https://www.alphaknockout.com

Mouse Rab39b Knockout Project (CRISPR/Cas9)

Objective: To create a Rab39b knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rab39b (NCBI Reference Sequence: NM_175122 ; Ensembl: ENSMUSG00000031202 ) is located on Mouse X. 2 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 2 (Transcript: ENSMUST00000033545). Exon 1~2 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 1 starts from about 0.16% of the coding region. Exon 1~2 covers 100.0% of the coding region. The size of effective KO region: ~3422 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

Legends Exon of mouse Rab39b 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 start codon 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 stop codon 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(34.05% 681) | C(18.35% 367) | T(24.65% 493) | G(22.95% 459)

Note: The 2000 bp section upstream of start codon 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(34.15% 683) | C(17.95% 359) | T(30.0% 600) | G(17.9% 358)

Note: The 2000 bp section downstream of stop codon 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% chrX - 75578008 75580007 2000 browser details YourSeq 442 112 859 2000 87.4% chrX - 153530274 153531186 913 browser details YourSeq 424 299 952 2000 86.9% chr5 + 101047598 101048198 601 browser details YourSeq 414 310 879 2000 89.3% chr6 - 113053255 113054024 770 browser details YourSeq 414 306 876 2000 88.9% chr13 - 109594297 109594890 594 browser details YourSeq 414 304 880 2000 88.9% chr18 + 11764766 11765367 602 browser details YourSeq 412 299 877 2000 88.6% chr13 + 81258903 81259514 612 browser details YourSeq 401 304 877 2000 89.0% chr14 + 73439072 73439653 582 browser details YourSeq 396 299 860 2000 92.0% chr18 + 63984320 63984914 595 browser details YourSeq 394 299 845 2000 88.4% chr7 - 75940747 75941317 571 browser details YourSeq 394 299 878 2000 89.5% chr16 - 42239934 42240528 595 browser details YourSeq 394 92 876 2000 86.0% chr13 + 64846701 64847461 761 browser details YourSeq 393 306 876 2000 87.8% chr5 - 107321101 107321705 605 browser details YourSeq 390 324 876 2000 87.1% chrX + 72565152 72565729 578 browser details YourSeq 389 306 877 2000 88.8% chr18 + 10427274 10427865 592 browser details YourSeq 387 299 877 2000 86.5% chr6 - 81234078 81234688 611 browser details YourSeq 386 299 783 2000 90.1% chr5 - 89961739 89962250 512 browser details YourSeq 386 307 878 2000 86.3% chr14 - 74471648 74472243 596 browser details YourSeq 386 304 876 2000 87.7% chr19 + 19139164 19139810 647 browser details YourSeq 383 299 876 2000 87.7% chr10 - 3676771 3677375 605

Note: The 2000 bp section upstream of start codon 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% chrX - 75572584 75574583 2000 browser details YourSeq 24 955 986 2000 96.2% chr3 - 96700930 96700962 33 browser details YourSeq 24 1517 1542 2000 96.2% chr5_JH584299_random + 572276 572301 26 browser details YourSeq 24 1517 1542 2000 96.2% chr5_JH584299_random + 932056 932081 26 browser details YourSeq 24 1517 1542 2000 96.2% chr5 + 95612691 95612716 26

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

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Gene and information: Rab39b RAB39B, member RAS oncogene family [ Mus musculus (house mouse) ] Gene ID: 67790, updated on 12-Aug-2019

Gene summary

Official Symbol Rab39b provided by MGI Official Full Name RAB39B, member RAS oncogene family provided by MGI Primary source MGI:MGI:1915040 See related Ensembl:ENSMUSG00000031202 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 6330580M05Rik Summary This gene encodes a member of the Ras-related small GTPases, which regulate membrane trafficking in organelles and Expression transport vesicles. This protein has been reported to be enriched in mouse brain, and specifically within neurons, and may play a role in synapse formation. In humans mutations in this gene are associated with X-linked cognitive disability. [provided by RefSeq, Jun 2013] Orthologs Biased expression in cerebellum adult (RPKM 6.5), CNS E18 (RPKM 6.4) and 6 other tissues See more human all

Genomic context

Location: X 38.26 cM; X A7.3 See Rab39b in Genome Data Viewer Exon count: 2

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (75572045..75578231, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (72817397..72823543, complement)

Chromosome X - NC_000086.7

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Transcript information: This gene has 1 transcript

Gene: Rab39b ENSMUSG00000031202

Description RAB39B, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:1915040] Gene Synonyms 6330580M05Rik Location Chromosome X: 75,572,046-75,578,231 reverse strand. GRCm38:CM001013.2 About this gene This gene has 1 transcript (splice variant), 241 orthologues, 62 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rab39b-201 ENSMUST00000033545.5 3401 213aa ENSMUSP00000033545.5 Protein coding CCDS30242 Q0PD14 Q8BHC1 TSL:1 GENCODE basic APPRIS P1

26.19 kb Forward strand 75.565Mb 75.570Mb 75.575Mb 75.580Mb 75.585Mb Gm15063-201 >lncRNA (Comprehensive set...

Contigs AL671860.6 > Genes (Comprehensive set... < Rab39b-201protein coding < Gm25421-201snRNA

Regulatory Build

75.565Mb 75.570Mb 75.575Mb 75.580Mb 75.585Mb Reverse strand 26.19 kb

Regulation Legend CTCF Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene

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Transcript: ENSMUST00000033545

< Rab39b-201protein coding

Reverse strand 6.19 kb

ENSMUSP00000033... TIGRFAM Small GTP-binding protein domain Superfamily P-loop containing nucleoside triphosphate hydrolase SMART SM00174

SM00175

SM00173

SM00176 Prints PR00449 Pfam Small GTPase PROSITE profiles PS51419 PANTHER PTHR24073:SF916

PTHR24073 Gene3D 3.40.50.300 CDD Rab39

Scale bar 0 20 40 60 80 100 120 140 160 180 213

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

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