https://www.alphaknockout.com
Mouse Mrap2 Knockout Project (CRISPR/Cas9)
Objective: To create a Mrap2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Mrap2 gene (NCBI Reference Sequence: NM_001177731 ; Ensembl: ENSMUSG00000042761 ) is located on Mouse chromosome 9. 5 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000179313). Exon 3 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: Mice homozygous for a knock-out allele develop severe obesity at a young age. Obesity develops early during ad libitum feeding before the onset of hyperphagia, persists in mutant mice pair-fed to a normal dietary intake, and is abolished only by underfeeding.
Exon 3 starts from the coding region. Exon 3 covers 21.42% of the coding region. The size of effective KO region: ~134 bp. The KO region does not have any other known gene.
Page 1 of 8 https://www.alphaknockout.com
Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 3 5
Legends Exon of mouse Mrap2 Knockout region
Page 2 of 8 https://www.alphaknockout.com
Overview of the Dot Plot (up) Window size: 15 bp
Forward Reverse Complement
Sequence 12
Note: The 2000 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 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.
Page 3 of 8 https://www.alphaknockout.com
Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(24.6% 492) | C(23.35% 467) | T(28.6% 572) | G(23.45% 469)
Note: The 2000 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(21.35% 427) | T(28.95% 579) | G(20.65% 413)
Note: The 2000 bp section downstream 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.
Page 4 of 8 https://www.alphaknockout.com
BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr9 + 87167527 87169526 2000 browser details YourSeq 137 652 911 2000 88.8% chr3 - 31126780 31127061 282 browser details YourSeq 137 653 905 2000 89.1% chr4 + 46676004 46676260 257 browser details YourSeq 130 625 893 2000 86.5% chr17 + 74321155 74321457 303 browser details YourSeq 127 592 892 2000 90.0% chr13 + 46667406 46667719 314 browser details YourSeq 120 620 892 2000 89.3% chr12 + 80301912 80302184 273 browser details YourSeq 118 657 895 2000 83.1% chr2 + 122184081 122184307 227 browser details YourSeq 112 669 851 2000 82.9% chr17 + 93169621 93169788 168 browser details YourSeq 109 652 896 2000 81.3% chr11 - 76889575 76889805 231 browser details YourSeq 94 717 911 2000 79.2% chr2 - 70092069 70092233 165 browser details YourSeq 89 596 902 2000 91.0% chr12 + 39974819 39975131 313 browser details YourSeq 89 740 902 2000 87.3% chr1 + 171713291 171713459 169 browser details YourSeq 81 641 899 2000 80.6% chr14 - 21813833 21814084 252 browser details YourSeq 80 165 361 2000 90.1% chr8 + 117690677 118055716 365040 browser details YourSeq 77 706 898 2000 87.5% chr6 - 77102731 77103402 672 browser details YourSeq 74 692 899 2000 71.4% chr16 - 20977560 20977706 147 browser details YourSeq 74 640 839 2000 88.6% chr16 + 33640607 33640817 211 browser details YourSeq 72 652 889 2000 79.2% chr6 + 124943539 124943761 223 browser details YourSeq 71 652 903 2000 92.9% chr9 - 102389290 102389548 259 browser details YourSeq 71 561 758 2000 79.1% chr1 - 165761301 165761461 161
Note: The 2000 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% chr9 + 87169660 87171659 2000 browser details YourSeq 170 346 846 2000 85.1% chr3 - 138128281 138128616 336 browser details YourSeq 124 372 514 2000 96.4% chr11 - 76181061 76181203 143 browser details YourSeq 120 345 504 2000 93.5% chr10 - 127115747 127115907 161 browser details YourSeq 118 782 1331 2000 93.9% chr1 - 7849733 8207655 357923 browser details YourSeq 118 346 485 2000 94.1% chrX + 103951486 103951626 141 browser details YourSeq 108 369 732 2000 77.4% chr8 - 106565854 106566080 227 browser details YourSeq 107 346 486 2000 94.3% chr5 - 27580400 27580542 143 browser details YourSeq 106 346 514 2000 85.1% chr1 - 4874525 4874677 153 browser details YourSeq 104 373 506 2000 94.9% chr2 - 19999318 19999466 149 browser details YourSeq 104 346 493 2000 93.3% chr11 + 57844185 57844333 149 browser details YourSeq 101 1176 1331 2000 99.1% chr1 - 70120453 70370178 249726 browser details YourSeq 100 369 506 2000 93.3% chr12 - 54846549 54846690 142 browser details YourSeq 99 346 485 2000 93.1% chr14 - 47697871 47698012 142 browser details YourSeq 98 378 738 2000 76.7% chr12 - 22750052 22750188 137 browser details YourSeq 98 346 496 2000 86.5% chr1 - 35900573 35900724 152 browser details YourSeq 97 1232 1350 2000 95.3% chr8 - 102078676 102078949 274 browser details YourSeq 97 1221 1329 2000 91.1% chr13 + 62341589 62341690 102 browser details YourSeq 96 1227 1332 2000 90.9% chr5 - 106241912 106242009 98 browser details YourSeq 96 1225 1331 2000 92.1% chr1 - 143253228 143253330 103
Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
Page 5 of 8 https://www.alphaknockout.com
Gene and protein information: Mrap2 melanocortin 2 receptor accessory protein 2 [ Mus musculus (house mouse) ] Gene ID: 244958, updated on 24-Oct-2019
Gene summary
Official Symbol Mrap2 provided by MGI Official Full Name melanocortin 2 receptor accessory protein 2 provided by MGI Primary source MGI:MGI:3609239 See related Ensembl:ENSMUSG00000042761 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 BB633055 Expression Biased expression in cerebellum adult (RPKM 1.9), frontal lobe adult (RPKM 0.7) and 5 other tissues See more Orthologs human all
Genomic context
Location: 9; 9 E3.1 See Mrap2 in Genome Data Viewer Exon count: 9
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (87144236..87184051)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (87039141..87078880)
Chromosome 9 - NC_000075.6
Page 6 of 8 https://www.alphaknockout.com
Transcript information: This gene has 3 transcripts
Gene: Mrap2 ENSMUSG00000042761
Description melanocortin 2 receptor accessory protein 2 [Source:MGI Symbol;Acc:MGI:3609239] Location Chromosome 9: 87,144,306-87,184,045 forward strand. GRCm38:CM001002.2 About this gene This gene has 3 transcripts (splice variants), 140 orthologues, is a member of 1 Ensembl protein family and is associated with 30 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Mrap2-202 ENSMUST00000113149.7 2112 207aa ENSMUSP00000108774.1 Protein coding CCDS52878 D3Z1Q2 TSL:5 GENCODE basic APPRIS P1
Mrap2-203 ENSMUST00000179313.2 2039 207aa ENSMUSP00000135904.1 Protein coding CCDS52878 D3Z1Q2 TSL:2 GENCODE basic APPRIS P1
Mrap2-201 ENSMUST00000049457.13 1931 207aa ENSMUSP00000046271.7 Protein coding CCDS52878 D3Z1Q2 TSL:3 GENCODE basic APPRIS P1
59.74 kb Forward strand
87.14Mb 87.15Mb 87.16Mb 87.17Mb 87.18Mb 87.19Mb Genes (Comprehensive set... Mrap2-201 >protein coding
Mrap2-202 >protein coding
Mrap2-203 >protein coding
Contigs AC168882.3 > AC125370.4 >
Genes < Cep162-201protein coding (Comprehensive set...
< Gm37012-201TEC
Regulatory Build
87.14Mb 87.15Mb 87.16Mb 87.17Mb 87.18Mb 87.19Mb Reverse strand 59.74 kb
Regulation Legend CTCF Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding merged Ensembl/Havana
Non-Protein Coding
processed transcript
Page 7 of 8 https://www.alphaknockout.com
Transcript: ENSMUST00000179313
39.70 kb Forward strand
Mrap2-203 >protein coding
ENSMUSP00000135... Transmembrane heli... Pfam Melanocortin-2 receptor accessory protein family
PANTHER PTHR28675:SF1
Melanocortin-2 receptor accessory protein family
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend
missense variant synonymous variant
Scale bar 0 20 40 60 80 100 120 140 160 180 207
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
Page 8 of 8