https://www.alphaknockout.com Mouse Gjd4 Knockout Project (CRISPR/Cas9) Objective: To create a Gjd4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gjd4 gene (NCBI Reference Sequence: NM_153086 ; Ensembl: ENSMUSG00000036855 ) is located on Mouse chromosome 18. 2 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 2 (Transcript: ENSMUST00000041007). Exon 1~2 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 accelerated muscle regeneration following BaCl2 injection. Exon 1 starts from about 0.09% of the coding region. Exon 1~2 covers 100.0% of the coding region. The size of effective KO region: ~2632 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 Legends Exon of mouse Gjd4 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. 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 stop codon 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(27.3% 546) | C(21.8% 436) | T(30.5% 610) | G(20.4% 408) 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(28.65% 573) | C(20.7% 414) | T(29.35% 587) | G(21.3% 426) 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% chr18 - 9282619 9284618 2000 browser details YourSeq 118 1040 1288 2000 82.6% chr3 - 138018106 138018366 261 browser details YourSeq 106 1040 1349 2000 89.0% chrX - 12450777 12451121 345 browser details YourSeq 88 1112 1312 2000 81.0% chr2 - 110400386 110400581 196 browser details YourSeq 85 1209 1350 2000 89.8% chr5 + 3292928 3293117 190 browser details YourSeq 83 1066 1288 2000 81.7% chr1 - 33368659 33368883 225 browser details YourSeq 80 1014 1287 2000 90.0% chr1 + 58109941 58110230 290 browser details YourSeq 76 1212 1349 2000 76.2% chr5 + 138712432 138712567 136 browser details YourSeq 76 1129 1288 2000 82.8% chr11 + 17048614 17048855 242 browser details YourSeq 72 1111 1350 2000 76.2% chr10 + 53106440 53106666 227 browser details YourSeq 71 1103 1288 2000 78.4% chr6 - 129685846 129686028 183 browser details YourSeq 68 1040 1308 2000 87.1% chr9 - 40712563 40712912 350 browser details YourSeq 68 1056 1287 2000 91.1% chr11 - 60724050 60724280 231 browser details YourSeq 67 1154 1350 2000 85.2% chr1 - 170289961 170290152 192 browser details YourSeq 66 1213 1312 2000 83.0% chr9 + 48302591 48302690 100 browser details YourSeq 66 1251 1343 2000 88.7% chr17 + 63579190 63796076 216887 browser details YourSeq 66 1039 1343 2000 81.2% chr12 + 42511982 42512281 300 browser details YourSeq 65 1110 1288 2000 83.2% chr10 - 61985491 61985660 170 browser details YourSeq 65 1209 1288 2000 92.5% chr10 + 92201661 92201743 83 browser details YourSeq 60 1027 1304 2000 82.0% chr1 - 134015747 134016014 268 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% chr18 - 9277985 9279984 2000 browser details YourSeq 416 349 1252 2000 86.5% chrX - 22636231 22637240 1010 browser details YourSeq 391 345 1167 2000 87.0% chrX - 139581855 139582721 867 browser details YourSeq 377 345 1261 2000 89.4% chr2 - 135503897 135505237 1341 browser details YourSeq 375 346 1251 2000 87.4% chr5 + 77103910 77104874 965 browser details YourSeq 367 594 1279 2000 87.3% chr6 - 73500307 73501095 789 browser details YourSeq 334 487 1279 2000 88.2% chrX - 98423101 98423994 894 browser details YourSeq 330 345 1261 2000 87.1% chr6 - 128460369 128461396 1028 browser details YourSeq 326 417 1278 2000 89.1% chr8 - 107783673 107784710 1038 browser details YourSeq 310 492 1279 2000 86.6% chrX - 111739108 111740495 1388 browser details YourSeq 306 482 1280 2000 88.5% chr2 - 56546618 56547459 842 browser details YourSeq 303 656 1163 2000 87.2% chrX + 139587002 139587578 577 browser details YourSeq 302 503 1101 2000 82.7% chr6 - 9635134 9635819 686 browser details YourSeq 301 414 1260 2000 87.9% chr3 + 142705240 142706143 904 browser details YourSeq 299 345 1045 2000 85.4% chr18 + 43308513 43309294 782 browser details YourSeq 298 459 1228 2000 86.8% chr4 + 55103428 55104258 831 browser details YourSeq 286 636 1164 2000 89.7% chrX + 61624725 61625345 621 browser details YourSeq 283 362 1215 2000 89.0% chr15 + 76567040 76568083 1044 browser details YourSeq 282 345 1149 2000 87.7% chr15 + 18936334 18937307 974 browser details YourSeq 280 492 1209 2000 85.9% chr9 - 28848230 28849042 813 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: Gjd4 gap junction protein, delta 4 [ Mus musculus (house mouse) ] Gene ID: 225152, updated on 12-Aug-2019 Gene summary Official Symbol Gjd4 provided by MGI Official Full Name gap junction protein, delta 4 provided by MGI Primary source MGI:MGI:2444990 See related Ensembl:ENSMUSG00000036855 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Cx39; 9430022F06Rik Expression Biased expression in limb E14.5 (RPKM 1.3), CNS E11.5 (RPKM 0.2) and 2 other tissues See more Orthologs human all Genomic context Location: 18; 18 A1 See Gjd4 in Genome Data Viewer Exon count: 3 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (9278124..9289808, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (9278605..9282807, complement) Chromosome 18 - NC_000084.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 1 transcript Gene: Gjd4 ENSMUSG00000036855 Description gap junction protein, delta 4 [Source:MGI Symbol;Acc:MGI:2444990] Gene Synonyms 9430022F06Rik, Cx39, connexin 39 Location Chromosome 18: 9,278,607-9,282,809 reverse strand. GRCm38:CM001011.2 About this gene This gene has 1 transcript (splice variant), 205 orthologues, 19 paralogues, is a member of 1 Ensembl protein family and is associated with 5 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Gjd4-201 ENSMUST00000041007.3 2661 364aa ENSMUSP00000035472.3 Protein coding CCDS29049 Q8BSD4 TSL:1 GENCODE basic APPRIS P1 24.20 kb Forward strand 9.27Mb 9.28Mb 9.29Mb Contigs AC108777.7 > Genes (Comprehensive set... < Gjd4-201protein coding Regulatory Build 9.27Mb 9.28Mb 9.29Mb Reverse strand 24.20 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000041007 < Gjd4-201protein coding Reverse strand 4.20 kb ENSMUSP00000035... Transmembrane heli... MobiDB lite Low complexity (Seg) SMART Connexin, N-terminal Gap junction protein, cysteine-rich domain Prints Connexin Pfam Connexin, N-terminal PROSITE patterns Connexin, conserved site Connexin, conserved site PANTHER PTHR11984:SF3 Connexin Gene3D Connexin, N-terminal domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 364 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.