https://www.alphaknockout.com
Mouse Gorab Conditional Knockout Project (CRISPR/Cas9)
Objective: To create a Gorab conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Gorab gene (NCBI Reference Sequence: NM_178883 ; Ensembl: ENSMUSG00000040124 ) is located on Mouse chromosome 1. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000045138). 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 Gorab gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-30J21 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: Mice homozygous for a null gene trap allele exhibit hunched posture, craniofacial abnormalities, neonatal lethality, respiratory distress, skin edema, decreased hair follicles, fewer dermal condensates and papillae, and impaired formation of primary cilia on dermal condensate cells.
Exon 2 starts from about 5.62% 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: 6353 bp, and the size of intron 2 for 3'-loxP site insertion: 2119 bp. The size of effective cKO region: ~858 bp. The cKO region does not have any other known gene.
Page 1 of 7 https://www.alphaknockout.com
Overview of the Targeting Strategy
Wildtype allele gRNA region 5' gRNA region 3'
1 2 3 5 Targeting vector
Targeted allele
Constitutive KO allele (After Cre recombination)
Legends Exon of mouse Gorab Homology arm cKO region loxP site
Page 2 of 7 https://www.alphaknockout.com
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(7358bp) | A(29.91% 2201) | C(18.52% 1363) | T(30.48% 2243) | G(21.08% 1551)
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.
Page 3 of 7 https://www.alphaknockout.com
BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr1 - 163397420 163400419 3000 browser details YourSeq 65 1826 2040 3000 91.1% chr13 + 46597512 46597769 258 browser details YourSeq 44 827 972 3000 94.0% chr14 + 99463455 99463615 161 browser details YourSeq 44 1880 1953 3000 90.6% chr11 + 71746846 71746929 84 browser details YourSeq 44 828 950 3000 90.8% chr11 + 51973526 51973981 456 browser details YourSeq 43 907 967 3000 75.6% chr12 - 78763682 78763730 49 browser details YourSeq 41 827 944 3000 91.9% chr1 + 60025054 60025171 118 browser details YourSeq 37 907 954 3000 79.5% chr16 - 65982776 65982815 40 browser details YourSeq 36 907 954 3000 77.0% chr18 - 84732919 84732957 39 browser details YourSeq 36 1920 1995 3000 84.8% chr11 - 48527446 48527520 75 browser details YourSeq 36 828 954 3000 84.8% chr1 - 77519527 77519652 126 browser details YourSeq 36 908 952 3000 97.4% chr14 + 58687530 58687581 52 browser details YourSeq 35 2018 2073 3000 94.9% chr6 - 136558811 136558867 57 browser details YourSeq 35 1978 2054 3000 89.2% chr13 + 29033283 29033357 75 browser details YourSeq 35 907 953 3000 75.7% chr11 + 78681306 78681342 37 browser details YourSeq 33 886 967 3000 80.5% chr1 - 128566998 128567076 79 browser details YourSeq 32 920 955 3000 94.5% chr6 - 88130636 88130671 36 browser details YourSeq 32 923 956 3000 97.1% chr11 - 88325817 88325850 34 browser details YourSeq 32 923 958 3000 88.6% chr1 - 75307956 75307990 35 browser details YourSeq 32 923 954 3000 100.0% chr5 + 72609196 72609227 32
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% chr1 - 163393562 163396561 3000 browser details YourSeq 57 74 154 3000 95.6% chr10 - 121704044 121704187 144 browser details YourSeq 53 106 169 3000 96.5% chr16 - 36095450 36095521 72 browser details YourSeq 50 106 184 3000 96.5% chr10 + 83048739 83048937 199 browser details YourSeq 48 106 162 3000 86.6% chr11 + 112957753 112957805 53 browser details YourSeq 43 107 154 3000 89.2% chr18 - 7911890 7911935 46 browser details YourSeq 43 80 157 3000 88.0% chr16 - 9359064 9359139 76 browser details YourSeq 43 1289 1376 3000 88.5% chr1 + 189951647 189951733 87 browser details YourSeq 40 82 139 3000 90.7% chr14 - 80510241 80510296 56 browser details YourSeq 39 111 153 3000 95.4% chr2 - 158839177 158839219 43 browser details YourSeq 39 116 187 3000 95.5% chr10 - 24202757 24202846 90 browser details YourSeq 37 1277 1324 3000 93.2% chr18 - 66646810 66646873 64 browser details YourSeq 36 111 152 3000 92.9% chr15 - 16571247 16571288 42 browser details YourSeq 36 106 148 3000 82.1% chr12 - 72260951 72260989 39 browser details YourSeq 36 91 153 3000 90.0% chr1 + 186399421 186399482 62 browser details YourSeq 35 106 147 3000 83.8% chr12 - 14873316 14873353 38 browser details YourSeq 34 106 140 3000 100.0% chr11 - 81629477 81629551 75 browser details YourSeq 34 135 182 3000 94.6% chr1 + 34638308 34638356 49 browser details YourSeq 32 74 129 3000 94.3% chr14 + 23833460 23833515 56 browser details YourSeq 30 140 179 3000 94.0% chr17 - 76411255 76411304 50
Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.
Page 4 of 7 https://www.alphaknockout.com
Gene and protein information: Gorab golgin, RAB6-interacting [ Mus musculus (house mouse) ] Gene ID: 98376, updated on 10-Oct-2019
Gene summary
Official Symbol Gorab provided by MGI Official Full Name golgin, RAB6-interacting provided by MGI Primary source MGI:MGI:2138271 See related Ensembl:ENSMUSG00000040124 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 NTKL-BP1; Scyl1bp1; SCYL1-BP1 Expression Broad expression in limb E14.5 (RPKM 5.8), CNS E11.5 (RPKM 5.3) and 24 other tissues See more Orthologs human all
Genomic context
Location: 1; 1 H2.1 See Gorab in Genome Data Viewer
Exon count: 6
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 1 NC_000067.6 (163384903..163403669, complement)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 1 NC_000067.5 (165315040..165333772, complement)
Chromosome 1 - NC_000067.6
Page 5 of 7 https://www.alphaknockout.com
Transcript information: This gene has 3 transcripts
Gene: Gorab ENSMUSG00000040124
Description golgin, RAB6-interacting [Source:MGI Symbol;Acc:MGI:2138271] Gene Synonyms NTKL-BP1, Scyl1bp1 Location Chromosome 1: 163,384,908-163,403,669 reverse strand. GRCm38:CM000994.2 About this gene This gene has 3 transcripts (splice variants), 193 orthologues, is a member of 1 Ensembl protein family and is associated with 10 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Gorab- ENSMUST00000045138.5 2538 368aa ENSMUSP00000036253.4 Protein coding CCDS15429 Q8BRM2 TSL:1 201 GENCODE basic APPRIS P1
Gorab- ENSMUST00000186402.1 2663 161aa ENSMUSP00000140320.1 Nonsense mediated - A0A087WQS3 TSL:1 203 decay
Gorab- ENSMUST00000185299.1 724 No - Retained intron - - TSL:2 202 protein
38.76 kb Forward strand 163.38Mb 163.39Mb 163.40Mb 163.41Mb Genes Gm24940-201 >snRNA (Comprehensive set...
Contigs AC113511.7 > Genes < Gorab-201protein coding (Comprehensive set...
< Gorab-203nonsense mediated decay
< Gorab-202retained intron
Regulatory Build
163.38Mb 163.39Mb 163.40Mb 163.41Mb Reverse strand 38.76 kb
Regulation Legend
Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
merged Ensembl/Havana
Non-Protein Coding
RNA gene processed transcript
Page 6 of 7 https://www.alphaknockout.com
Transcript: ENSMUST00000045138
< Gorab-201protein coding
Reverse strand 18.76 kb
ENSMUSP00000036... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam RAB6-interacting golgin PANTHER PTHR21470:SF2
RAB6-interacting golgin
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend frameshift variant missense variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 368
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
Page 7 of 7