https://www.alphaknockout.com

Mouse Sdk2 Knockout Project (CRISPR/Cas9)

Objective: To create a Sdk2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Sdk2 gene (NCBI Reference Sequence: NM_172800 ; Ensembl: ENSMUSG00000041592 ) is located on Mouse chromosome 11. 45 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 45 (Transcript: ENSMUST00000041627). Exon 2 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 exhibit impaired interconnectvity between VG3 amacrine cells and W3B retinal ganglion cells.

Exon 2 starts from about 1.18% of the coding region. Exon 2 covers 2.45% of the coding region. The size of effective KO region: ~160 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 gRNA region 5' gRNA region 3'

1 2 45

Legends Exon of mouse Sdk2 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 2 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 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.

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(21.4% 428) | C(28.15% 563) | T(25.05% 501) | G(25.4% 508)

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(23.9% 478) | C(24.95% 499) | T(23.3% 466) | G(27.85% 557)

Note: The 2000 bp section downstream 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.

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% chr11 - 113943279 113945278 2000 browser details YourSeq 42 1481 1595 2000 92.0% chr1 - 118643919 118644046 128 browser details YourSeq 40 1449 1555 2000 74.4% chr18 + 16639111 16639223 113 browser details YourSeq 37 1534 1592 2000 75.0% chr4 + 130366998 130367046 49 browser details YourSeq 37 1312 1595 2000 59.7% chr2 + 119176667 119176844 178 browser details YourSeq 35 1481 1554 2000 94.9% chr1 - 128508178 128508257 80 browser details YourSeq 35 1481 1604 2000 97.4% chr14 + 72349128 72349329 202 browser details YourSeq 32 1535 1595 2000 90.0% chr15 + 73522114 73522191 78 browser details YourSeq 30 1449 1494 2000 82.7% chr16 - 14294941 14294986 46 browser details YourSeq 29 1447 1497 2000 90.4% chr17 - 6036053 6036102 50 browser details YourSeq 29 1443 1495 2000 94.2% chr15 + 40638934 40638986 53 browser details YourSeq 28 1473 1502 2000 100.0% chr13 - 46486562 46486877 316 browser details YourSeq 27 1304 1335 2000 96.6% chr10 - 23685665 23685697 33 browser details YourSeq 27 1482 1517 2000 83.9% chr16 + 9453833 9453866 34 browser details YourSeq 26 1546 1573 2000 96.5% chr4 - 22205670 22205697 28 browser details YourSeq 26 1538 1569 2000 83.9% chr2 - 165925636 165925666 31 browser details YourSeq 26 1482 1520 2000 71.5% chr13 - 52297960 52297988 29 browser details YourSeq 25 1537 1569 2000 88.9% chr10 - 59818805 59818836 32 browser details YourSeq 25 1481 1514 2000 85.2% chr17 + 35493455 35493486 32 browser details YourSeq 25 1481 1513 2000 96.3% chr1 + 36085893 36085926 34

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% chr11 - 113941119 113943118 2000 browser details YourSeq 72 1372 1658 2000 80.5% chr6 - 98732950 98733297 348 browser details YourSeq 58 1345 1444 2000 82.3% chr17 - 52130033 52130111 79 browser details YourSeq 49 1372 1421 2000 100.0% chr11 - 11899195 11899246 52 browser details YourSeq 48 1383 1443 2000 96.2% chr5 + 111926413 111926567 155 browser details YourSeq 47 1373 1517 2000 71.7% chr17 - 72023049 72023141 93 browser details YourSeq 46 1377 1577 2000 77.4% chr8 - 68701087 68701269 183 browser details YourSeq 43 1379 1421 2000 100.0% chr16 - 61046668 61046710 43 browser details YourSeq 39 1585 1667 2000 73.5% chr12 + 16371441 16371523 83 browser details YourSeq 31 1631 1665 2000 94.3% chr2 + 155939061 155939095 35 browser details YourSeq 26 1721 1752 2000 90.7% chr17 - 34691345 34691376 32 browser details YourSeq 26 1373 1400 2000 100.0% chr6 + 60843377 60843409 33 browser details YourSeq 26 1373 1398 2000 100.0% chr5 + 107003953 107003978 26 browser details YourSeq 24 1432 1455 2000 100.0% chr11 - 11899286 11899309 24 browser details YourSeq 23 1464 1486 2000 100.0% chr10 - 30457654 30457676 23 browser details YourSeq 23 1698 1721 2000 100.0% chr11 + 61185061 61185107 47 browser details YourSeq 22 1941 1962 2000 100.0% chr5 - 135135051 135135072 22 browser details YourSeq 22 611 632 2000 100.0% chr14 - 56487395 56487416 22 browser details YourSeq 21 45 65 2000 100.0% chr11 - 116805761 116805781 21 browser details YourSeq 21 1625 1653 2000 86.3% chr9 + 30894605 30894633 29

Note: The 2000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 5 of 8 https://www.alphaknockout.com

Gene and protein information: Sdk2 sidekick cell adhesion molecule 2 [ Mus musculus (house mouse) ] Gene ID: 237979, updated on 12-Aug-2019

Gene summary

Official Symbol Sdk2 provided by MGI Official Full Name sidekick cell adhesion molecule 2 provided by MGI Primary source MGI:MGI:2443847 See related Ensembl:ENSMUSG00000041592 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 Sdk-2; mKIAA1514; 4632412F08Rik; 5330435L01Rik Expression Broad expression in limb E14.5 (RPKM 3.2), frontal lobe adult (RPKM 1.8) and 22 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 E2 See Sdk2 in Genome Data Viewer Exon count: 47

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (113776374..114067029, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (113642104..113927265, complement)

Chromosome 11 - NC_000077.6

Page 6 of 8 https://www.alphaknockout.com

Transcript information: This gene has 6 transcripts

Gene: Sdk2 ENSMUSG00000041592

Description sidekick cell adhesion molecule 2 [Source:MGI Symbol;Acc:MGI:2443847] Gene Synonyms 4632412F08Rik, 5330435L01Rik Location Chromosome 11: 113,776,374-114,067,046 reverse strand. GRCm38:CM001004.2 About this gene This gene has 6 transcripts (splice variants), 239 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Sdk2-201 ENSMUST00000041627.13 11287 2176aa ENSMUSP00000038972.7 Protein coding CCDS25605 Q6V4S5 TSL:1 GENCODE basic APPRIS P1

Sdk2-203 ENSMUST00000141943.1 2783 895aa ENSMUSP00000116872.1 Protein coding - B1ASR7 CDS 3' incomplete TSL:1

Sdk2-205 ENSMUST00000155651.1 4155 No protein - lncRNA - - TSL:1

Sdk2-204 ENSMUST00000144301.1 3083 No protein - lncRNA - - TSL:1

Sdk2-202 ENSMUST00000125879.1 681 No protein - lncRNA - - TSL:3

Sdk2-206 ENSMUST00000156955.1 546 No protein - lncRNA - - TSL:3

310.67 kb Forward strand 113.8Mb 113.9Mb 114.0Mb Contigs AL603706.13 > AL663117.14 > AL627323.6 > Genes (Comprehensive set... < Sdk2-201protein coding

< Sdk2-206lncRNA < Sdk2-203protein coding

< Sdk2-202lncRNA < Sdk2-205lncRNA

< Sdk2-204lncRNA

Regulatory Build

113.8Mb 113.9Mb 114.0Mb Reverse strand 310.67 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

RNA gene

Page 7 of 8 https://www.alphaknockout.com

Transcript: ENSMUST00000041627

< Sdk2-201protein coding

Reverse strand 289.85 kb

ENSMUSP00000038... Transmembrane heli... PDB-ENSP mappings MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily

Fibronectin type III superfamily SMART Immunoglobulin subtype Fibronectin type III

Immunoglobulin subtype 2 Prints PR00014 Pfam PF13927 Fibronectin type III

Immunoglobulin I-set PROSITE profiles Immunoglobulin-like domain Fibronectin type III PANTHER PTHR13817

PTHR13817:SF59 Gene3D Immunoglobulin-like fold CDD cd00096 Fibronectin type III

All sequence SNPs/i... Sequence variants (dbSNP and all other sources)

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2176

We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.

Page 8 of 8