https://www.alphaknockout.com
Mouse Rabgef1 Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Rabgef1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Rabgef1 gene (NCBI Reference Sequence: NM_019983 ; Ensembl: ENSMUSG00000025340 ) is located on Mouse chromosome 5. 8 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000026390). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Rabgef1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-151E24 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: Nullizygous mutations can cause neonatal or postnatal lethality associated with severe skin inflammation, high mast cell numbers and serum levels of IgE and histamine, and enhanced mast cell degranulation and release of mediators and cytokines in response to high affinity IgE receptor aggregation.
Exon 2 starts from about 12.22% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 3290 bp, and the size of intron 2 for 3'-loxP site insertion: 6906 bp. The size of effective cKO region: ~667 bp. The cKO region does not have any other known gene.
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Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 8 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Rabgef1 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. 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(7167bp) | A(26.12% 1872) | C(20.82% 1492) | T(28.58% 2048) | G(24.49% 1755)
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% chr5 + 130187628 130190627 3000 browser details YourSeq 480 766 1261 3000 99.0% chr10 + 94199079 94220949 21871 browser details YourSeq 345 379 1739 3000 91.1% chr8 - 25207493 25745658 538166 browser details YourSeq 287 277 1732 3000 89.6% chr7 + 130112782 130286293 173512 browser details YourSeq 262 1278 1732 3000 87.7% chr10 - 57729088 57729644 557 browser details YourSeq 254 1274 1714 3000 88.5% chr18 + 82450967 82451581 615 browser details YourSeq 242 1274 1708 3000 86.6% chr16 - 8552870 8553414 545 browser details YourSeq 235 1274 1732 3000 87.9% chr15 - 90857093 90857671 579 browser details YourSeq 235 340 728 3000 89.4% chr8 + 117150527 117151082 556 browser details YourSeq 225 1274 1754 3000 87.4% chr2 + 28383966 28384478 513 browser details YourSeq 221 1295 1773 3000 87.0% chr10 + 45831999 45832612 614 browser details YourSeq 217 1274 1750 3000 88.9% chr3 + 32926544 32927062 519 browser details YourSeq 217 1274 1771 3000 88.2% chr16 + 5325632 5326176 545 browser details YourSeq 213 1274 1675 3000 88.8% chr10 - 95030883 95031379 497 browser details YourSeq 212 1284 1732 3000 87.4% chr18 - 74998352 74998793 442 browser details YourSeq 211 1313 1706 3000 88.9% chr5 - 149674159 149674654 496 browser details YourSeq 210 1274 1732 3000 87.8% chr5 + 89615536 89616011 476 browser details YourSeq 207 341 676 3000 88.6% chr4 + 44288360 44288711 352 browser details YourSeq 206 1285 1708 3000 87.6% chr6 - 22831382 22831903 522 browser details YourSeq 206 1276 1691 3000 89.6% chr16 - 93823589 93824102 514
Note: The 3000 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 3000 1 3000 3000 100.0% chr5 + 130191295 130194294 3000 browser details YourSeq 240 307 2366 3000 92.1% chr11 - 3236892 3460044 223153 browser details YourSeq 146 299 462 3000 92.6% chr15 - 39100918 39101078 161 browser details YourSeq 145 299 459 3000 93.1% chr15 - 100017950 100018107 158 browser details YourSeq 144 299 462 3000 92.0% chr11 - 117111862 117112022 161 browser details YourSeq 143 299 462 3000 92.5% chr9 - 110804061 110804222 162 browser details YourSeq 141 304 462 3000 92.4% chr4 - 117207048 117207203 156 browser details YourSeq 141 304 462 3000 92.4% chr17 - 26706497 26706652 156 browser details YourSeq 141 304 462 3000 96.2% chr13 - 44542286 44542447 162 browser details YourSeq 140 299 462 3000 90.7% chr17 - 74203688 74203848 161 browser details YourSeq 139 283 462 3000 89.7% chr5 - 31169354 31169517 164 browser details YourSeq 137 304 462 3000 91.1% chr7 - 110016877 110017032 156 browser details YourSeq 137 304 462 3000 91.0% chr11 + 67810198 67810353 156 browser details YourSeq 136 304 462 3000 91.7% chr7 - 140977216 140977372 157 browser details YourSeq 136 304 462 3000 90.2% chr6 - 113505495 113505647 153 browser details YourSeq 136 299 462 3000 90.6% chr2 - 65514019 65514168 150 browser details YourSeq 136 299 459 3000 93.1% chr2 + 73766990 73767151 162 browser details YourSeq 135 299 457 3000 94.2% chrX - 162388518 162388687 170 browser details YourSeq 134 299 462 3000 92.5% chr4 - 11128925 11129092 168 browser details YourSeq 134 304 449 3000 97.9% chr10 - 61421468 61421613 146
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
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Gene and protein information: Rabgef1 RAB guanine nucleotide exchange factor (GEF) 1 [ Mus musculus (house mouse) ] Gene ID: 56715, updated on 12-Aug-2019
Gene summary
Official Symbol Rabgef1 provided by MGI Official Full Name RAB guanine nucleotide exchange factor (GEF) 1 provided by MGI Primary source MGI:MGI:1929459 See related Ensembl:ENSMUSG00000025340 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 Rin2; Rab5ef; Rabex5 Expression Ubiquitous expression in bladder adult (RPKM 13.1), CNS E18 (RPKM 5.9) and 28 other tissues See more Orthologs human all
Genomic context
Location: 5; 5 G1.3 See Rabgef1 in Genome Data Viewer
Exon count: 10
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (130171791..130214342)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (130663050..130690207)
Chromosome 5 - NC_000071.6
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Transcript information: This gene has 9 transcripts
Gene: Rabgef1 ENSMUSG00000025340
Description RAB guanine nucleotide exchange factor (GEF) 1 [Source:MGI Symbol;Acc:MGI:1929459] Gene Synonyms Ras negative regulator Rabex-5 Location Chromosome 5: 130,171,798-130,214,342 forward strand. GRCm38:CM000998.2 About this gene This gene has 9 transcripts (splice variants), 228 orthologues, 6 paralogues, is a member of 1 Ensembl protein family and is associated with 14 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Rabgef1- ENSMUST00000026390.7 2828 491aa ENSMUSP00000026390.7 Protein coding CCDS19708 Q9JM13 TSL:1 201 GENCODE basic APPRIS P1
Rabgef1- ENSMUST00000119797.7 2690 491aa ENSMUSP00000114103.1 Protein coding CCDS19708 Q9JM13 TSL:1 203 GENCODE basic APPRIS P1
Rabgef1- ENSMUST00000119027.7 912 221aa ENSMUSP00000112537.1 Protein coding - D3YWB4 CDS 3' 202 incomplete TSL:5
Rabgef1- ENSMUST00000148264.7 828 250aa ENSMUSP00000119245.1 Protein coding - D3Z2L2 CDS 3' 208 incomplete TSL:5
Rabgef1- ENSMUST00000143865.1 426 22aa ENSMUSP00000118590.1 Nonsense mediated - F6VIJ0 CDS 5' 206 decay incomplete TSL:2
Rabgef1- ENSMUST00000147493.1 880 No - Retained intron - - TSL:2 207 protein
Rabgef1- ENSMUST00000143727.1 812 No - Retained intron - - TSL:2 205 protein
Rabgef1- ENSMUST00000125937.1 739 No - Retained intron - - TSL:5 204 protein
Rabgef1- ENSMUST00000202729.1 628 No - lncRNA - - TSL:3 209 protein
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62.55 kb Forward strand 130.17Mb 130.18Mb 130.19Mb 130.20Mb 130.21Mb 130.22Mb Genes (Comprehensive set... Rabgef1-203 >protein coding Tmem248-205 >protein coding
Rabgef1-208 >protein coding Rabgef1-206 >nonsense mediated decay
Rabgef1-201 >protein coding Tmem248-201 >protein coding
Rabgef1-202 >protein coding Rabgef1-207 >retained intron
Rabgef1-209 >lncRNA Tmem248-204 >protein coding
Rabgef1-205 >retained intron Rabgef1-204 >retained intron Tmem248-203 >protein coding
Gm15920-201 >processed pseudogene Tmem248-202 >protein coding
Contigs < AC122339.3 AC113029.23 >
Genes < Gm15921-201processed pseudogene (Comprehensive set...
< Gm6598-201lncRNA
Regulatory Build
130.17Mb 130.18Mb 130.19Mb 130.20Mb 130.21Mb 130.22Mb Reverse strand 62.55 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
pseudogene processed transcript RNA gene
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Transcript: ENSMUST00000026390
27.18 kb Forward strand
Rabgef1-201 >protein coding
ENSMUSP00000026... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57716 VPS9 domain superfamily
SMART Zinc finger, A20-type VPS9 domain
Pfam Zinc finger, A20-type RABX5, catalytic core helical domain
VPS9 domain PROSITE profiles Zinc finger, A20-type VPS9 domain
PANTHER PTHR23101:SF107
PTHR23101 Gene3D 1.10.246.120 VPS9 domain superfamily
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend frameshift variant missense variant synonymous variant
Scale bar 0 60 120 180 240 300 360 420 491
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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