details YourSeq 131 1115 1314 3000 82.3% chr1 + 178209534 178209732 199 browser details YourSeq 130 1113 1309 3000 89.3% chr16 - 50744843 50745054 212 browser details YourSeq 130 1138 1305 3000 93.4% chr14 - 71965183 72096096 130914 browser details YourSeq 129 1115 1308 3000 86.1% chr9 - 72130419 72130654 236 browser details YourSeq 129 1117 1290 3000 89.6% chr15 - 79009244 79009420 177 browser details YourSeq 129 1113 1311 3000 85.8% chr4 + 121066150 121066350 201 browser details YourSeq 129 1110 1305 3000 87.0% chr4 + 114717311 114717515 205 Note: The 3000 bp section downstream of Exon 11 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Kif1c kinesin family member 1C [ Mus musculus (house mouse) ] Gene ID: 16562, updated on 12-Aug-2019 Gene summary Official Symbol Kif1c provided by MGI Official Full Name kinesin family member 1C provided by MGI Primary source MGI:MGI:1098260 See related Ensembl:ENSMUSG00000020821 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 Ltxs1; Orch3; D11Bwg1349e; B430105J22Rik Expression Ubiquitous expression in heart adult (RPKM 50.0), colon adult (RPKM 41.5) and 27 other tissues See more Orthologs human all Genomic context Location: 11; 11 B3 See Kif1c in Genome Data Viewer Exon count: 24 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (70700396..70731970) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (70514050..70545472) Chromosome 11 - NC_000077.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Kif1c ENSMUSG00000020821 Description kinesin family member 1C [Source:MGI Symbol;Acc:MGI:1098260] Gene Synonyms B430105J22Rik, D11Bwg1349e, N-3 kinsin, Orch3 Location Chromosome 11: 70,700,548-70,731,964 forward strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 175 orthologues, 43 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Kif1c-203 ENSMUST00000102554.7 6684 1100aa ENSMUSP00000099614.1 Protein coding CCDS24965 O35071 TSL:5 GENCODE basic APPRIS P2 Kif1c-204 ENSMUST00000137119.2 6596 1100aa ENSMUSP00000123242.2 Protein coding CCDS24965 O35071 TSL:5 GENCODE basic APPRIS P2 Kif1c-202 ENSMUST00000094499.10 5168 1100aa ENSMUSP00000092075.4 Protein coding CCDS24965 O35071 TSL:1 GENCODE basic APPRIS P2 Kif1c-201 ENSMUST00000072187.11 5046 1112aa ENSMUSP00000072048.5 Protein coding - E9Q9B0 TSL:1 GENCODE basic APPRIS ALT2 Kif1c-205 ENSMUST00000152618.8 1943 425aa ENSMUSP00000136258.1 Protein coding - Q3V3Y9 TSL:1 GENCODE basic Page 6 of 8 https://www.alphaknockout.com 51.42 kb Forward strand 70.70Mb 70.71Mb 70.72Mb 70.73Mb 70.74Mb Genes (Comprehensive set... Kif1c-203 >protein coding Kif1c-205 >protein coding Kif1c-202 >protein coding Kif1c-201 >protein coding Kif1c-204 >protein coding Mir6925-201 >miRNA Contigs AL596117.19 > Genes < Inca1-201protein coding (Comprehensive set... < Inca1-205protein coding < Inca1-202protein coding < Inca1-203protein coding < Inca1-204protein coding Regulatory Build 70.70Mb 70.71Mb 70.72Mb 70.73Mb 70.74Mb Reverse strand 51.42 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000094499 29.83 kb Forward strand Kif1c-202 >protein coding ENSMUSP00000092... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily P-loop containing nucleoside triphosphate hydrolase SMAD/FHA domain superfamily SMART Kinesin motor domain Prints Kinesin motor domain Pfam Kinesin motor domain Kinesin-associated Forkhead-associated (FHA) domain PROSITE profiles Kinesin motor domain PROSITE patterns Kinesin motor domain, conserved site PANTHER Kinesin-like protein PTHR24115:SF322 Gene3D Kinesin motor domain superfamily 2.60.200.20 CDD cd01365 Forkhead-associated (FHA) domain 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 1000 1100 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.