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Mouse Cdc16 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Cdc16 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cdc16 gene (NCBI Reference Sequence: NM_027276 ; Ensembl: ENSMUSG00000038416 ) is located on Mouse chromosome 8. 18 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 18 (Transcript: ENSMUST00000043962). Exon 6~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Cdc16 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-20K21 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 6 starts from about 20.54% of the coding region. The knockout of Exon 6~8 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 1733 bp, and the size of intron 8 for 3'-loxP site insertion: 1715 bp. The size of effective cKO region: ~2352 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 5 6 7 8 9 18 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Cdc16 Homology arm cKO region loxP site
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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(8852bp) | A(25.45% 2253) | C(20.37% 1803) | T(32.31% 2860) | G(21.87% 1936)
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.
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BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 + 13759602 13762601 3000 browser details YourSeq 286 218 2449 3000 93.0% chr11 + 3277613 3377672 100060 browser details YourSeq 208 267 1303 3000 94.9% chr19 + 7209316 7508313 298998 browser details YourSeq 171 255 1239 3000 94.8% chr11 - 87158174 87179943 21770 browser details YourSeq 165 211 417 3000 89.1% chr11 + 32736819 32737014 196 browser details YourSeq 155 234 414 3000 97.0% chr9 - 62383591 62383790 200 browser details YourSeq 155 78 395 3000 89.7% chr2 - 121775025 121775338 314 browser details YourSeq 153 78 391 3000 92.3% chr10 + 42292215 42292735 521 browser details YourSeq 152 217 399 3000 90.2% chr1 + 132517339 132517513 175 browser details YourSeq 150 234 399 3000 95.8% chr3 - 54623285 54623453 169 browser details YourSeq 150 212 399 3000 88.7% chr17 + 45755252 45755422 171 browser details YourSeq 148 234 395 3000 96.3% chr2 - 160485030 160485193 164 browser details YourSeq 147 231 407 3000 91.9% chr6 - 5589471 5589646 176 browser details YourSeq 147 218 391 3000 94.6% chr12 + 56287883 56288077 195 browser details YourSeq 144 237 399 3000 94.5% chr7 - 48133304 48133466 163 browser details YourSeq 144 250 409 3000 96.2% chr4 - 11315720 11315887 168 browser details YourSeq 143 75 391 3000 96.8% chr1 - 86131504 86132050 547 browser details YourSeq 141 236 395 3000 95.0% chr3 - 146793038 146793202 165 browser details YourSeq 140 235 395 3000 93.8% chr12 + 25491382 25491543 162 browser details YourSeq 139 235 395 3000 93.2% chr2 + 24937726 24937886 161
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% chr8 + 13764954 13767953 3000 browser details YourSeq 353 394 1092 3000 93.2% chr1 - 182117097 182352202 235106 browser details YourSeq 317 647 2887 3000 89.6% chr10 - 43184870 43561846 376977 browser details YourSeq 301 440 1091 3000 86.1% chr19 + 3827180 3827785 606 browser details YourSeq 263 655 1262 3000 89.7% chr2 + 31941438 31942025 588 browser details YourSeq 252 640 1091 3000 88.0% chr15 - 100630642 100630996 355 browser details YourSeq 239 678 1092 3000 87.1% chr11 + 73042280 73042591 312 browser details YourSeq 233 647 1092 3000 94.4% chr3 - 51320420 51320938 519 browser details YourSeq 232 662 1094 3000 93.0% chr19 + 32681599 32682233 635 browser details YourSeq 222 639 1092 3000 86.6% chr19 - 6122986 6123368 383 browser details YourSeq 212 443 1093 3000 81.1% chr15 + 102318026 102318311 286 browser details YourSeq 207 956 2888 3000 91.2% chr10 + 127431580 127971454 539875 browser details YourSeq 200 681 1091 3000 94.7% chr8 + 105265654 105266164 511 browser details YourSeq 198 950 2878 3000 91.3% chr5 + 65933200 66396724 463525 browser details YourSeq 187 441 1091 3000 85.8% chr5 + 65541070 65541550 481 browser details YourSeq 176 445 1091 3000 82.7% chr9 - 113683171 113683560 390 browser details YourSeq 166 904 1091 3000 94.2% chr11 - 103949832 103950019 188 browser details YourSeq 165 560 1090 3000 85.7% chr11 + 105260412 105260766 355 browser details YourSeq 162 904 1091 3000 94.6% chr6 - 149454866 149455054 189 browser details YourSeq 162 905 1118 3000 87.9% chr10 + 60241835 60242030 196
Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Cdc16 CDC16 cell division cycle 16 [ Mus musculus (house mouse) ] Gene ID: 69957, updated on 12-Aug-2019
Gene summary
Official Symbol Cdc16 provided by MGI Official Full Name CDC16 cell division cycle 16 provided by MGI Primary source MGI:MGI:1917207 See related Ensembl:ENSMUSG00000038416 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 APC6; 2700071J12Rik; 2810431D22Rik Expression Ubiquitous expression in limb E14.5 (RPKM 32.5), CNS E11.5 (RPKM 27.0) and 28 other tissues See more Orthologs human all
Genomic context
Location: 8; 8 A1.1 See Cdc16 in Genome Data Viewer
Exon count: 19
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (13757649..13781951)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (13757690..13781882)
Chromosome 8 - NC_000074.6
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Transcript information: This gene has 5 transcripts
Gene: Cdc16 ENSMUSG00000038416
Description CDC16 cell division cycle 16 [Source:MGI Symbol;Acc:MGI:1917207] Gene Synonyms 2700071J12Rik, 2810431D22Rik Location Chromosome 8: 13,757,676-13,781,938 forward strand. GRCm38:CM001001.2 About this gene This gene has 5 transcripts (splice variants), 203 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cdc16-201 ENSMUST00000043962.8 2301 620aa ENSMUSP00000047950.8 Protein coding CCDS22114 Q3TI84 Q8R349 TSL:1 GENCODE basic APPRIS P1
Cdc16-204 ENSMUST00000134645.7 926 85aa ENSMUSP00000147594.1 Protein coding - Q3TZ92 CDS 3' incomplete TSL:1
Cdc16-203 ENSMUST00000130173.8 923 188aa ENSMUSP00000147399.1 Protein coding - A0A1B0GR68 CDS 3' incomplete TSL:5
Cdc16-205 ENSMUST00000137360.1 884 No protein - Retained intron - - TSL:1
Cdc16-202 ENSMUST00000129872.1 811 No protein - Retained intron - - TSL:1
44.26 kb Forward strand 13.75Mb 13.76Mb 13.77Mb 13.78Mb 13.79Mb Genes (Comprehensive set... Cdc16-201 >protein coding Upf3a-201 >protein coding
Cdc16-203 >protein coding Cdc16-205 >retained intronUpf3a-203 >retained intron
Cdc16-204 >protein coding Upf3a-202 >retained intron
Cdc16-202 >retained intron
Contigs < AC134623.5 AC139186.4 > Regulatory Build
13.75Mb 13.76Mb 13.77Mb 13.78Mb 13.79Mb Reverse strand 44.26 kb
Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site
Gene Legend Protein Coding
merged Ensembl/Havana Ensembl protein coding
Non-Protein Coding
processed transcript
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Transcript: ENSMUST00000043962
24.26 kb Forward strand
Cdc16-201 >protein coding
ENSMUSP00000047... MobiDB lite Superfamily Tetratricopeptide-like helical domain superfamily SMART Tetratricopeptide repeat Pfam PF12895 PF13424
PROSITE profiles Tetratricopeptide repeat-containing domain
Tetratricopeptide repeat PANTHER PTHR12558:SF9
PTHR12558
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 480 540 620
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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