https://www.alphaknockout.com

Mouse Btg3 Knockout Project (CRISPR/Cas9)

Objective: To create a Btg3 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Btg3 (NCBI Reference Sequence: NM_009770 ; Ensembl: ENSMUSG00000022863 ) is located on Mouse 16. 5 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000023570). Exon 2~3 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 increased incidence of lung tumors.

Exon 2 starts from the coding region. Exon 2~3 covers 41.14% of the coding region. The size of effective KO region: ~4311 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 5

Legends Exon of mouse Btg3 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 2000 bp section downstream of Exon 3 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(25.3% 506) | C(20.3% 406) | T(34.2% 684) | G(20.2% 404)

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(27.9% 558) | C(20.45% 409) | T(30.3% 606) | G(21.35% 427)

Note: The 2000 bp section downstream of Exon 3 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% chr16 - 78373418 78375417 2000 browser details YourSeq 96 1106 1266 2000 96.3% chr15 + 91792885 91793118 234 browser details YourSeq 92 1166 1262 2000 98.0% chr9 + 41401606 41401717 112 browser details YourSeq 90 1184 1276 2000 99.0% chr11 + 61984775 61984891 117 browser details YourSeq 89 1152 1262 2000 95.9% chr1 - 71767529 71767661 133 browser details YourSeq 85 1178 1262 2000 100.0% chr7 + 26202685 26202769 85 browser details YourSeq 83 1178 1262 2000 98.9% chr8 + 39042291 39042375 85 browser details YourSeq 83 1178 1262 2000 98.9% chr17 + 43285003 43285087 85 browser details YourSeq 83 1178 1262 2000 98.9% chr13 + 20299927 20300011 85 browser details YourSeq 82 1178 1263 2000 98.9% chr15 + 85357425 85357526 102 browser details YourSeq 82 904 1262 2000 94.7% chr12 + 73130940 73131447 508 browser details YourSeq 81 1166 1262 2000 94.6% chr5 - 103082825 103083061 237 browser details YourSeq 81 1178 1262 2000 98.9% chr17 - 40837961 40838059 99 browser details YourSeq 81 1178 1262 2000 98.9% chr12 + 103986734 103986888 155 browser details YourSeq 81 1178 1262 2000 97.7% chr1 + 11339013 11339097 85 browser details YourSeq 78 1181 1271 2000 94.4% chr12 - 12524484 12524583 100 browser details YourSeq 78 1178 1262 2000 96.5% chr1 - 9581751 9581838 88 browser details YourSeq 77 1181 1268 2000 94.4% chr6 - 26082296 26082388 93 browser details YourSeq 77 1183 1262 2000 98.8% chr1 - 71767473 71767600 128 browser details YourSeq 76 1181 1266 2000 94.2% chr1 - 189961290 189961375 86

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% chr16 - 78367113 78369112 2000 browser details YourSeq 372 1316 1762 2000 95.2% chr2 - 51696040 51999520 303481 browser details YourSeq 372 1366 1887 2000 95.4% chr18 - 24829776 24830537 762 browser details YourSeq 367 1368 1762 2000 96.5% chr13 + 39514565 39514959 395 browser details YourSeq 366 1368 1761 2000 96.5% chr3 - 102904587 102904980 394 browser details YourSeq 366 1368 1761 2000 96.5% chr3 - 102906071 102906464 394 browser details YourSeq 366 1368 1761 2000 96.5% chr5 + 108493138 108493531 394 browser details YourSeq 366 1367 1768 2000 95.8% chr18 + 29113473 29113875 403 browser details YourSeq 366 1368 1761 2000 96.5% chr18 + 12713555 12713948 394 browser details YourSeq 365 1365 1762 2000 96.0% chr6 - 24143299 24143696 398 browser details YourSeq 365 1368 1762 2000 96.3% chr3 - 105332089 105332483 395 browser details YourSeq 365 1367 1761 2000 96.5% chr17 - 51851183 51851579 397 browser details YourSeq 365 1368 1761 2000 96.5% chr2 + 27787519 27787913 395 browser details YourSeq 365 1368 1759 2000 96.7% chr2 + 6132463 6132855 393 browser details YourSeq 365 1368 1762 2000 96.3% chr1 + 151744297 151744691 395 browser details YourSeq 365 1368 1774 2000 94.8% chr1 + 23236366 23236770 405 browser details YourSeq 364 1366 1763 2000 95.3% chr4 - 44946795 44947191 397 browser details YourSeq 364 1342 1761 2000 93.3% chr19 - 24240116 24240519 404 browser details YourSeq 364 1366 1760 2000 96.3% chr16 - 28492330 28492725 396 browser details YourSeq 364 1368 1762 2000 96.3% chr14 - 24417487 24417882 396

Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Btg3 BTG anti-proliferation factor 3 [ Mus musculus (house mouse) ] Gene ID: 12228, updated on 27-Aug-2019

Gene summary

Official Symbol Btg3 provided by MGI Official Full Name BTG anti-proliferation factor 3 provided by MGI Primary source MGI:MGI:109532 See related Ensembl:ENSMUSG00000022863 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 ANA; tob5 Summary This gene encodes B cell translocation gene 3, a member of the BTG gene family. This family is defined by a conserved N- Expression terminal domain, known to bind transcription factors, and a less conserved C-terminal domain. This protein is thought to have anti-proliferative properties, and may be involved in regulating the G1-S transition to suppress cell cycle progression. Mice deficient for this gene display an increased incidence of lung cancers, and many human lung cancer cells exhibit decreased levels of B cell translocation gene 3. Alternate splicing results in multiple transcript variants. A pseudogene of this gene is found on chromosome 17. [provided by RefSeq, Jul 2014] Orthologs Broad expression in genital fat pad adult (RPKM 36.7), placenta adult (RPKM 14.6) and 20 other tissues See more human all

Genomic context

Location: 16; 16 C3.1 See Btg3 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 16 NC_000082.6 (78359860..78377181, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 16 NC_000082.5 (78360105..78377030, complement)

Chromosome 16 - NC_000082.6

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Transcript information: This gene has 3 transcripts

Gene: Btg3 ENSMUSG00000022863

Description B cell translocation gene 3 [Source:MGI Symbol;Acc:MGI:109532] Gene Synonyms ANA, tob5 Location Chromosome 16: 78,332,637-78,377,192 reverse strand. GRCm38:CM001009.2 About this gene This gene has 3 transcripts (splice variants), 213 orthologues, 5 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Btg3-201 ENSMUST00000023570.13 1414 252aa ENSMUSP00000023570.7 Protein coding CCDS28277 P50615 Q52L83 TSL:1 GENCODE basic APPRIS P1

Btg3-203 ENSMUST00000231353.1 3013 152aa ENSMUSP00000156138.1 Protein coding - A0A338P787 CDS 5' incomplete

Btg3-202 ENSMUST00000148124.1 442 137aa ENSMUSP00000119706.1 Protein coding - D3Z1M9 CDS 3' incomplete TSL:5

64.56 kb Forward strand 78.33Mb 78.34Mb 78.35Mb 78.36Mb 78.37Mb 78.38Mb Cxadr-205 >protein coding (Comprehensive set...

Cxadr-201 >protein coding

Cxadr-202 >protein coding

Cxadr-204 >protein coding

Cxadr-206 >retained intron

Cxadr-203 >nonsense mediated decay

Contigs AC130844.3 >

Genes (Comprehensive set... < Btg3-203protein coding

< Gm49590-201TEC < Btg3-201protein coding

< Btg3-202protein coding

Regulatory Build

78.33Mb 78.34Mb 78.35Mb 78.36Mb 78.37Mb 78.38Mb Reverse strand 64.56 kb

Regulation Legend

CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000023570

< Btg3-201protein coding

Reverse strand 16.95 kb

ENSMUSP00000023... MobiDB lite Low complexity (Seg) Superfamily BTG-like domain superfamily SMART Anti-proliferative protein Prints Anti-proliferative protein Pfam Anti-proliferative protein PROSITE patterns Anti-proliferative protein

Anti-proliferative protein PANTHER PTHR22978

PTHR22978:SF6 Gene3D BTG-like domain superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 252

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

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