https://www.alphaknockout.com
Mouse Zfyve28 Knockout Project (CRISPR/Cas9)
Objective: To create a Zfyve28 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Zfyve28 gene (NCBI Reference Sequence: NM_001015039 ; Ensembl: ENSMUSG00000037224 ) is located on Mouse chromosome 5. 13 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 13 (Transcript: ENSMUST00000094868). Exon 2~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit normal kidney morphology and function.
Exon 2 starts from about 1.47% of the coding region. Exon 2~4 covers 17.75% of the coding region. The size of effective KO region: ~9029 bp. The KO 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 2 3 4 13
Legends Exon of mouse Zfyve28 Knockout region
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Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.
Overview of the Dot Plot (down) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 890 bp section downstream of Exon 4 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.
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Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(25.65% 513) | C(21.1% 422) | T(25.3% 506) | G(27.95% 559)
Note: The 2000 bp section upstream of Exon 2 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.
Overview of the GC Content Distribution (down) Window size: 300 bp
Sequence 12
Summary: Full Length(890bp) | A(22.7% 202) | C(20.9% 186) | T(24.38% 217) | G(32.02% 285)
Note: The 890 bp section downstream of Exon 4 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 2000 1 2000 2000 100.0% chr5 - 34243314 34245313 2000 browser details YourSeq 135 573 733 2000 92.0% chr10 - 41671538 41671693 156 browser details YourSeq 133 558 733 2000 95.4% chr10 - 93468193 93468393 201 browser details YourSeq 130 571 735 2000 95.1% chr17 + 89776004 89776223 220 browser details YourSeq 129 575 733 2000 88.2% chr4 - 134885624 134885768 145 browser details YourSeq 129 574 733 2000 90.3% chr4 + 76994986 76995139 154 browser details YourSeq 126 574 741 2000 94.8% chr11 + 18527426 18527619 194 browser details YourSeq 125 575 722 2000 92.7% chr5 - 61092735 61092877 143 browser details YourSeq 124 575 737 2000 90.5% chr5 + 50660651 50660807 157 browser details YourSeq 124 583 733 2000 95.2% chr4 + 141128856 141129218 363 browser details YourSeq 123 573 732 2000 88.3% chr18 - 80792734 80792873 140 browser details YourSeq 123 574 796 2000 94.6% chr12 + 72491859 72492475 617 browser details YourSeq 122 573 726 2000 89.7% chr6 - 140660037 140660181 145 browser details YourSeq 122 558 737 2000 83.4% chr3 + 13130937 13131102 166 browser details YourSeq 121 554 732 2000 92.4% chr8 + 111253069 111253581 513 browser details YourSeq 121 575 725 2000 88.5% chr18 + 79899187 79899317 131 browser details YourSeq 120 575 726 2000 88.6% chr7 - 39833780 39833917 138 browser details YourSeq 120 574 733 2000 87.8% chr2 - 97526508 97526656 149 browser details YourSeq 120 571 708 2000 93.0% chr15 - 28767531 28767661 131 browser details YourSeq 120 558 709 2000 97.1% chr10 - 93468168 93468417 250
Note: The 2000 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 890 1 890 890 100.0% chr5 - 34233395 34234284 890 browser details YourSeq 27 648 690 890 76.7% chr10 - 75280471 75280506 36 browser details YourSeq 25 802 831 890 82.2% chr11 + 115935861 115935888 28 browser details YourSeq 22 386 409 890 95.9% chr14 + 64191281 64191304 24 browser details YourSeq 21 643 663 890 100.0% chr11 - 106180468 106180488 21 browser details YourSeq 21 845 865 890 100.0% chr9 + 123399931 123399951 21 browser details YourSeq 20 674 693 890 100.0% chr1 - 40106323 40106342 20 browser details YourSeq 20 161 180 890 100.0% chr14 + 68325877 68325896 20
Note: The 890 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Zfyve28 zinc finger, FYVE domain containing 28 [ Mus musculus (house mouse) ] Gene ID: 231125, updated on 12-Aug-2019
Gene summary
Official Symbol Zfyve28 provided by MGI Official Full Name zinc finger, FYVE domain containing 28 provided by MGI Primary source MGI:MGI:2684992 See related Ensembl:ENSMUSG00000037224 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 Gm146; mKIAA1643; 9630058O20Rik Expression Biased expression in cerebellum adult (RPKM 4.9), cortex adult (RPKM 3.0) and 12 other tissues See more Orthologs human all
Genomic context
Location: 5; 5 B2 See Zfyve28 in Genome Data Viewer Exon count: 21
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (34194893..34288368, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (34537543..34630973, complement)
Chromosome 5 - NC_000071.6
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Transcript information: This gene has 6 transcripts
Gene: Zfyve28 ENSMUSG00000037224
Description zinc finger, FYVE domain containing 28 [Source:MGI Symbol;Acc:MGI:2684992] Gene Synonyms 9630058O20Rik Location Chromosome 5: 34,194,893-34,288,449 reverse strand. GRCm38:CM000998.2 About this gene This gene has 6 transcripts (splice variants), 201 orthologues, 12 paralogues, is a member of 1 Ensembl protein family and is associated with 12 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Zfyve28-201 ENSMUST00000094868.9 3983 905aa ENSMUSP00000092464.3 Protein coding CCDS39068 Q6ZPK7 TSL:1 GENCODE basic APPRIS P1
Zfyve28-202 ENSMUST00000114368.1 712 137aa ENSMUSP00000110008.1 Protein coding - D3Z3F0 CDS 3' incomplete TSL:3
Zfyve28-204 ENSMUST00000114370.7 1499 No protein - Retained intron - - TSL:1
Zfyve28-206 ENSMUST00000132104.1 666 No protein - Retained intron - - TSL:3
Zfyve28-203 ENSMUST00000114369.2 1086 No protein - lncRNA - - TSL:5
Zfyve28-205 ENSMUST00000130435.4 650 No protein - lncRNA - - TSL:3
113.56 kb Forward strand 34.20Mb 34.22Mb 34.24Mb 34.26Mb 34.28Mb Genes Gm42848-201 >lncRNA Cfap99-201 >protein coding (Comprehensive set...
Contigs < AC104889.10 Genes < Mxd4-201protein coding < Gm15513-201lncRNA< Zfyve28-203lncRNA (Comprehensive set...
< Mxd4-202protein coding < Zfyve28-206retained intro < Zfyve28-201protein coding < Zfyve28-205lncRNA < Zfyve28-204retained intron Regulatory Build 34.20Mb 34.22Mb 34.24Mb 34.26Mb 34.28Mb Reverse strand 113.56 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 RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000094868 < Zfyve28-201protein coding Reverse strand 93.43 kb ENSMUSP00000092... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Zinc finger, FYVE/PHD-type SMART FYVE zinc finger Pfam FYVE zinc finger PROSITE profiles Zinc finger, FYVE-related PANTHER PTHR46465 Gene3D Zinc finger, RING/FYVE/PHD-type CDD cd15731 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 80 160 240 320 400 480 560 640 720 800 905 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8