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Mouse Zmym2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Zmym2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Zmym2 gene (NCBI Reference Sequence: NM_029498 ; Ensembl: ENSMUSG00000021945 ) is located on Mouse chromosome 14. 24 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 24 (Transcript: ENSMUST00000022511). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Zmym2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-83G22 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 an ENU-induced mutation exhibit prenatal lethality.

Exon 4 starts from about 27.4% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 1591 bp, and the size of intron 4 for 3'-loxP site insertion: 838 bp. The size of effective cKO region: ~666 bp. The cKO region does not have any other known gene.

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Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 3 4 5 24 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Zmym2 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector.

Overview of the GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(7166bp) | A(26.89% 1927) | C(16.49% 1182) | T(37.44% 2683) | G(19.17% 1374)

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% chr14 + 56909703 56912702 3000 browser details YourSeq 280 1362 1663 3000 96.4% chr14 - 56798905 56799206 302 browser details YourSeq 263 422 1103 3000 89.1% chr1 + 167296442 167297188 747 browser details YourSeq 262 766 1103 3000 89.5% chr11 + 78869325 79226444 357120 browser details YourSeq 230 811 1103 3000 90.9% chr10 + 127315000 127325389 10390 browser details YourSeq 210 1375 1666 3000 87.8% chr8 + 21237168 21237462 295 browser details YourSeq 210 1375 1666 3000 87.8% chr8 + 21101145 21101439 295 browser details YourSeq 206 1375 1666 3000 87.1% chr8 + 21599399 21599693 295 browser details YourSeq 204 1375 1666 3000 86.7% chr8 + 21461452 21461746 295 browser details YourSeq 203 812 1103 3000 85.1% chr11 - 60824289 60824570 282 browser details YourSeq 196 772 1103 3000 89.9% chr2 - 150781411 150782049 639 browser details YourSeq 192 812 1089 3000 85.9% chr10 - 117460780 117461046 267 browser details YourSeq 191 1375 1619 3000 91.0% chr8 + 21708525 21708775 251 browser details YourSeq 190 812 1096 3000 90.0% chr11 + 110388338 110388737 400 browser details YourSeq 189 1375 1619 3000 90.6% chr8 + 21331442 21331692 251 browser details YourSeq 187 815 1103 3000 88.5% chr8 + 104289395 104289917 523 browser details YourSeq 170 842 1103 3000 91.7% chr11 - 61114924 61115281 358 browser details YourSeq 166 741 1103 3000 84.5% chr19 - 53111395 53111651 257 browser details YourSeq 160 812 1103 3000 91.7% chr13 - 104088239 104088765 527 browser details YourSeq 160 812 1091 3000 83.6% chr1 - 59618373 59618601 229

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% chr14 + 56913369 56916368 3000 browser details YourSeq 146 1 1534 3000 94.0% chr11 + 98735599 99233767 498169 browser details YourSeq 123 1452 1616 3000 88.4% chr10 + 127221946 127222109 164 browser details YourSeq 114 1462 1630 3000 84.9% chr4 - 139079350 139079515 166 browser details YourSeq 110 1454 1587 3000 91.1% chr17 + 51732953 51733086 134 browser details YourSeq 109 1445 1619 3000 84.7% chr2 + 132461561 132461796 236 browser details YourSeq 105 1454 1631 3000 90.7% chr1 + 59881226 59881419 194 browser details YourSeq 102 1452 1578 3000 92.5% chr11 - 62528268 62528730 463 browser details YourSeq 99 1475 1587 3000 92.0% chr12 - 33163686 33163797 112 browser details YourSeq 99 1456 1591 3000 86.7% chr13 + 17774856 17775001 146 browser details YourSeq 97 1450 1582 3000 86.5% chr4 + 135201320 135201452 133 browser details YourSeq 95 1448 1571 3000 88.8% chr9 - 56906557 56906681 125 browser details YourSeq 95 1452 1576 3000 88.0% chr10 - 32814508 32814632 125 browser details YourSeq 93 1459 1573 3000 90.5% chr16 - 29785318 29785432 115 browser details YourSeq 93 1452 1578 3000 86.7% chr16 - 8503685 8503811 127 browser details YourSeq 93 1471 1587 3000 89.8% chr5 + 147642216 147642332 117 browser details YourSeq 92 1452 1582 3000 83.6% chr11 - 57541486 57541614 129 browser details YourSeq 92 1456 1582 3000 86.7% chr10 + 21127356 21127486 131 browser details YourSeq 91 1448 1574 3000 83.2% chr14 + 30983294 30983418 125 browser details YourSeq 90 1458 1578 3000 88.4% chr10 + 95286695 95286814 120

Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Zmym2 zinc finger, MYM-type 2 [ Mus musculus (house mouse) ] Gene ID: 76007, updated on 24-Oct-2019

Gene summary

Official Symbol Zmym2 provided by MGI Official Full Name zinc finger, MYM-type 2 provided by MGI Primary source MGI:MGI:1923257 See related Ensembl:ENSMUSG00000021945 Gene type protein coding RefSeq status REVIEWED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as FIM; MYM; RAMP; SCLL; Zfp198; Znf198; 5830413P05Rik Summary This gene encodes a protein that contains nine MYM-type zinc finger motifs. Expression of this gene may mediate the Expression inhibition of hematopoietic cell development during ontogeny, and the encoded protein may also play a role in transforming growth factor-beta signaling as a Smad binding protein. [provided by RefSeq, Feb 2011] Orthologs Broad expression in CNS E11.5 (RPKM 12.5), CNS E14 (RPKM 11.4) and 25 other tissues See more human all

Genomic context

Location: 14; 14 C3 See Zmym2 in Genome Data Viewer

Exon count: 27

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (56886529..56962358)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (57506631..57579186)

Chromosome 14 - NC_000080.6

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Transcript information: This gene has 7 transcripts

Gene: Zmym2 ENSMUSG00000021945

Description zinc finger, MYM-type 2 [Source:MGI Symbol;Acc:MGI:1923257] Gene Synonyms FIM, MYM, RAMP, SCLL, Zfp198 Location Chromosome 14: 56,886,653-56,962,701 forward strand. GRCm38:CM001007.2 About this gene This gene has 7 transcripts (splice variants), 203 orthologues, 13 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Zmym2-201 ENSMUST00000022511.9 6974 1376aa ENSMUSP00000022511.8 Protein coding CCDS49506 Q9CU65 TSL:1 GENCODE basic APPRIS P1

Zmym2-202 ENSMUST00000223669.1 1281 290aa ENSMUSP00000152925.1 Protein coding - A0A286YCD5 GENCODE basic

Zmym2-203 ENSMUST00000223965.1 772 No protein - Retained intron - - -

Zmym2-207 ENSMUST00000226025.1 607 No protein - Retained intron - - -

Zmym2-206 ENSMUST00000225393.1 332 No protein - Retained intron - - -

Zmym2-204 ENSMUST00000224922.1 2829 No protein - lncRNA - - -

Zmym2-205 ENSMUST00000225282.1 741 No protein - lncRNA - - -

96.05 kb Forward strand 56.88Mb 56.90Mb 56.92Mb 56.94Mb 56.96Mb Genes (Comprehensive set... Zmym2-202 >protein coding Zmym2-204 >lncRNA

Zmym2-201 >protein coding

Zmym2-206 >retained intron

Zmym2-205 >lncRNAZmym2-203 >retained intron

Zmym2-207 >retained intron

Contigs < AC154361.2 < AC154303.2 Regulatory Build

56.88Mb 56.90Mb 56.92Mb 56.94Mb 56.96Mb Reverse strand 96.05 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000022511

74.88 kb Forward strand

Zmym2-201 >protein coding

ENSMUSP00000022... MobiDB lite Low complexity (Seg) Superfamily SSF57716 SMART TRASH domain Pfam Zinc finger, MYM-type Protein of unknown function DUF3504 PANTHER PTHR45736

PTHR45736:SF6

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

Variant Legend missense variant synonymous variant

Scale bar 0 200 400 600 800 1000 1376

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

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