https://www.alphaknockout.com

Mouse Atp6v1g3 Knockout Project (CRISPR/Cas9)

Objective: To create a Atp6v1g3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Atp6v1g3 (NCBI Reference Sequence: NM_177397 ; Ensembl: ENSMUSG00000026394 ) is located on Mouse 1. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000027643). Exon 2 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:

Exon 2 starts from about 23.45% of the coding region. Exon 2 covers 28.53% of the coding region. The size of effective KO region: ~101 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

Legends Exon of mouse Atp6v1g3 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. 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(32.95% 659) | C(18.5% 370) | T(27.6% 552) | G(20.95% 419)

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(2000bp) | A(31.25% 625) | C(20.2% 404) | T(31.4% 628) | G(17.15% 343)

Note: The 2000 bp section downstream 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.

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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% chr1 + 138281667 138283666 2000 browser details YourSeq 50 1848 1947 2000 84.8% chr13 + 74344692 74344795 104 browser details YourSeq 39 435 523 2000 93.4% chr11 + 43435956 43436217 262 browser details YourSeq 35 553 608 2000 97.4% chr3 + 74182961 74183072 112 browser details YourSeq 35 462 531 2000 85.8% chr2 + 127223480 127223548 69 browser details YourSeq 32 1750 1812 2000 86.4% chr15 - 76235308 76235371 64 browser details YourSeq 30 462 522 2000 89.5% chr13 - 107189549 107189611 63 browser details YourSeq 30 1644 1673 2000 100.0% chr11 - 110282958 110282987 30 browser details YourSeq 30 1781 1816 2000 91.7% chr11 - 77414574 77414609 36 browser details YourSeq 30 1780 1829 2000 80.0% chr13 + 53287121 53287170 50 browser details YourSeq 29 1918 1948 2000 96.8% chr11 - 22777856 22777886 31 browser details YourSeq 29 1780 1814 2000 91.5% chr17 + 65420956 65420990 35 browser details YourSeq 29 462 523 2000 74.6% chr12 + 85384585 85384648 64 browser details YourSeq 29 1641 1671 2000 96.8% chr1 + 131585438 131585468 31 browser details YourSeq 28 1641 1668 2000 100.0% chr6 + 42525446 42525473 28 browser details YourSeq 28 462 495 2000 91.2% chr18 + 65657885 65657918 34 browser details YourSeq 28 1916 1947 2000 93.8% chr11 + 62057836 62057867 32 browser details YourSeq 27 1780 1829 2000 76.8% chr19 - 6678443 6678491 49 browser details YourSeq 27 1780 1812 2000 91.0% chr1 - 184364184 184364216 33 browser details YourSeq 27 1843 1871 2000 96.6% chr3 + 89085945 89085973 29

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 2000 1 2000 2000 100.0% chr1 + 138283768 138285767 2000 browser details YourSeq 95 640 835 2000 90.9% chr11 + 33507216 33507446 231 browser details YourSeq 67 675 1188 2000 69.3% chr10 - 104653583 104653748 166 browser details YourSeq 66 691 855 2000 93.5% chr1 + 59514586 59514753 168 browser details YourSeq 65 675 852 2000 92.2% chr11 + 94908695 94909140 446 browser details YourSeq 63 647 827 2000 91.0% chr11 + 105233299 105233797 499 browser details YourSeq 58 648 833 2000 95.4% chr1 - 125543611 125543888 278 browser details YourSeq 58 647 738 2000 83.2% chr1 - 122129139 122129237 99 browser details YourSeq 58 646 735 2000 92.7% chr1 - 16554990 16555085 96 browser details YourSeq 58 646 783 2000 94.1% chr12 + 59080788 59080964 177 browser details YourSeq 57 641 807 2000 94.0% chr11 + 120355079 120355254 176 browser details YourSeq 55 646 723 2000 87.7% chr3 - 58447635 58447717 83 browser details YourSeq 55 719 802 2000 83.4% chr13 - 43611516 43611600 85 browser details YourSeq 55 649 728 2000 92.4% chr11 + 6170587 6170670 84 browser details YourSeq 54 703 789 2000 92.1% chr5 - 31676392 31676494 103 browser details YourSeq 52 646 725 2000 93.4% chr11 - 68860909 68860993 85 browser details YourSeq 52 675 775 2000 94.9% chr18 + 77871126 77871232 107 browser details YourSeq 52 646 738 2000 90.7% chr12 + 98341761 98341860 100 browser details YourSeq 52 687 773 2000 89.4% chr1 + 192517832 192517923 92 browser details YourSeq 49 648 754 2000 98.1% chr4 + 124761752 124761866 115

Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Atp6v1g3 ATPase, H+ transporting, lysosomal V1 subunit G3 [ Mus musculus (house mouse) ] Gene ID: 338375, updated on 12-Aug-2019

Gene summary

Official Symbol Atp6v1g3 provided by MGI Official Full Name ATPase, H+ transporting, lysosomal V1 subunit G3 provided by MGI Primary source MGI:MGI:2450548 See related Ensembl:ENSMUSG00000026394 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 Expression Biased expression in kidney adult (RPKM 3.8) and genital fat pad adult (RPKM 1.2) See more Orthologs human all

Genomic context

Location: 1; 1 E4 See Atp6v1g3 in Genome Data Viewer

Exon count: 3

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (138273738..138289462)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (140170315..140186039)

Chromosome 1 - NC_000067.6

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Transcript information: This gene has 1 transcript

Gene: Atp6v1g3 ENSMUSG00000026394

Description ATPase, H+ transporting, lysosomal V1 subunit G3 [Source:MGI Symbol;Acc:MGI:2450548] Location : 138,273,738-138,289,462 forward strand. GRCm38:CM000994.2 About this gene This gene has 1 transcript (splice variant), 130 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Atp6v1g3-201 ENSMUST00000027643.5 1887 118aa ENSMUSP00000027643.4 Protein coding CCDS15331 Q8BMC1 TSL:1 GENCODE basic APPRIS P1

35.73 kb Forward strand

138.27Mb 138.28Mb 138.29Mb (Comprehensive set... Atp6v1g3-201 >protein coding

Contigs AC116868.9 > Regulatory Build

138.27Mb 138.28Mb 138.29Mb Reverse strand 35.73 kb

Regulation Legend

CTCF Enhancer Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana

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Transcript: ENSMUST00000027643

15.72 kb Forward strand

Atp6v1g3-201 >protein coding

ENSMUSP00000027... MobiDB lite Coiled-coils (Ncoils) TIGRFAM Vacuolar (H+)-ATPase G subunit Pfam Vacuolar (H+)-ATPase G subunit PANTHER PTHR12713:SF5

Vacuolar (H+)-ATPase G subunit Gene3D 1.20.5.620

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant synonymous variant

Scale bar 0 10 20 30 40 50 60 70 80 90 100 118

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

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