https://www.alphaknockout.com Mouse Mad2l1bp Knockout Project (CRISPR/Cas9) Objective: To create a Mad2l1bp knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mad2l1bp gene (NCBI Reference Sequence: NM_025649 ; Ensembl: ENSMUSG00000034509 ) is located on Mouse chromosome 17. 3 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 3 (Transcript: ENSMUST00000171172). Exon 1~3 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: Exon 1 starts from about 0.12% of the coding region. Exon 1~3 covers 100.0% of the coding region. The size of effective KO region: ~5721 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 2 3 Legends Exon of mouse Mad2l1bp 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 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. 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(26.75% 535) | C(21.65% 433) | T(28.55% 571) | G(23.05% 461) 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(23.95% 479) | C(23.55% 471) | T(30.35% 607) | G(22.15% 443) 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. 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% chr17 - 46153498 46155497 2000 browser details YourSeq 203 104 715 2000 81.7% chr3 - 107915172 107915479 308 browser details YourSeq 195 1352 1671 2000 87.7% chr15 - 34223981 34224478 498 browser details YourSeq 182 1340 1659 2000 89.2% chr17 + 28783119 28783469 351 browser details YourSeq 172 1337 1719 2000 91.4% chr16 - 96572533 96572925 393 browser details YourSeq 165 874 1486 2000 86.8% chr7 - 128469100 128469680 581 browser details YourSeq 153 1495 1762 2000 85.2% chr5 - 126676991 126677233 243 browser details YourSeq 153 61 250 2000 92.8% chr1 - 128059960 128060316 357 browser details YourSeq 153 1337 1590 2000 87.3% chr1 + 131900495 131901046 552 browser details YourSeq 149 1332 1513 2000 90.8% chr1 - 59865917 59866083 167 browser details YourSeq 147 1419 1753 2000 87.4% chr11 + 23079929 23080529 601 browser details YourSeq 144 1521 1762 2000 88.7% chr14 + 45671626 45671934 309 browser details YourSeq 142 1332 1492 2000 94.5% chr12 + 4886106 4886267 162 browser details YourSeq 140 146 711 2000 83.4% chr4 + 132307255 132307708 454 browser details YourSeq 140 1333 1488 2000 92.6% chr19 + 14669795 14669942 148 browser details YourSeq 140 1474 1764 2000 93.3% chr16 + 29012781 29013092 312 browser details YourSeq 139 1332 1486 2000 95.5% chr7 - 49121821 49121978 158 browser details YourSeq 138 1337 1489 2000 95.4% chr9 - 119356785 119357287 503 browser details YourSeq 138 1338 1484 2000 97.3% chr9 + 15290998 15291152 155 browser details YourSeq 138 1332 1479 2000 97.3% chr12 + 101034891 101035055 165 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% chr17 - 46145775 46147774 2000 browser details YourSeq 137 942 1096 2000 95.4% chr5 + 114657097 114657261 165 browser details YourSeq 136 941 1085 2000 95.2% chr17 - 68721770 68721912 143 browser details YourSeq 132 946 1086 2000 94.3% chr2 - 50339430 50339567 138 browser details YourSeq 130 929 1085 2000 94.5% chr11 - 95665276 95665441 166 browser details YourSeq 130 946 1084 2000 94.9% chr19 + 8841809 8841945 137 browser details YourSeq 130 942 1086 2000 93.1% chr11 + 70692499 70692641 143 browser details YourSeq 129 941 1086 2000 92.4% chr7 - 65408401 65408544 144 browser details YourSeq 129 942 1085 2000 93.0% chr9 + 44891758 44891899 142 browser details YourSeq 128 943 1085 2000 93.0% chr1 - 11314627 11314767 141 browser details YourSeq 128 942 1084 2000 93.0% chr2 + 38798373 38798513 141 browser details YourSeq 128 942 1084 2000 93.0% chr17 + 84812231 84812371 141 browser details YourSeq 128 939 1083 2000 92.4% chr13 + 52957518 52957660 143 browser details YourSeq 128 941 1085 2000 92.4% chr13 + 14914327 14914469 143 browser details YourSeq 127 940 1084 2000 93.8% chrX - 94407497 94407641 145 browser details YourSeq 127 942 1085 2000 92.3% chr4 - 22189404 22189545 142 browser details YourSeq 127 942 1084 2000 91.4% chr17 - 12189767 12189905 139 browser details YourSeq 127 949 1086 2000 94.9% chr10 - 70606582 70606717 136 browser details YourSeq 127 938 1085 2000 91.0% chr19 + 7305316 7305460 145 browser details YourSeq 127 946 1085 2000 93.5% chr17 + 64541489 64541626 138 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Mad2l1bp MAD2L1 binding protein [ Mus musculus (house mouse) ] Gene ID: 66591, updated on 10-Oct-2019 Gene summary Official Symbol Mad2l1bp provided by MGI Official Full Name MAD2L1 binding protein provided by MGI Primary source MGI:MGI:1913841 See related Ensembl:ENSMUSG00000034509 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 CMT2A; CMT2B; C76045; Mad2lbp; AU018945; AW550977; mKIAA0110; 0610009D16Rik; 2510031P20Rik Expression Ubiquitous expression in liver E14.5 (RPKM 16.5), large intestine adult (RPKM 15.4) and 28 other tissues See more Orthologs human all Genomic context Location: 17; 17 C See Mad2l1bp in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (46147385..46153551, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (46284334..46290500, complement) Chromosome 17 - NC_000083.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Mad2l1bp ENSMUSG00000034509 Description MAD2L1 binding protein [Source:MGI Symbol;Acc:MGI:1913841] Gene Synonyms 0610009D16Rik, 2510031P20Rik, CMT2A, CMT2B Location Chromosome 17: 46,147,387-46,153,553 reverse strand. GRCm38:CM001010.2 About this gene This gene has 1 transcript (splice variant), 197 orthologues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mad2l1bp-201 ENSMUST00000171172.2 1272 276aa ENSMUSP00000125915.1 Protein coding CCDS28821 Q9DCX1 TSL:1 GENCODE basic APPRIS P1 26.17 kb Forward strand 46.14Mb 46.15Mb 46.16Mb Genes Gtpbp2-211 >nonsense mediated decay (Comprehensive set... Gtpbp2-209 >retained intron Gtpbp2-214 >retained intron Gtpbp2-201 >protein coding Gtpbp2-217 >protein coding Gtpbp2-206 >protein coding Gtpbp2-203 >lncRNA Gtpbp2-204 >lncRNA Gtpbp2-216 >lncRNA Gtpbp2-215 >lncRNA Contigs AC110562.17 > < AC116739.13 Genes < Rsph9-201protein coding < Mad2l1bp-201protein coding (Comprehensive set... Regulatory Build 46.14Mb 46.15Mb 46.16Mb Reverse strand 26.17 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000171172 < Mad2l1bp-201protein coding Reverse strand 6.17 kb ENSMUSP00000125... MobiDB lite Pfam Mad1/Cdc20-bound-Mad2 binding protein PANTHER PTHR15681 Gene3D 3.30.900.20 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 276 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.