https://www.alphaknockout.com

Mouse Adcy7 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Adcy7 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Adcy7 (NCBI Reference Sequence: NM_007406 ; Ensembl: ENSMUSG00000031659 ) is located on Mouse 8. 28 exons are identified, with the ATG start codon in exon 4 and the TGA stop codon in exon 28 (Transcript: ENSMUST00000169037). Exon 6~8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Adcy7 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-116K3 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: Mice homozygous for disruptions in this gene display some degree of pre- or postnatal lethality. Surviving adults for one allele appear to be normal. Heterozygous females for one allele display decreased depression related behaviors.

Exon 6 starts from about 11.4% of the coding region. The knockout of Exon 6~8 will result in frameshift of the gene. The size of intron 5 for 5'-loxP site insertion: 847 bp, and the size of intron 8 for 3'-loxP site insertion: 1052 bp. The size of effective cKO region: ~1956 bp. The cKO 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 5 6 7 8 9 28 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Adcy7 Homology arm cKO region loxP site

Page 2 of 8 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(8456bp) | A(22.71% 1920) | C(24.81% 2098) | T(24.65% 2084) | G(27.84% 2354)

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 8 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% chr8 + 88306443 88309442 3000 browser details YourSeq 269 580 1025 3000 89.6% chr5 + 136350380 136350802 423 browser details YourSeq 261 597 1029 3000 87.1% chr6 - 128484104 128484428 325 browser details YourSeq 257 577 1018 3000 88.4% chr13 - 100801712 100802059 348 browser details YourSeq 253 589 1027 3000 95.1% chrX - 137169459 137170132 674 browser details YourSeq 249 608 1045 3000 88.9% chr10 + 80045164 80045518 355 browser details YourSeq 241 575 1017 3000 89.4% chr4 + 132363933 132364345 413 browser details YourSeq 228 648 1040 3000 93.9% chr10 - 7745454 7745878 425 browser details YourSeq 227 653 1026 3000 92.9% chr1 - 75425476 75426135 660 browser details YourSeq 216 816 1065 3000 93.7% chr11 - 107023583 107023829 247 browser details YourSeq 212 615 1018 3000 88.3% chr1 - 59518233 59518539 307 browser details YourSeq 210 776 1031 3000 96.1% chr7 - 81583013 81583382 370 browser details YourSeq 209 589 1017 3000 94.1% chr19 - 43758399 43759081 683 browser details YourSeq 209 591 1007 3000 87.5% chr2 + 6085291 6085566 276 browser details YourSeq 208 759 1032 3000 98.2% chr10 - 117495190 117495821 632 browser details YourSeq 202 821 1043 3000 94.5% chr14 + 54600061 54600278 218 browser details YourSeq 201 842 1190 3000 95.5% chr18 + 77738783 77739406 624 browser details YourSeq 200 715 1025 3000 95.9% chr16 + 14304706 14305425 720 browser details YourSeq 198 821 1033 3000 97.2% chr1 - 108109026 108109239 214 browser details YourSeq 198 815 1046 3000 95.5% chr3 + 127439413 127439944 532

Note: The 3000 bp section upstream of Exon 6 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% chr8 + 88311399 88314398 3000 browser details YourSeq 117 327 505 3000 90.0% chr3 - 86691176 86691376 201 browser details YourSeq 117 329 504 3000 89.4% chr13 + 37317653 37317828 176 browser details YourSeq 107 343 505 3000 88.8% chr1 + 194326723 194326884 162 browser details YourSeq 106 319 505 3000 90.1% chr4 - 125892248 125892438 191 browser details YourSeq 106 327 500 3000 84.3% chr7 + 81524543 81524727 185 browser details YourSeq 101 345 505 3000 85.7% chr12 + 76069476 76069636 161 browser details YourSeq 94 366 505 3000 89.2% chr13 - 44233205 44233345 141 browser details YourSeq 93 366 504 3000 87.4% chr12 + 91508346 91508483 138 browser details YourSeq 93 341 505 3000 88.6% chr12 + 5300408 5300643 236 browser details YourSeq 93 366 510 3000 87.1% chr10 + 64132543 64132685 143 browser details YourSeq 92 357 499 3000 84.8% chr11 - 65499523 65499664 142 browser details YourSeq 92 348 503 3000 88.0% chr2 + 72747643 72747797 155 browser details YourSeq 91 328 504 3000 88.1% chr10 - 99248015 99248213 199 browser details YourSeq 90 336 456 3000 85.1% chr17 - 88516058 88516174 117 browser details YourSeq 90 345 501 3000 85.2% chr15 - 66313625 66313773 149 browser details YourSeq 90 338 505 3000 85.9% chr7 + 30051974 30052141 168 browser details YourSeq 90 343 505 3000 86.2% chr13 + 42263300 42263472 173 browser details YourSeq 89 326 505 3000 83.8% chr9 - 116452561 116452736 176 browser details YourSeq 89 343 505 3000 86.8% chr9 - 66263853 66264013 161

Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 8 https://www.alphaknockout.com

Gene and protein information: Adcy7 adenylate 7 [ Mus musculus (house mouse) ] Gene ID: 11513, updated on 12-Aug-2019

Gene summary

Official Symbol Adcy7 provided by MGI Official Full Name adenylate cyclase 7 provided by MGI Primary source MGI:MGI:102891 See related Ensembl:ENSMUSG00000031659 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 AA407758 Expression Broad expression in spleen adult (RPKM 30.9), lung adult (RPKM 18.8) and 19 other tissuesS ee more Orthologs human all

Genomic context

Location: 8 C3; 8 43.06 cM See Adcy7 in Genome Data Viewer

Exon count: 36

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (88272190..88329965)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (90796302..90853861)

Chromosome 8 - NC_000074.6

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

Transcript information: This gene has 6 transcripts

Gene: Adcy7 ENSMUSG00000031659

Description adenylate cyclase 7 [Source:MGI Symbol;Acc:MGI:102891] Location Chromosome 8: 88,272,403-88,329,962 forward strand. GRCm38:CM001001.2 About this gene This gene has 6 transcripts (splice variants), 233 orthologues, 20 paralogues, is a member of 1 Ensembl and is associated with 2 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Adcy7- ENSMUST00000171456.8 5951 1099aa ENSMUSP00000132528.1 Protein coding CCDS22509 P51829 TSL:1 204 GENCODE basic APPRIS P1

Adcy7- ENSMUST00000098521.3 5937 1099aa ENSMUSP00000096122.2 Protein coding CCDS22509 P51829 TSL:1 201 GENCODE basic APPRIS P1

Adcy7- ENSMUST00000168545.7 5937 1099aa ENSMUSP00000129252.1 Protein coding CCDS22509 P51829 TSL:1 202 GENCODE basic APPRIS P1

Adcy7- ENSMUST00000169037.8 5198 1099aa ENSMUSP00000130594.1 Protein coding CCDS22509 P51829 TSL:1 203 GENCODE basic APPRIS P1

Adcy7- ENSMUST00000210688.1 2387 428aa ENSMUSP00000148207.1 Nonsense mediated - A0A1B0GT56 CDS 5' 205 decay incomplete TSL:1

Adcy7- ENSMUST00000211311.1 936 No - Retained intron - - TSL:1 206 protein

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

77.56 kb Forward strand

88.28Mb 88.30Mb 88.32Mb Adcy7-203 >protein coding (Comprehensive set...

Adcy7-202 >protein coding

Adcy7-204 >protein coding

Adcy7-201 >protein coding

Adcy7-206 >retained intron Adcy7-205 >nonsense mediated decay

Contigs AC155170.3 > Genes < Brd7-204retained intron (Comprehensive set...

< Brd7-201protein coding

< Brd7-202lncRNA

< Brd7-206lncRNA

< Brd7-207lncRNA

< Brd7-205lncRNA

Regulatory Build

88.28Mb 88.30Mb 88.32Mb Reverse strand 77.56 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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

Transcript: ENSMUST00000169037

56.64 kb Forward strand

Adcy7-203 >protein coding

ENSMUSP00000130... Transmembrane heli... MobiDB lite Low complexity (Seg) Superfamily Nucleotide cyclase SMART class-3/4/guanylyl cyclase Pfam Adenylyl cyclase class-3/4/guanylyl cyclase

Adenylate cyclase, N-terminal Adenylate cyclase, conserved domain PROSITE profiles Adenylyl cyclase class-3/4/guanylyl cyclase PROSITE patterns Adenylyl cyclase class-4/guanylyl cyclase, conserved site PIRSF Adenylate cyclase PANTHER PTHR45627

PTHR45627:SF9 Gene3D Nucleotide cyclase CDD cd07302

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

Variant Legend inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 100 200 300 400 500 600 700 800 900 1099

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

Page 8 of 8