https://www.alphaknockout.com

Mouse Fra10ac1 Knockout Project (CRISPR/Cas9)

Objective: To create a Fra10ac1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Fra10ac1 (NCBI Reference Sequence: NM_001081075 ; Ensembl: ENSMUSG00000054237 ) is located on Mouse 19. 14 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 14 (Transcript: ENSMUST00000067167). Exon 2~6 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:

Exon 2 starts from the coding region. Exon 2~6 covers 40.21% of the coding region. The size of effective KO region: ~7540 bp. The KO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 5 6 14

Legends Exon of mouse Fra10ac1 Knockout region

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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 928 bp section downstream of Exon 6 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.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(29.15% 583) | C(19.3% 386) | T(29.65% 593) | G(21.9% 438)

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(928bp) | A(27.05% 251) | C(16.27% 151) | T(35.67% 331) | G(21.01% 195)

Note: The 928 bp section downstream of Exon 6 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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr19 - 38221914 38223913 2000 browser details YourSeq 25 557 589 2000 96.3% chr5 - 81868233 81868266 34 browser details YourSeq 24 712 735 2000 100.0% chr7 + 119663744 119663767 24 browser details YourSeq 22 1250 1271 2000 100.0% chr8 - 86861216 86861237 22 browser details YourSeq 22 1046 1071 2000 84.0% chr1 - 12232511 12232535 25 browser details YourSeq 21 1652 1672 2000 100.0% chr5 + 61792531 61792551 21 browser details YourSeq 21 991 1011 2000 100.0% chr17 + 75867671 75867691 21

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 928 1 928 928 100.0% chr19 - 38213446 38214373 928 browser details YourSeq 77 253 434 928 70.5% chr16 + 92017599 92017777 179 browser details YourSeq 65 15 100 928 94.6% chr11 + 77802019 77802123 105 browser details YourSeq 58 289 430 928 91.4% chr14 - 54538404 54538816 413 browser details YourSeq 55 330 430 928 91.1% chr18 + 53383606 53383710 105 browser details YourSeq 54 17 79 928 96.7% chr1 - 74166300 74166438 139 browser details YourSeq 52 295 430 928 80.0% chr4 - 136039754 136039876 123 browser details YourSeq 50 301 430 928 79.0% chr5 + 117920512 117920626 115 browser details YourSeq 49 27 78 928 98.1% chr1 - 135005156 135005225 70 browser details YourSeq 48 289 433 928 82.5% chr2 + 84581366 84581504 139 browser details YourSeq 48 240 345 928 91.4% chr17 + 68244554 68244680 127 browser details YourSeq 47 263 433 928 94.3% chr12 - 44864215 44864504 290 browser details YourSeq 45 291 430 928 92.5% chr2 + 180921795 180921936 142 browser details YourSeq 44 49 107 928 94.0% chr2 - 49547286 49547345 60 browser details YourSeq 43 111 430 928 58.9% chr19 - 56706411 56706478 68 browser details YourSeq 42 330 431 928 91.4% chr2 + 32793736 32793836 101 browser details YourSeq 42 296 363 928 80.9% chr1 + 93105206 93105273 68 browser details YourSeq 41 338 440 928 85.2% chr2 - 14928952 14929051 100 browser details YourSeq 41 337 432 928 86.7% chr5 + 34769354 34769446 93 browser details YourSeq 41 337 412 928 86.7% chr12 + 66824516 66824589 74

Note: The 928 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Fra10ac1 FRA10AC1 homolog (human) [ Mus musculus (house mouse) ] Gene ID: 70567, updated on 12-Aug-2019

Gene summary

Official Symbol Fra10ac1 provided by MGI Official Full Name FRA10AC1 homolog (human) provided by MGI Primary source MGI:MGI:1917817 See related Ensembl:ENSMUSG00000054237 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 AI448293; 5730455O13Rik Expression Ubiquitous expression in CNS E11.5 (RPKM 10.1), CNS E14 (RPKM 7.3) and 27 other tissues See more Orthologs human all

Genomic context

Location: 19; 19 C2 See Fra10ac1 in Genome Data Viewer Exon count: 15

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (38188477..38224630, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (38262967..38298622, complement)

Chromosome 19 - NC_000085.6

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Transcript information: This gene has 3 transcripts

Gene: Fra10ac1 ENSMUSG00000054237

Description FRA10AC1 homolog (human) [Source:MGI Symbol;Acc:MGI:1917817] Gene Synonyms 5730455O13Rik Location Chromosome 19: 38,188,481-38,224,156 reverse strand. GRCm38:CM001012.2 About this gene This gene has 3 transcripts (splice variants), 197 orthologues, 5 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Fra10ac1-201 ENSMUST00000067167.5 1402 315aa ENSMUSP00000070534.5 Protein coding CCDS37972 Q8BP78 TSL:1 GENCODE basic APPRIS P1

Fra10ac1-203 ENSMUST00000238154.1 1884 No protein - Retained intron - - -

Fra10ac1-202 ENSMUST00000238007.1 1010 No protein - Retained intron - - -

55.68 kb Forward strand

38.18Mb 38.19Mb 38.20Mb 38.21Mb 38.22Mb 38.23Mb Pde6c-202 >protein coding (Comprehensive set...

Pde6c-201 >protein coding

Contigs < AC112153.4

Genes (Comprehensive set... < Fra10ac1-201protein coding

< Fra10ac1-202retained intron

< Fra10ac1-203retained intron

Regulatory Build

38.18Mb 38.19Mb 38.20Mb 38.21Mb 38.22Mb 38.23Mb Reverse strand 55.68 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000067167

< Fra10ac1-201protein coding

Reverse strand 35.66 kb

ENSMUSP00000070... MobiDB lite Low complexity (Seg) Pfam Folate-sensitive fragile site protein Fra10Ac1 PANTHER PTHR11567:SF25

PTHR11567

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 315

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

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