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Mouse Bcl2l13 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Bcl2l13 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Bcl2l13 gene (NCBI Reference Sequence: NM_153516 ; Ensembl: ENSMUSG00000009112 ) is located on Mouse chromosome 6. 7 exons are identified, with the ATG start codon in exon 2 and the TAA stop codon in exon 7 (Transcript: ENSMUST00000009256). 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 Bcl2l13 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-385F20 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 2 starts from about 100% 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: 12098 bp, and the size of intron 2 for 3'-loxP site insertion: 14238 bp. The size of effective cKO region: ~612 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 2 7 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Bcl2l13 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(7112bp) | A(26.69% 1898) | C(21.02% 1495) | T(31.96% 2273) | G(20.33% 1446)

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% chr6 + 120845256 120848255 3000 browser details YourSeq 264 1005 1596 3000 93.5% chr13 + 64316926 64317883 958 browser details YourSeq 263 1025 1595 3000 92.1% chr10 - 117064448 117065576 1129 browser details YourSeq 260 1015 1744 3000 83.5% chr8 + 107166843 107167433 591 browser details YourSeq 259 1023 1596 3000 87.0% chr4 + 141765416 141765765 350 browser details YourSeq 253 1050 2039 3000 84.7% chr11 - 88921639 88922421 783 browser details YourSeq 250 1025 1596 3000 90.1% chr11 - 51590428 51798550 208123 browser details YourSeq 244 1055 1596 3000 88.0% chr3 + 94934978 94935357 380 browser details YourSeq 243 1024 1595 3000 87.2% chr10 - 81211023 81211459 437 browser details YourSeq 227 1025 1596 3000 85.0% chr15 - 99000002 99000308 307 browser details YourSeq 219 1023 1590 3000 84.4% chr9 - 44893143 44893449 307 browser details YourSeq 204 1028 1596 3000 83.8% chr12 - 84298485 84298915 431 browser details YourSeq 190 1097 1596 3000 85.0% chr17 - 29196510 29196918 409 browser details YourSeq 187 1405 2052 3000 88.3% chr19 - 44541295 44541959 665 browser details YourSeq 181 1122 1596 3000 92.5% chr13 - 54901380 54902041 662 browser details YourSeq 174 1086 1595 3000 83.8% chr5 + 135292729 135293154 426 browser details YourSeq 173 1404 2072 3000 82.2% chr11 - 119361490 119361725 236 browser details YourSeq 173 944 1596 3000 83.9% chr17 + 85096963 85097181 219 browser details YourSeq 172 1112 1597 3000 90.6% chr2 - 146789656 146790278 623 browser details YourSeq 172 1409 1619 3000 91.4% chr15 + 99009278 99009944 667

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% chr6 + 120848868 120851867 3000 browser details YourSeq 1341 450 2659 3000 90.0% chr6 + 133873078 133875044 1967 browser details YourSeq 1255 468 2660 3000 89.5% chr3 + 123770870 123772669 1800 browser details YourSeq 1202 455 2618 3000 88.3% chr17 - 36589187 36591012 1826 browser details YourSeq 1169 473 2659 3000 88.2% chr10 - 86773008 86774771 1764 browser details YourSeq 1099 591 2614 3000 87.7% chrX + 127091391 127440441 349051 browser details YourSeq 861 1053 2659 3000 88.7% chr2 - 52748388 52749650 1263 browser details YourSeq 829 1482 2659 3000 90.6% chrX - 36686105 36687326 1222 browser details YourSeq 814 1482 2644 3000 88.1% chr1 - 146659083 146660260 1178 browser details YourSeq 812 1483 2662 3000 88.2% chr8 + 4600031 4601138 1108 browser details YourSeq 811 1482 2660 3000 89.3% chr12 + 59254989 59256085 1097 browser details YourSeq 810 1482 2644 3000 89.1% chr2 - 89452598 89453729 1132 browser details YourSeq 809 1485 2661 3000 89.3% chr17 + 17656199 17657431 1233 browser details YourSeq 808 1488 2795 3000 87.8% chr12 - 64808125 64809382 1258 browser details YourSeq 805 1482 2658 3000 87.9% chr11 - 110123063 110124279 1217 browser details YourSeq 786 1483 2659 3000 88.5% chr14 + 73103683 73104803 1121 browser details YourSeq 782 1482 2635 3000 88.8% chr2 + 111601506 111602686 1181 browser details YourSeq 779 1482 2660 3000 87.0% chr3 - 103847525 103848675 1151 browser details YourSeq 778 1489 2659 3000 88.3% chr1 - 174433476 174434727 1252 browser details YourSeq 769 1482 2661 3000 88.3% chr13 + 36253036 36254142 1107

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

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Gene and protein information: Bcl2l13 BCL2-like 13 (apoptosis facilitator) [ Mus musculus (house mouse) ] Gene ID: 94044, updated on 10-Oct-2019

Gene summary

Official Symbol Bcl2l13 provided by MGI Official Full Name BCL2-like 13 (apoptosis facilitator) provided by MGI Primary source MGI:MGI:2136959 See related Ensembl:ENSMUSG00000009112 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 Mil1; Mil-1; BCL-RAMBO; E430016C20Rik Expression Ubiquitous expression in heart adult (RPKM 9.6), subcutaneous fat pad adult (RPKM 9.3) and 28 other tissues See more Orthologs human all

Genomic context

Location: 6 F1; 6 57.01 cM See Bcl2l13 in Genome Data Viewer

Exon count: 7

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (120836206..120892842)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (120786248..120842860)

Chromosome 6 - NC_000072.6

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Transcript information: This gene has 4 transcripts

Gene: Bcl2l13 ENSMUSG00000009112

Description BCL2-like 13 (apoptosis facilitator) [Source:MGI Symbol;Acc:MGI:2136959] Gene Synonyms BCL-RAMBO, E430016C20Rik, Mil-1, Mil1 Location Chromosome 6: 120,836,212-120,892,842 forward strand. GRCm38:CM000999.2 About this gene This gene has 4 transcripts (splice variants), 205 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Bcl2l13-201 ENSMUST00000009256.3 6925 434aa ENSMUSP00000009256.2 Protein coding CCDS20485 P59017 TSL:1 GENCODE basic APPRIS P1

Bcl2l13-202 ENSMUST00000203037.2 408 75aa ENSMUSP00000145390.1 Protein coding - A0A0N4SW63 CDS 3' incomplete TSL:5

Bcl2l13-203 ENSMUST00000203584.2 349 51aa ENSMUSP00000145341.1 Protein coding - A0A0N4SW24 CDS 3' incomplete TSL:2

Bcl2l13-204 ENSMUST00000204004.1 2710 No protein - Retained intron - - TSL:1

76.63 kb Forward strand 120.84Mb 120.86Mb 120.88Mb 120.90Mb Genes (Comprehensive set... Bcl2l13-204 >retained intron

Bcl2l13-203 >protein coding

Bcl2l13-202 >protein coding

Bcl2l13-201 >protein coding

Contigs AC084273.20 > < AC083894.21

Genes < Bid-204nonsense mediated decay (Comprehensive set...

< Bid-205retained intron

< Bid-201protein coding

< Bid-203protein coding

Regulatory Build

120.84Mb 120.86Mb 120.88Mb 120.90Mb Reverse strand 76.63 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

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Transcript: ENSMUST00000009256

56.60 kb Forward strand

Bcl2l13-201 >protein coding

ENSMUSP00000009... Transmembrane heli... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily Blc2-like superfamily Pfam Blc2 family PROSITE profiles Bcl2-like PANTHER Bcl-2-like protein 13 Gene3D Blc2-like superfamily

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 434

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

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