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Mouse Rab14 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Rab14 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Rab14 gene (NCBI Reference Sequence: NM_026697 ; Ensembl: ENSMUSG00000026878 ) is located on Mouse chromosome 2. 8 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000028238). Exon 3~4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rab14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-198N5 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 3 starts from about 8.22% of the coding region. The knockout of Exon 3~4 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1055 bp, and the size of intron 4 for 3'-loxP site insertion: 3159 bp. The size of effective cKO region: ~2166 bp. The cKO 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 4 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Rab14 Homology arm cKO region loxP site

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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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(8666bp) | A(30.38% 2633) | C(15.79% 1368) | T(33.81% 2930) | G(20.02% 1735)

Note: The sequence of homologous arms and cKO region 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 3000 1 3000 3000 100.0% chr2 - 35191816 35194815 3000 browser details YourSeq 125 386 572 3000 91.9% chr15 - 4095627 4096183 557 browser details YourSeq 124 371 692 3000 78.9% chrX - 86348721 86348924 204 browser details YourSeq 123 369 527 3000 87.5% chr11 - 14687808 14687964 157 browser details YourSeq 118 364 521 3000 89.3% chr5 + 35749734 35750034 301 browser details YourSeq 118 372 527 3000 84.8% chr3 + 90224183 90224333 151 browser details YourSeq 118 372 515 3000 91.6% chr15 + 45673456 45673600 145 browser details YourSeq 117 371 527 3000 83.8% chr16 + 79950145 79950293 149 browser details YourSeq 116 371 521 3000 89.7% chr11 - 110508456 110508613 158 browser details YourSeq 115 371 522 3000 90.2% chr1 - 146143830 146475562 331733 browser details YourSeq 114 371 520 3000 85.0% chr17 - 92548129 92548274 146 browser details YourSeq 114 370 512 3000 86.2% chr15 - 76819458 76819594 137 browser details YourSeq 114 370 512 3000 86.1% chr12 - 105779479 105779614 136 browser details YourSeq 114 368 521 3000 84.2% chr1 - 75500953 75501091 139 browser details YourSeq 113 369 522 3000 83.4% chr4 - 8678400 8678543 144 browser details YourSeq 113 372 512 3000 90.6% chr6 + 86561710 86561852 143 browser details YourSeq 111 370 522 3000 82.1% chr5 - 6365716 6365860 145 browser details YourSeq 111 369 527 3000 82.2% chr18 - 65847969 65848123 155 browser details YourSeq 110 380 521 3000 88.3% chr6 - 43092325 43092465 141 browser details YourSeq 110 371 512 3000 89.8% chr12 - 92670973 92671114 142

Note: The 3000 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 3000 1 3000 3000 100.0% chr2 - 35186650 35189649 3000 browser details YourSeq 108 2292 2429 3000 89.8% chr6 + 32123363 32123505 143 browser details YourSeq 106 2290 2452 3000 90.1% chr14 + 103443923 103444452 530 browser details YourSeq 104 2296 2428 3000 89.4% chr12 - 88503186 88503319 134 browser details YourSeq 104 2294 2458 3000 89.4% chr3 + 123011872 123012331 460 browser details YourSeq 103 2290 2423 3000 90.6% chr1 + 185530107 185530242 136 browser details YourSeq 102 2290 2716 3000 86.9% chr9 - 118262687 118263191 505 browser details YourSeq 102 2294 2424 3000 88.5% chr2 + 105970267 105970394 128 browser details YourSeq 101 2290 2425 3000 88.4% chr4 + 64560242 64560374 133 browser details YourSeq 100 2290 2424 3000 88.6% chr2 - 78936722 78936854 133 browser details YourSeq 99 2291 2426 3000 90.3% chr5 - 47174482 47174687 206 browser details YourSeq 98 2293 2420 3000 90.0% chr2 + 84510609 84510741 133 browser details YourSeq 98 2273 2408 3000 89.5% chr10 + 114451447 114451884 438 browser details YourSeq 98 2290 2423 3000 90.4% chr10 + 55349053 55349189 137 browser details YourSeq 97 2296 2426 3000 85.4% chr4 - 79118470 79118599 130 browser details YourSeq 97 2292 2431 3000 85.4% chr1 - 95269050 95269184 135 browser details YourSeq 97 2290 2424 3000 88.7% chr1 - 73325990 73326121 132 browser details YourSeq 97 2292 2429 3000 90.2% chr1 + 188293378 188293520 143 browser details YourSeq 96 2290 2423 3000 86.3% chr15 - 41766104 41766235 132 browser details YourSeq 96 2292 2426 3000 88.7% chr18 + 72422556 72422690 135

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Rab14 RAB14, member RAS oncogene family [ Mus musculus (house mouse) ] Gene ID: 68365, updated on 12-Aug-2019

Gene summary

Official Symbol Rab14 provided by MGI Official Full Name RAB14, member RAS oncogene family provided by MGI Primary source MGI:MGI:1915615 See related Ensembl:ENSMUSG00000026878 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 AI314285; AI649155; 0610030G24Rik; 2810475J17Rik; A830021G03Rik; D030017L14Rik Expression Ubiquitous expression in adrenal adult (RPKM 151.4), stomach adult (RPKM 60.7) and 28 other tissues See more Orthologs human all

Genomic context

Location: 2; 2 B See Rab14 in Genome Data Viewer

Exon count: 8

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (35180205..35201120, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (35035725..35056640, complement)

Chromosome 2 - NC_000068.7

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Transcript information: This gene has 13 transcripts

Gene: Rab14 ENSMUSG00000026878

Description RAB14, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:1915615] Gene Synonyms 0610030G24Rik, 2810475J17Rik, A830021G03Rik, D030017L14Rik Location Chromosome 2: 35,180,205-35,201,120 reverse strand. GRCm38:CM000995.2 About this gene This gene has 13 transcripts (splice variants), 203 orthologues, 62 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Rab14- ENSMUST00000028238.14 3081 215aa ENSMUSP00000028238.8 Protein coding CCDS15959 Q50HX4 TSL:1 201 Q91V41 GENCODE basic APPRIS P1

Rab14- ENSMUST00000230751.1 925 197aa ENSMUSP00000155278.1 Protein coding - Q50HX3 GENCODE basic 213

Rab14- ENSMUST00000230657.1 415 83aa ENSMUSP00000155540.1 Protein coding - A0A2R8VHW9 CDS 5' 212 incomplete

Rab14- ENSMUST00000201896.1 5003 No - Retained - - TSL:NA 210 protein intron

Rab14- ENSMUST00000113025.1 4346 No - Retained - - TSL:1 202 protein intron

Rab14- ENSMUST00000201694.1 3717 No - Retained - - TSL:NA 209 protein intron

Rab14- ENSMUST00000202602.3 800 No - Retained - - TSL:1 211 protein intron

Rab14- ENSMUST00000155483.7 731 No - Retained - - TSL:3 208 protein intron

Rab14- ENSMUST00000137709.1 657 No - Retained - - TSL:2 204 protein intron

Rab14- ENSMUST00000142015.1 639 No - Retained - - TSL:3 205 protein intron

Rab14- ENSMUST00000155359.2 494 No - lncRNA - - TSL:2 207 protein

Rab14- ENSMUST00000148543.1 400 No - lncRNA - - TSL:3 206 protein

Rab14- ENSMUST00000126224.1 355 No - lncRNA - - TSL:3 203 protein

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40.92 kb Forward strand

35.18Mb 35.19Mb 35.20Mb 35.21Mb Genes Cntrl-202 >protein coding (Comprehensive set...

Cntrl-212 >protein coding

Cntrl-203 >protein coding

Cntrl-206 >protein coding

Cntrl-210 >retained intron

Contigs AL773523.4 > Genes (Comprehensive set... < Rab14-202retained intron

< Rab14-201protein coding

< Rab14-204retained intron < Rab14-206lncRNA

< Rab14-213protein coding

< Rab14-212protein coding < Rab14-207lncRNA

< Rab14-205retained intron < Rab14-210retained intron

< Rab14-211retained intron

< Rab14-208retained intron < Rab14-203lncRNA

< Rab14-209retained intron

Regulatory Build

35.18Mb 35.19Mb 35.20Mb 35.21Mb Reverse strand 40.92 kb

Regulation Legend CTCF Enhancer Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000028238

< Rab14-201protein coding

Reverse strand 20.92 kb

ENSMUSP00000028... MobiDB lite Coiled-coils (Ncoils) TIGRFAM Small GTP-binding protein domain Superfamily P-loop containing nucleoside triphosphate hydrolase SMART SM00176

SM00173

SM00175

SM00174 Prints PR00449 Pfam Small GTPase PROSITE profiles PS51419 PANTHER Ras-related protein Rab14

PTHR24073 Gene3D 3.40.50.300 CDD Ras-related protein Rab14

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

Variant Legend splice region variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 180 215

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

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