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Mouse Ammecr1l Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Ammecr1l conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Ammecr1l gene (NCBI Reference Sequence: NM_153515 ; Ensembl: ENSMUSG00000041915 ) is located on Mouse chromosome 18. 8 exons are identified, with the ATG start codon in exon 3 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000115808). Exon 4~6 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ammecr1l gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-37K14 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 4 starts from about 43.87% of the coding region. The knockout of Exon 4~6 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 2121 bp, and the size of intron 6 for 3'-loxP site insertion: 2779 bp. The size of effective cKO region: ~2342 bp. The cKO 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 3 4 5 6 8 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Ammecr1l Homology arm cKO region loxP site

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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(8842bp) | A(29.09% 2572) | C(19.26% 1703) | T(28.21% 2494) | G(23.44% 2073)

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.

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BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr18 + 31770987 31773986 3000 browser details YourSeq 291 755 1128 3000 88.4% chr15 + 19749461 19749831 371 browser details YourSeq 231 829 1115 3000 91.1% chr1 + 110600020 110600300 281 browser details YourSeq 174 91 2264 3000 94.1% chr11 - 51544431 51766971 222541 browser details YourSeq 128 1788 2305 3000 82.4% chr12 - 17569007 17569451 445 browser details YourSeq 126 1801 2314 3000 82.0% chr1 + 59805825 59806113 289 browser details YourSeq 123 1767 2249 3000 83.6% chr8 - 4218976 4219387 412 browser details YourSeq 102 1805 2290 3000 74.1% chr11 + 101420960 101421325 366 browser details YourSeq 93 1847 2315 3000 90.0% chr11 - 82715771 82716307 537 browser details YourSeq 86 2189 2317 3000 84.8% chr17 + 87117489 87117622 134 browser details YourSeq 84 1784 1910 3000 90.6% chr10 - 127192248 127192384 137 browser details YourSeq 82 1917 2317 3000 70.0% chr11 + 87265567 87265707 141 browser details YourSeq 81 2189 2314 3000 83.7% chr11 + 120505062 120505398 337 browser details YourSeq 79 2189 2323 3000 84.7% chrX + 41939248 41939366 119 browser details YourSeq 79 1792 1912 3000 94.5% chr1 + 160866114 160866262 149 browser details YourSeq 78 1791 1910 3000 97.6% chr15 - 102253339 102253465 127 browser details YourSeq 78 2221 2323 3000 91.6% chr15 - 95464151 95464258 108 browser details YourSeq 78 2221 2323 3000 92.7% chr10 - 4365554 4366078 525 browser details YourSeq 77 2221 2321 3000 91.4% chr18 - 82446269 82446373 105 browser details YourSeq 77 1792 1910 3000 96.5% chr12 + 102717994 102718119 126

Note: The 3000 bp section upstream of Exon 4 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% chr18 + 31776329 31779328 3000 browser details YourSeq 147 1661 2194 3000 90.7% chr2 + 34692037 34692572 536 browser details YourSeq 132 1661 1982 3000 91.9% chr12 + 102298612 102298958 347 browser details YourSeq 131 1661 1855 3000 86.7% chr12 + 79159433 79159626 194 browser details YourSeq 128 1686 1983 3000 92.3% chr18 + 31991311 31991755 445 browser details YourSeq 122 1655 1853 3000 85.8% chr9 - 96103441 96103635 195 browser details YourSeq 122 1661 1982 3000 90.8% chr4 + 124697135 124697461 327 browser details YourSeq 120 1520 1824 3000 92.2% chr4 + 118298749 118299288 540 browser details YourSeq 119 1661 1825 3000 84.8% chr1 - 86050526 86050686 161 browser details YourSeq 119 1714 1982 3000 84.4% chr15 + 85848689 85848941 253 browser details YourSeq 117 1674 1859 3000 85.9% chr13 + 63408703 63408909 207 browser details YourSeq 116 1548 1956 3000 90.3% chr9 - 108755969 108756544 576 browser details YourSeq 116 1716 2194 3000 92.6% chr8 + 70265213 70265714 502 browser details YourSeq 114 1661 1823 3000 87.9% chr7 + 121967210 121967374 165 browser details YourSeq 114 1661 1851 3000 83.8% chr1 + 177500757 177500936 180 browser details YourSeq 113 1661 1819 3000 83.7% chr16 - 96149854 96150006 153 browser details YourSeq 111 1661 1968 3000 93.8% chr4 - 133348936 133349523 588 browser details YourSeq 107 1667 1852 3000 81.7% chr2 - 30805942 30806109 168 browser details YourSeq 106 1662 1818 3000 85.5% chr4 - 94718416 94718567 152 browser details YourSeq 104 1661 2093 3000 86.6% chr2 + 38559355 38559856 502

Note: The 3000 bp section downstream of Exon 6 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Ammecr1l AMME chromosomal region gene 1-like [ Mus musculus (house mouse) ] Gene ID: 225339, updated on 13-Aug-2019

Gene summary

Official Symbol Ammecr1l provided by MGI Official Full Name AMME chromosomal region gene 1-like provided by MGI Primary source MGI:MGI:2442711 See related Ensembl:ENSMUSG00000041915 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 AU022236; AU040755; AW111353; 5430429D03Rik; E230022H04Rik Expression Ubiquitous expression in testis adult (RPKM 16.4), limb E14.5 (RPKM 8.4) and 28 other tissues See more Orthologs human all

Genomic context

Location: 18; 18 B1 See Ammecr1l in Genome Data Viewer

Exon count: 12

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 18 NC_000084.6 (31758504..31784083)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 18 NC_000084.5 (31919534..31942397)

Chromosome 18 - NC_000084.6

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Transcript information: This gene has 5 transcripts

Gene: Ammecr1l ENSMUSG00000041915

Description AMME chromosomal region gene 1-like [Source:MGI Symbol;Acc:MGI:2442711] Gene Synonyms 5430429D03Rik, E230022H04Rik Location Chromosome 18: 31,759,826-31,782,743 forward strand. GRCm38:CM001011.2 About this gene This gene has 5 transcripts (splice variants), 145 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Ammecr1l- ENSMUST00000115808.3 3385 310aa ENSMUSP00000111475.2 Protein coding CCDS37753 Q8JZZ6 TSL:1 201 GENCODE basic APPRIS P1

Ammecr1l- ENSMUST00000234413.1 3181 310aa ENSMUSP00000156982.1 Protein coding CCDS37753 Q8JZZ6 GENCODE basic 203 APPRIS P1

Ammecr1l- ENSMUST00000234191.1 3174 300aa ENSMUSP00000157050.1 Protein coding - A0A3Q4EHL9 GENCODE basic 202

Ammecr1l- ENSMUST00000234441.1 2898 No protein - Retained intron - - - 204

Ammecr1l- ENSMUST00000234500.1 2689 No protein - Retained intron - - - 205

42.92 kb Forward strand 31.75Mb 31.76Mb 31.77Mb 31.78Mb 31.79Mb Genes (Comprehensive set... Ammecr1l-205 >retained intron Polr2d-201 >protein coding

Ammecr1l-201 >protein coding Polr2d-202 >protein coding

Ammecr1l-204 >retained intron

Ammecr1l-203 >protein coding

Ammecr1l-202 >protein coding

Contigs < AC161511.3

Genes < Gm26533-201lncRNA (Comprehensive set...

Regulatory Build

31.75Mb 31.76Mb 31.77Mb 31.78Mb 31.79Mb Reverse strand 42.92 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000115808

22.86 kb Forward strand

Ammecr1l-201 >protein coding

ENSMUSP00000111... MobiDB lite TIGRFAM AMMECR1 Superfamily AMMECR1 domain superfamily Pfam AMMECR1 domain PROSITE profiles AMMECR1 domain PANTHER AMMECR1

PTHR13016:SF1 Gene3D AMMECR1, N-terminal 3.30.1490.150

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 310

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

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