https://www.alphaknockout.com

Mouse Med13 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Med13 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Med13 (NCBI Reference Sequence: NM_001080931 ; Ensembl: ENSMUSG00000034297 ) is located on Mouse 11. 30 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 30 (Transcript: ENSMUST00000043624). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Med13 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-338C9 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 a conditional allele exhibited in the heart exhibit increased susceptibility to obesity and worsened glucose intolerance when fed a high fat diet.

Exon 2 starts from about 1.03% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 1951 bp, and the size of intron 2 for 3'-loxP site insertion: 9245 bp. The size of effective cKO region: ~735 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele gRNA region 5' gRNA region 3'

1 2 30 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Homology arm Exon of mouse Med13 cKO region loxP site

Page 2 of 7 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(7235bp) | A(22.64% 1638) | C(21.05% 1523) | T(33.52% 2425) | G(22.79% 1649)

Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector.

Page 3 of 7 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% chr11 - 86355759 86358758 3000 browser details YourSeq 47 127 522 3000 63.0% chr18 - 58196631 58196850 220 browser details YourSeq 43 880 958 3000 90.8% chr18 + 75310999 75311078 80 browser details YourSeq 38 1042 1112 3000 78.3% chr13 - 58127692 58127755 64 browser details YourSeq 30 123 163 3000 75.0% chr5 - 38610616 38610648 33 browser details YourSeq 29 851 885 3000 91.5% chr1 - 31834211 31834245 35 browser details YourSeq 26 867 895 3000 85.2% chrX - 105048864 105048890 27 browser details YourSeq 26 1729 1764 3000 96.6% chr1 - 41799876 41799913 38 browser details YourSeq 26 368 399 3000 86.7% chr5 + 52191876 52191906 31 browser details YourSeq 26 1311 1341 3000 93.4% chr17 + 79020554 79020586 33 browser details YourSeq 25 891 917 3000 88.5% chr6 - 57708636 57708661 26 browser details YourSeq 25 363 394 3000 75.9% chr17 + 13066459 13066487 29 browser details YourSeq 23 1632 1657 3000 96.0% chr18 - 82376531 82376556 26 browser details YourSeq 23 918 940 3000 100.0% chr10 - 41017865 41017887 23 browser details YourSeq 22 2707 2728 3000 100.0% chr11 + 77158436 77158457 22 browser details YourSeq 21 853 873 3000 100.0% chr7 - 11772061 11772081 21 browser details YourSeq 21 2725 2745 3000 100.0% chr1 + 69376206 69376226 21

Note: The 3000 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 3000 1 3000 3000 100.0% chr11 - 86352024 86355023 3000 browser details YourSeq 173 2109 2307 3000 96.3% chr11 - 86353788 86353986 199 browser details YourSeq 173 1038 1236 3000 96.3% chr11 - 86352717 86352915 199 browser details YourSeq 42 387 661 3000 95.7% chr5 - 114992454 114992732 279 browser details YourSeq 36 2764 2897 3000 97.4% chr11 - 54724127 54724292 166 browser details YourSeq 34 807 920 3000 90.5% chr2 - 155925552 155925667 116 browser details YourSeq 33 1573 1620 3000 72.3% chr12 + 39155559 39155594 36 browser details YourSeq 32 386 708 3000 45.8% chr14 - 54251305 54251339 35 browser details YourSeq 30 807 852 3000 87.5% chr8 + 120706704 120706747 44 browser details YourSeq 29 1204 1295 3000 62.2% chr12 - 15487940 15488005 66 browser details YourSeq 28 1594 1621 3000 100.0% chr7 + 16142090 16142117 28 browser details YourSeq 27 629 663 3000 96.6% chrX - 166403458 166403493 36 browser details YourSeq 25 1577 1613 3000 81.9% chr18 - 34939515 34939550 36 browser details YourSeq 25 647 675 3000 85.8% chr16 - 11223041 11223068 28 browser details YourSeq 24 634 663 3000 77.8% chr9 - 108685501 108685527 27 browser details YourSeq 24 1268 1295 3000 92.9% chr16 + 6492725 6492752 28 browser details YourSeq 23 1509 1535 3000 92.6% chr11 + 103374276 103374302 27 browser details YourSeq 22 237 260 3000 87.0% chr14 + 72244115 72244137 23 browser details YourSeq 22 239 262 3000 87.0% chr10 + 122733311 122733333 23

Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and information: Med13 complex subunit 13 [ Mus musculus (house mouse) ] Gene ID: 327987, updated on 10-Oct-2019

Gene summary

Official Symbol Med13 provided by MGI Official Full Name mediator complex subunit 13 provided by MGI Primary source MGI:MGI:3029632 See related Ensembl:ENSMUSG00000034297 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 Thrap1; Trap240; D030023K18; 1110067M05Rik Expression Ubiquitous expression in thymus adult (RPKM 6.7), whole brain E14.5 (RPKM 6.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 11; 11 C See Med13 in Genome Data Viewer

Exon count: 30

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (86267033..86357596, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (86079217..86171027, complement)

Chromosome 11 - NC_000077.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 4 transcripts

Gene: Med13 ENSMUSG00000034297

Description mediator complex subunit 13 [Source:MGI Symbol;Acc:MGI:3029632] Gene Synonyms 1110067M05Rik, Thrap1, Trap240 Location Chromosome 11: 86,267,033-86,357,602 reverse strand. GRCm38:CM001004.2 About this gene This gene has 4 transcripts (splice variants), 264 orthologues, 1 paralogue and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Med13-201 ENSMUST00000043624.8 10546 2171aa ENSMUSP00000044268.8 Protein coding CCDS36267 Q5SWW4 TSL:2 GENCODE basic APPRIS P1

Med13-203 ENSMUST00000140514.1 738 No protein - Retained intron - - TSL:3

Med13-204 ENSMUST00000144983.1 481 No protein - Retained intron - - TSL:2

Med13-202 ENSMUST00000136622.7 540 No protein - lncRNA - - TSL:5

110.57 kb Forward strand

86.26Mb 86.28Mb 86.30Mb 86.32Mb 86.34Mb 86.36Mb Brip1os-202 >lncRNA Gm25427-201 >misc RNA (Comprehensive set...

Contigs < AL592065.7 AL596256.14 >

Genes (Comprehensive set... < Ints2-201protein coding< Med13-202lncRNA < Med13-204retained intron

< Ints2-207retained intron < Med13-203retained intron

< Ints2-209protein coding

< Ints2-205protein coding

< Ints2-208protein coding

< Ints2-204retained intron

< Ints2-210lncRNA

< Med13-201protein coding

Regulatory Build

86.26Mb 86.28Mb 86.30Mb 86.32Mb 86.34Mb 86.36Mb Reverse strand 110.57 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

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000043624

< Med13-201protein coding

Reverse strand 90.57 kb

ENSMUSP00000044... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Mediator complex, subunit Med13, N-terminal, metazoa/fungi MID domain of medPIWI

Mediator complex subunit Med13, C-terminal PANTHER PTHR10791:SF51

PTHR10791

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

Variant Legend missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1600 1800 2171

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

Page 7 of 7