https://www.alphaknockout.com Mouse Stab2 Knockout Project (CRISPR/Cas9) Objective: To create a Stab2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stab2 gene (NCBI Reference Sequence: NM_138673 ; Ensembl: ENSMUSG00000035459 ) is located on Mouse chromosome 10. 69 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 69 (Transcript: ENSMUST00000035288). Exon 2~3 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 knock-out alleles exhibit no gross abnormaities. Mice homozygous for one null allele display elevated serum hyaluronic acid levels and decreased metastasis. Exon 2 starts from about 1.38% of the coding region. Exon 2~3 covers 3.26% of the coding region. The size of effective KO region: ~6291 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 69 Legends Exon of mouse Stab2 Knockout region Page 2 of 9 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 2 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 Exon 3 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 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(30.95% 619) | C(21.65% 433) | T(25.9% 518) | G(21.5% 430) Note: The 2000 bp section upstream of Exon 2 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(29.65% 593) | C(20.95% 419) | T(28.1% 562) | G(21.3% 426) 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 9 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% chr10 - 87003096 87005095 2000 browser details YourSeq 67 1433 1989 2000 70.7% chr14 + 58130802 58130936 135 browser details YourSeq 64 1425 1499 2000 86.8% chr2 + 158917817 158917884 68 browser details YourSeq 60 1428 1497 2000 89.9% chr4 - 56673840 56673908 69 browser details YourSeq 59 1428 1500 2000 92.7% chr9 + 96516798 96516872 75 browser details YourSeq 54 1426 1499 2000 82.6% chr17 + 16150099 16150165 67 browser details YourSeq 52 1432 1490 2000 86.8% chr9 - 117742893 117742945 53 browser details YourSeq 51 1436 1500 2000 96.3% chr18 - 19517318 19517385 68 browser details YourSeq 50 1433 1496 2000 96.4% chr3 - 109330045 109330120 76 browser details YourSeq 50 1426 1491 2000 82.8% chr17 - 43557079 43557140 62 browser details YourSeq 49 1433 1499 2000 94.6% chr2 + 24810775 24810865 91 browser details YourSeq 48 1435 1490 2000 86.8% chr2 - 62311239 62311291 53 browser details YourSeq 48 1428 1488 2000 81.5% chr17 - 70522653 70522706 54 browser details YourSeq 48 1435 1491 2000 84.4% chr11 + 109148491 109148541 51 browser details YourSeq 47 1426 1483 2000 94.4% chr15 - 78233861 78233919 59 browser details YourSeq 46 1428 1484 2000 94.2% chr4 - 58649513 58649576 64 browser details YourSeq 45 1453 1501 2000 97.9% chr12 - 101806221 101806283 63 browser details YourSeq 44 1432 1481 2000 94.0% chr16 - 72154178 72154227 50 browser details YourSeq 44 1436 1497 2000 79.3% chr14 + 114721951 114722006 56 browser details YourSeq 43 1433 1485 2000 95.7% chr15 - 17625914 17625966 53 Note: The 2000 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 2000 1 2000 2000 100.0% chr10 - 86994805 86996804 2000 browser details YourSeq 138 638 1034 2000 90.2% chr10 + 10974431 11002243 27813 browser details YourSeq 123 469 777 2000 93.7% chr7 - 44887985 44888480 496 browser details YourSeq 122 549 805 2000 83.0% chr1 - 21522655 21522852 198 browser details YourSeq 115 627 1245 2000 91.3% chr11 - 57679840 57804872 125033 browser details YourSeq 110 638 799 2000 84.7% chrX - 94406237 94406390 154 browser details YourSeq 110 638 799 2000 84.2% chr11 + 46643745 46643896 152 browser details YourSeq 110 638 777 2000 89.1% chr1 + 192910497 192910635 139 browser details YourSeq 108 638 779 2000 88.3% chr7 - 112860705 112860842 138 browser details YourSeq 107 638 779 2000 86.4% chr11 - 3311564 3311701 138 browser details YourSeq 106 638 778 2000 86.9% chr13 - 65362445 65362581 137 browser details YourSeq 106 638 791 2000 88.5% chr10 - 58558139 58558301 163 browser details YourSeq 106 638 778 2000 89.0% chr1 - 132396193 132396336 144 browser details YourSeq 106 638 779 2000 87.9% chr11 + 86678463 86678602 140 browser details YourSeq 106 647 1035 2000 86.4% chr10 + 3738506 3738914 409 browser details YourSeq 105 638 776 2000 88.0% chr13 - 106831456 106831590 135 browser details YourSeq 105 638 777 2000 87.5% chr11 - 50081239 50081375 137 browser details YourSeq 105 638 798 2000 85.0% chr1 - 93538902 93539046 145 browser details YourSeq 104 633 779 2000 86.1% chr11 - 5696400 5696544 145 browser details YourSeq 104 638 778 2000 91.4% chr5 + 139116744 139116885 142 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Stab2 stabilin 2 [ Mus musculus (house mouse) ] Gene ID: 192188, updated on 12-Aug-2019 Gene summary Official Symbol Stab2 provided by MGI Official Full Name stabilin 2 provided by MGI Primary source MGI:MGI:2178743 See related Ensembl:ENSMUSG00000035459 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 FELL; FEEL-2; STAB-2; MFEEL-2 Expression Biased expression in spleen adult (RPKM 16.0), liver E18 (RPKM 6.2) and 8 other tissues See more Orthologs human all Genomic context Location: 10; 10 C1 See Stab2 in Genome Data Viewer Exon count: 74 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (86841194..87008038, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (86303955..86470687, complement) Chromosome 10 - NC_000076.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Stab2 ENSMUSG00000035459 Description stabilin 2 [Source:MGI Symbol;Acc:MGI:2178743] Gene Synonyms FEEL-2, STAB-2 Location Chromosome 10: 86,841,198-87,008,025 reverse strand. GRCm38:CM001003.2 About this gene This gene has 7 transcripts (splice variants), 226 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stab2- ENSMUST00000035288.16 8228 2559aa ENSMUSP00000048309.8 Protein coding CCDS36021 E5RKF9 TSL:1 201 Q8R4U0 GENCODE basic APPRIS P1 Stab2- ENSMUST00000219341.2 3502 870aa ENSMUSP00000151465.2 Nonsense mediated - A0A1W2P6Y4 CDS 5' 205 decay incomplete TSL:5 Stab2- ENSMUST00000219612.1 502 No - Retained intron - - TSL:3 206 protein Stab2- ENSMUST00000219659.1 442 No - Retained intron - - TSL:5 207 protein Stab2- ENSMUST00000218408.1 1237 No - lncRNA - - TSL:1 203 protein Stab2- ENSMUST00000218366.1 720 No - lncRNA - - TSL:1 202 protein Stab2- ENSMUST00000219280.1 523 No - lncRNA - - TSL:3 204 protein Page 7 of 9 https://www.alphaknockout.com 186.83 kb Forward strand 86.85Mb 86.90Mb 86.95Mb 87.00Mb Genes Nt5dc3-201 >protein coding Gm16280-201 >lncRNA Gm16271-201 >lncRNA (Comprehensive set... Nt5dc3-202 >retained intron Gm16270-201 >transcribed processed pseudogene Gm16269-201 >processed pseudogene Gm49358-201 >protein coding Gm16268-201 >lncRNA Contigs < AC112790.5 < AC025501.19 Genes (Comprehensive set... < Stab2-201protein coding < Stab2-205nonsense mediated decay < Stab2-203lncRNA < Stab2-204lncRNA < Stab2-206retained intron < Stab2-202lncRNA < Stab2-207retained intron Regulatory Build 86.85Mb 86.90Mb 86.95Mb 87.00Mb Reverse strand 186.83 kb Regulation Legend CTCF Enhancer Open Chromatin 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 Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000035288 < Stab2-201protein coding Reverse strand 166.83 kb ENSMUSP00000048... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily SSF57196 C-type lectin fold FAS1 domain superfamily SMART EGF-like domain Link domain EGF-like calcium-binding domain FAS1 domain Laminin EGF domain Pfam FAS1 domain Link domain EGF domain PROSITE profiles FAS1 domain EGF-like domain Link domain PROSITE patterns EGF-like, conserved site Link domain EGF-like, conserved site PANTHER PTHR24038 PTHR24038:SF0 Gene3D 2.170.300.10 C-type lectin-like/link domain superfamily FAS1 domain superfamily 2.10.25.10 CDD cd00054 cd00055 All sequence SNPs/i..