https://www.alphaknockout.com

Mouse Ndufb6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ndufb6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ndufb6 gene (NCBI Reference Sequence: NM_001033305 ; Ensembl: ENSMUSG00000071014 ) is located on Mouse chromosome 4. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000095128). Exon 1~2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ndufb6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-167E21 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 1~2 covers 71.09% of the coding region. Start codon is in exon 1, and stop codon is in exon 4. The size of intron 2 for 3'-loxP site insertion: 4798 bp. The size of effective cKO region: ~1945 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

gRNA region

Wildtype allele A T

5' G gRNA region 3'

1 2 4

Targeting vector A T G

Targeted allele A T G

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Ndufb6 cKO region loxP site

Page 2 of 7 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(8185bp) | A(27.57% 2257) | C(20.21% 1654) | T(29.02% 2375) | G(23.2% 1899)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

Page 3 of 7 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% chr4 - 40279338 40282337 3000 browser details YourSeq 381 43 603 3000 84.7% chr19 + 30370776 30371343 568 browser details YourSeq 354 42 588 3000 82.8% chrX - 139680275 139680822 548 browser details YourSeq 353 43 592 3000 85.0% chrX - 103147331 103147876 546 browser details YourSeq 350 43 589 3000 83.4% chr15 + 94534234 94534790 557 browser details YourSeq 348 43 592 3000 82.7% chr5 - 91921457 91921995 539 browser details YourSeq 340 42 560 3000 88.0% chr8 - 80209466 80536269 326804 browser details YourSeq 340 43 592 3000 83.1% chr4 - 116527507 116528054 548 browser details YourSeq 340 45 579 3000 86.8% chr11 + 111052094 111052666 573 browser details YourSeq 337 45 592 3000 83.2% chr19 - 35978519 35979109 591 browser details YourSeq 333 43 592 3000 88.3% chr11 + 94887657 94888225 569 browser details YourSeq 333 44 572 3000 84.0% chr1 + 38194934 38195460 527 browser details YourSeq 328 43 592 3000 84.2% chrX + 86477767 86478391 625 browser details YourSeq 325 75 610 3000 87.1% chr8 + 112030461 112030995 535 browser details YourSeq 323 43 605 3000 86.5% chr7 - 109304954 109305532 579 browser details YourSeq 323 43 602 3000 88.8% chr3 - 85507188 85507749 562 browser details YourSeq 323 74 592 3000 85.6% chr14 + 87321305 87321835 531 browser details YourSeq 322 43 580 3000 84.5% chr4 - 93918058 94303923 385866 browser details YourSeq 320 41 571 3000 83.1% chr4 - 5216342 5216862 521 browser details YourSeq 320 44 592 3000 84.2% chr13 - 14224258 14224819 562

Note: The 3000 bp section upstream of Exon 1 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% chr4 - 40274403 40277402 3000 browser details YourSeq 166 2734 3000 3000 91.7% chr11 + 97880879 97881142 264 browser details YourSeq 154 2801 3000 3000 94.8% chr11 + 70620329 70620820 492 browser details YourSeq 149 2801 3000 3000 97.5% chr11 - 6425869 6426358 490 browser details YourSeq 142 2801 3000 3000 95.5% chr12 + 117423948 117424315 368 browser details YourSeq 142 2809 3000 3000 91.0% chr10 + 59946991 59947170 180 browser details YourSeq 137 2801 3000 3000 96.1% chr10 - 80041702 80041998 297 browser details YourSeq 133 2812 3000 3000 94.0% chr10 + 71323425 71323615 191 browser details YourSeq 131 2810 3000 3000 98.6% chr1 - 135396934 135397302 369 browser details YourSeq 128 2713 3000 3000 89.4% chr16 - 64337517 64337750 234 browser details YourSeq 124 2877 3000 3000 100.0% chr17 - 34624583 34624706 124 browser details YourSeq 124 2877 3000 3000 100.0% chr14 - 54600143 54600266 124 browser details YourSeq 124 2874 3000 3000 99.3% chr12 - 76855337 76855464 128 browser details YourSeq 124 2877 3000 3000 100.0% chr11 - 117137812 117137935 124 browser details YourSeq 124 2877 3000 3000 100.0% chr15 + 54924672 54924795 124 browser details YourSeq 123 2876 3000 3000 99.2% chr15 + 89153211 89153335 125 browser details YourSeq 122 2877 3000 3000 99.2% chr17 - 48766162 48766285 124 browser details YourSeq 122 2877 3000 3000 99.2% chr17 - 11986122 11986245 124 browser details YourSeq 122 2877 3000 3000 99.2% chr16 - 90270004 90270127 124 browser details YourSeq 122 2877 3000 3000 99.2% chr15 - 83015813 83015936 124

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Ndufb6 NADH:ubiquinone oxidoreductase subunit B6 [ Mus musculus (house mouse) ] Gene ID: 230075, updated on 12-Aug-2019

Gene summary

Official Symbol Ndufb6 provided by MGI Official Full Name NADH:ubiquinone oxidoreductase subunit B6 provided by MGI Primary source MGI:MGI:2684983 See related Ensembl:ENSMUSG00000071014 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Gm137; CI-B17 Summary This gene encodes a subunit of complex I (NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain. This Expression complex functions in electron transport and generation of a proton gradient across the inner mitochondrial membrane to drive ATP synthesis. Data from human cell lines suggests that the encoded subunit may be required for electron transfer activity. [provided by RefSeq, Aug 2015] Orthologs Ubiquitous expression in heart adult (RPKM 89.9), kidney adult (RPKM 75.1) and 28 other tissues See more human all

Genomic context

Location: 4; 4 A5 See Ndufb6 in Genome Data Viewer

Exon count: 4

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (40270591..40279421, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (40217696..40226401, complement)

Chromosome 4 - NC_000070.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 2 transcripts

Gene: Ndufb6 ENSMUSG00000071014

Description NADH:ubiquinone oxidoreductase subunit B6 [Source:MGI Symbol;Acc:MGI:2684983] Gene Synonyms LOC230075 Location Chromosome 4: 40,270,591-40,279,421 reverse strand. GRCm38:CM000997.2 About this gene This gene has 2 transcripts (splice variants), 194 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ndufb6-201 ENSMUST00000095128.9 659 128aa ENSMUSP00000092746.3 Protein coding CCDS18045 A2AP31 Q3UIU2 TSL:1 GENCODE basic APPRIS P1

Ndufb6-202 ENSMUST00000108108.2 480 97aa ENSMUSP00000103743.2 Protein coding - A2AP32 TSL:3 GENCODE basic

28.83 kb Forward strand 40.265Mb 40.270Mb 40.275Mb 40.280Mb 40.285Mb Genes Smim27-201 >protein coding (Comprehensive set...

Smim27-202 >lncRNA

Contigs AL831793.4 >

Genes (Comprehensive set... < Topors-201protein coding < Ndufb6-201protein coding

< Ndufb6-202protein coding

Regulatory Build

40.265Mb 40.270Mb 40.275Mb 40.280Mb 40.285Mb Reverse strand 28.83 kb

Regulation Legend CTCF Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000095128

< Ndufb6-201protein coding

Reverse strand 8.83 kb

ENSMUSP00000092... Transmembrane heli... PDB-ENSP mappings Low complexity (Seg) Pfam NADH dehydrogenase 1, beta subcomplex, subunit 6 PANTHER NADH dehydrogenase 1, beta subcomplex, subunit 6

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 128

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

Page 7 of 7