http://beta.alphaknockout.cyagen.net

Mouse Rragc Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Rragc gene ( NCBI Reference Sequence: NM_017475.2 ; Ensembl: ENSMUSG00000028646 ) is located on mouse chromosome 4. 7 exons are identified , with the ATG start codon in exon 1 and the TAG stop codon in exon 7 (Transcript Rragc- 201: ENSMUST00000030399). Exon 3~5 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 3 starts from about 36.77% of the coding region. Exon 3~5 covers 38.36% of the coding region. The size of effective KO region: ~3939 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of RNA splicing and protein translation can not be predicted at existing technological level. Since the null allele lead to embronic lethalty, we highly recommond you thinking of generation a conditional knockout model. You can get the KO type by crossing with a deleter mice.

Page 1 of 9 http://beta.alphaknockout.cyagen.net

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 3 4 5 7

Legends Exon of mouse Rragc Knockout region

Page 2 of 9 http://beta.alphaknockout.cyagen.net

Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 898 bp section upstream 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.

Overview of the Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 5 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.

Page 3 of 9 http://beta.alphaknockout.cyagen.net

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

Sequence 12

Summary: Full Length(898bp) | A(25.39% 228) | C(18.04% 162) | G(21.71% 195) | T(34.86% 313)

Note: The 898 bp section upstream 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.

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

Sequence 12

Summary: Full Length(2000bp) | A(29.05% 581) | C(19.75% 395) | G(22.45% 449) | T(28.75% 575)

Note: The 2000 bp section downstream of Exon 5 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.

Page 4 of 9 http://beta.alphaknockout.cyagen.net

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 898 1 898 898 100.0% chr4 + 123920054 123920951 898 browser details YourSeq 23 875 898 898 100.0% chr1 + 22299809 22299898 90 browser details YourSeq 22 1 22 898 100.0% chr8 - 104078000 104078021 22 browser details YourSeq 22 440 461 898 100.0% chr17_JH584309_alt - 5495 5516 22 What is chro m_alt? browser details YourSeq 22 440 461 898 100.0% chr17_JH584266_alt - 4106140 4106161 22 What is chro m_alt? browser details YourSeq 21 550 571 898 100.0% chr10 - 65362354 65362376 23 browser details YourSeq 21 354 374 898 100.0% chr13 + 33835956 33835976 21 browser details YourSeq 21 610 630 898 100.0% chr12_GL456074_alt + 67221 67241 21 What is chro m_alt? browser details YourSeq 21 877 897 898 100.0% chr10 + 128959870 128959890 21 browser details YourSeq 20 341 360 898 100.0% chr11 - 65695380 65695399 20 browser details YourSeq 20 38 57 898 100.0% chr10 + 55436229 55436248 20

Note: The 898 bp section upstream of Exon 3 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% chr4 + 123924891 123926890 2000 browser details YourSeq 340 1289 1754 2000 90.4% chr8 - 78485974 78486459 486 browser details YourSeq 336 1288 1755 2000 90.0% chr6 + 145266351 145266838 488 browser details YourSeq 333 1288 1749 2000 90.2% chr16 + 30139410 30139887 478 browser details YourSeq 327 1289 1756 2000 89.5% chr12 + 110315942 110316429 488 browser details YourSeq 326 1288 1749 2000 89.8% chr5 - 137992992 137993473 482 browser details YourSeq 319 1299 1749 2000 90.9% chr12 + 85255462 85255948 487 browser details YourSeq 316 1287 1754 2000 89.8% chr18 - 65418910 65419397 488 browser details YourSeq 313 1290 1754 2000 90.3% chr4 + 91037853 91038334 482 browser details YourSeq 310 1288 1754 2000 87.6% chr18 + 35770269 35770727 459 browser details YourSeq 308 1291 1754 2000 88.0% chr5 - 30240015 30240494 480 browser details YourSeq 308 1287 1749 2000 90.6% chr18 - 30419628 30420110 483 browser details YourSeq 302 1288 1746 2000 90.0% chr5 - 66174911 66175393 483 browser details YourSeq 299 1291 1756 2000 88.7% chr19 + 61134789 61135246 458 browser details YourSeq 297 1291 1740 2000 89.9% chr15 + 90723589 90724046 458 browser details YourSeq 293 1289 1741 2000 90.4% chr6 - 88232330 88232785 456 browser details YourSeq 290 989 1741 2000 91.7% chr9 + 44212781 44410188 197408 browser details YourSeq 288 1206 1747 2000 90.0% chrX + 134551758 134552304 547 browser details YourSeq 268 1288 1754 2000 87.3% chr8 - 25788851 25789315 465 browser details YourSeq 258 1288 1747 2000 88.9% chr4 + 28612277 28612804 528

Note: The 2000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 9 http://beta.alphaknockout.cyagen.net Gene and protein information: Rragc Ras-related GTP binding C [ Mus musculus (house mouse) ] Gene ID: 54170, updated on 23-Jun-2021

Gene summary

Official Symbol Rragc provided by MGI Official Full Name Ras-related GTP binding C provided by MGI Primary source MGI:MGI:1858751 See related Ensembl:ENSMUSG00000028646 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 Gtr; Gtr2; RAGC; TIB92; TIB929; YGR163; YGR163W; AU041672 Expression Ubiquitous expression in placenta adult (RPKM 33.1), subcutaneous fat pad adult (RPKM 30.2) and 28 other tissues See Orthologs more human all NEW Try the new Gene table Try the new Transcript table

Genomic context

Location: 4; 4 D2.2 See Rragc in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

109 current GRCm39 (GCF_000001635.27) 4 NC_000070.7 (123811226..123830790)

108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (123917433..123936997)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (123594676..123614240)

Chromosome 4 - NC_000070.7

Page 6 of 9 http://beta.alphaknockout.cyagen.net

Transcript information: This gene has 5 transcripts

Gene: Rragc ENSMUSG00000028646

Description Ras-related GTP binding C [Source:MGI Symbol;Acc:MGI:1858751] Gene Synonyms Gtr2, TIB929, YGR163W Location Chromosome 4: 123,811,239-123,830,790 forward strand. GRCm39:CM000997.3 About this gene This gene has 5 transcripts (splice variants), 283 orthologues and 3 paralogues. Transcripts

UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match

Rragc- ENSMUST00000030399.7 2593 398aa ENSMUSP00000030399.7 Protein coding CCDS18622 Q99K70-1 GENCODE 201 basic APPRIS P1 TSL:1

Rragc- ENSMUST00000155757.8 2950 182aa ENSMUSP00000115232.2 Nonsense mediated - Q99K70-2 TSL:1 205 decay

Rragc- ENSMUST00000155454.2 599 No - Processed transcript - - TSL:2 204 protein

Rragc- ENSMUST00000143635.2 377 No - Processed transcript - - TSL:3 203 protein

Rragc- ENSMUST00000133445.8 642 No - Retained intron - - TSL:2 202 protein

Page 7 of 9 http://beta.alphaknockout.cyagen.net

39.55 kb Forward strand 123.81Mb 123.82Mb 123.83Mb 123.84Mb Genes (Comprehensive set... Mycbp-201 >protein coding Rragc-201 >protein coding

Mycbp-202 >protein coding Rragc-205 >nonsense mediated decay

Mycbp-204 >processed transcript Rragc-203 >processed transcript

Mycbp-203 >processed transcript Rragc-202 >retained intron

Rragc-204 >processed transcript

Gm12902-201 >processed pseudogene

Contigs AL606962.22 > Genes < Gm12905-201lncRNA < Gm12905-203lncRNA (Comprehensive set...

< Gm12905-204retained intron

Regulatory Build

123.81Mb 123.82Mb 123.83Mb 123.84Mb Reverse strand 39.55 kb

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene pseudogene

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Page 8 of 9 http://beta.alphaknockout.cyagen.net

Transcript: ENSMUST00000030399

19.55 kb Forward strand

Rragc-201 >protein coding

ENSMUSP00000030... MobiDB lite Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase Pfam Gtr1/RagA G protein PANTHER Gtr1/RagA G protein

PTHR11259:SF6 Gene3D 3.40.50.300 3.30.450.190

CDD RagC/D

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

Variant Legend missense variant

Scale bar 0 40 80 120 160 200 240 280 320 398

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

Page 9 of 9