https://www.alphaknockout.com Mouse Gosr2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Gosr2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gosr2 gene (NCBI Reference Sequence: NM_019650 ; Ensembl: ENSMUSG00000020946 ) is located on Mouse chromosome 11. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000021329). 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 Gosr2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-72N11 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: Exon 2 starts from about 4.72% 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: 8310 bp, and the size of intron 2 for 3'-loxP site insertion: 1621 bp. The size of effective cKO region: ~565 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 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Gosr2 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. 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 GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7065bp) | A(22.25% 1572) | C(22.45% 1586) | T(32.74% 2313) | G(22.56% 1594) 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% chr11 - 103689570 103692569 3000 browser details YourSeq 176 548 955 3000 77.9% chrX - 104910113 104910519 407 browser details YourSeq 176 273 895 3000 85.0% chr1 - 189950913 189951743 831 browser details YourSeq 164 440 948 3000 82.1% chr9 - 79137689 79652722 515034 browser details YourSeq 164 458 951 3000 84.8% chr12 - 99576791 99577302 512 browser details YourSeq 150 580 949 3000 79.4% chr14 - 37245293 37245643 351 browser details YourSeq 145 609 950 3000 86.3% chr1 + 192209242 192209580 339 browser details YourSeq 144 599 962 3000 81.9% chr6 - 135038184 135038547 364 browser details YourSeq 132 551 955 3000 83.2% chrX + 152072598 152072980 383 browser details YourSeq 125 592 951 3000 75.3% chr1 - 151275962 151276297 336 browser details YourSeq 121 475 949 3000 85.9% chr13 - 110101359 110101833 475 browser details YourSeq 117 550 895 3000 87.0% chr13 - 75059310 75059651 342 browser details YourSeq 115 511 953 3000 82.9% chrX + 162376808 162379861 3054 browser details YourSeq 115 612 895 3000 77.3% chrX + 70811130 70811409 280 browser details YourSeq 114 551 952 3000 78.5% chrX - 144563968 144564353 386 browser details YourSeq 107 790 962 3000 92.8% chr8 + 89837761 89837950 190 browser details YourSeq 105 499 821 3000 82.9% chr1 + 54382073 54382373 301 browser details YourSeq 101 551 835 3000 83.5% chrX - 151766907 151767302 396 browser details YourSeq 98 708 955 3000 78.0% chr1 - 82057068 82057301 234 browser details YourSeq 97 609 953 3000 89.6% chr13 + 37380961 37381519 559 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% chr11 - 103686005 103689004 3000 browser details YourSeq 130 1121 1308 3000 86.2% chr17 - 41901906 41902090 185 browser details YourSeq 122 1129 1295 3000 93.1% chr1 - 134517443 134517672 230 browser details YourSeq 121 1110 1279 3000 91.3% chr17 - 67669653 67669830 178 browser details YourSeq 120 1110 1279 3000 88.6% chr17 - 40936576 40936740 165 browser details YourSeq 118 1128 1279 3000 92.6% chr16 - 30546721 30546871 151 browser details YourSeq 118 1118 1279 3000 91.0% chr17 + 6102755 6102916 162 browser details YourSeq 118 1109 1279 3000 86.5% chr10 + 100521005 100521169 165 browser details YourSeq 117 1128 1279 3000 90.3% chr13 + 24412229 24412377 149 browser details YourSeq 116 1126 1279 3000 87.7% chr2 - 158038795 158038932 138 browser details YourSeq 116 1126 1279 3000 90.6% chr11 + 87268308 87268459 152 browser details YourSeq 115 1110 1279 3000 85.9% chr1 - 115417551 115417711 161 browser details YourSeq 115 1126 1279 3000 92.8% chr11 + 79313514 79313667 154 browser details YourSeq 114 1125 1273 3000 87.1% chr15 + 99971911 99972055 145 browser details YourSeq 113 1126 1279 3000 92.6% chr12 - 5168997 5169173 177 browser details YourSeq 113 1128 1279 3000 92.5% chr10 - 62440635 62658879 218245 browser details YourSeq 113 1134 1278 3000 88.3% chr1 - 85719690 85719826 137 browser details YourSeq 113 1121 1279 3000 87.5% chr4 + 152511046 152511200 155 browser details YourSeq 111 1138 1280 3000 94.5% chr13 - 48310266 48310419 154 browser details YourSeq 111 1124 1273 3000 87.8% chr13 + 23558043 23558196 154 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: Gosr2 golgi SNAP receptor complex member 2 [ Mus musculus (house mouse) ] Gene ID: 56494, updated on 14-Aug-2019 Gene summary Official Symbol Gosr2 provided by MGI Official Full Name golgi SNAP receptor complex member 2 provided by MGI Primary source MGI:MGI:1927204 See related Ensembl:ENSMUSG00000020946 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 Gs27; SNARE; C76855; membrin; 2310032N09Rik Expression Ubiquitous expression in testis adult (RPKM 36.5), genital fat pad adult (RPKM 36.3) and 28 other tissues See more Orthologs human all Genomic context Location: 11 E1; 11 67.43 cM See Gosr2 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (103676849..103697723, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (103538163..103559024, complement) Chromosome 11 - NC_000077.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 3 transcripts Gene: Gosr2 ENSMUSG00000020946 Description golgi SNAP receptor complex member 2 [Source:MGI Symbol;Acc:MGI:1927204] Gene Synonyms 2310032N09Rik, GS27, SNARE, membrin Location Chromosome 11: 103,676,849-103,697,898 reverse strand. GRCm38:CM001004.2 About this gene This gene has 3 transcripts (splice variants), 176 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 3 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Gosr2-201 ENSMUST00000021329.13 3266 212aa ENSMUSP00000021329.7 Protein coding CCDS25521 O35166 TSL:1 GENCODE basic APPRIS P1 Gosr2-202 ENSMUST00000107013.2 1044 165aa ENSMUSP00000102627.2 Protein coding - A2A9I0 TSL:2 GENCODE basic Gosr2-203 ENSMUST00000145265.1 917 No protein - lncRNA - - TSL:2 41.05 kb Forward strand 103.67Mb 103.68Mb 103.69Mb 103.70Mb Genes C130046K22Rik-201 >lncRNA (Comprehensive set... C130046K22Rik-202 >lncRNA Contigs AL627252.16 > Genes (Comprehensive set... < Gosr2-201protein coding < Gosr2-202protein coding < Gosr2-203lncRNA Regulatory Build 103.67Mb 103.68Mb 103.69Mb 103.70Mb Reverse strand 41.05 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 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000021329 < Gosr2-201protein coding Reverse strand 21.05 kb ENSMUSP00000021... Transmembrane heli... Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF58038 Pfam PF12352 PIRSF GOSR2/Membrin/Bos1 PANTHER PTHR21230:SF40 PTHR21230 Gene3D 1.20.5.110 CDD cd15863 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend synonymous variant Scale bar 0 20 40 60 80 100 120 140 160 180 212 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.