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Mouse Vps29 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Vps29 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Vps29 gene (NCBI Reference Sequence: NM_019780 ; Ensembl: ENSMUSG00000029462 ) is located on Mouse chromosome 5. 4 exons are identified, with the ATG start codon in exon 1 and the TAA stop codon in exon 4 (Transcript: ENSMUST00000155671). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Vps29 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-97B19 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 0.73% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5536 bp, and the size of intron 3 for 3'-loxP site insertion: 533 bp. The size of effective cKO region: ~2760 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 2 3 4 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Vps29 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(9259bp) | A(26.68% 2470) | C(20.96% 1941) | T(32.14% 2976) | G(20.22% 1872)

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% chr5 + 122356764 122359763 3000 browser details YourSeq 237 800 2639 3000 90.0% chr11 + 80203039 80535701 332663 browser details YourSeq 158 896 2533 3000 90.9% chr3 - 34633261 34881156 247896 browser details YourSeq 156 772 1213 3000 91.1% chr19 - 5709478 5709962 485 browser details YourSeq 155 830 1180 3000 92.9% chr7 - 28938257 28938633 377 browser details YourSeq 148 851 1236 3000 84.7% chr11 + 101255281 101255600 320 browser details YourSeq 112 1058 2639 3000 87.4% chr9 - 121747676 121900009 152334 browser details YourSeq 108 851 1185 3000 79.9% chr15 - 102344390 102344649 260 browser details YourSeq 96 835 1011 3000 83.7% chr8 - 107892821 107892989 169 browser details YourSeq 89 845 955 3000 86.6% chr16 + 20557023 20557126 104 browser details YourSeq 87 859 955 3000 94.9% chr9 - 113623392 113623488 97 browser details YourSeq 83 851 975 3000 82.2% chr14 + 108698261 108698384 124 browser details YourSeq 79 2488 2614 3000 90.8% chr4 + 148133832 148390541 256710 browser details YourSeq 73 1140 1644 3000 79.4% chr11 + 96082739 96083200 462 browser details YourSeq 69 1140 2533 3000 98.6% chr12 - 31061538 31372974 311437 browser details YourSeq 66 851 927 3000 93.6% chr11 + 85036990 85037067 78 browser details YourSeq 62 1135 1236 3000 94.3% chr6 - 129549798 129549910 113 browser details YourSeq 60 2412 2522 3000 95.5% chr18 - 6151373 6151485 113 browser details YourSeq 60 1605 1684 3000 90.8% chr17 - 28980546 28980645 100 browser details YourSeq 58 2387 2533 3000 90.5% chr5 + 54384915 54385076 162

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% chr5 + 122362523 122365522 3000 browser details YourSeq 275 2058 2399 3000 91.2% chrX - 38506023 38506371 349 browser details YourSeq 250 1320 2629 3000 91.7% chr10 - 78035947 78133206 97260 browser details YourSeq 199 1273 1638 3000 93.2% chr17 + 45496555 45497137 583 browser details YourSeq 196 1190 1580 3000 93.4% chr4 - 149326748 149729196 402449 browser details YourSeq 188 1093 1577 3000 88.3% chr11 + 78043600 78044359 760 browser details YourSeq 178 1278 1622 3000 89.4% chr17 - 46603573 46603922 350 browser details YourSeq 175 1118 1461 3000 82.6% chr16 + 33220387 33220651 265 browser details YourSeq 174 811 1452 3000 89.2% chr13 - 95485143 95485787 645 browser details YourSeq 170 1208 1463 3000 91.8% chr4 - 126983856 126984416 561 browser details YourSeq 167 1270 1466 3000 90.2% chr14 + 11773884 11774076 193 browser details YourSeq 167 1273 1954 3000 81.2% chr11 + 68575035 68575388 354 browser details YourSeq 165 1209 1475 3000 87.0% chr11 + 99204907 99205154 248 browser details YourSeq 161 1103 1436 3000 91.3% chr17 - 7525057 7525604 548 browser details YourSeq 160 1269 1660 3000 85.1% chr5 + 31459943 31460128 186 browser details YourSeq 158 1271 1458 3000 93.9% chr10 + 69518112 69518308 197 browser details YourSeq 157 1270 1458 3000 91.6% chr5 - 129861770 129861958 189 browser details YourSeq 157 1292 1622 3000 90.3% chr11 - 75129626 75130209 584 browser details YourSeq 157 1208 1459 3000 92.1% chr10 - 77028088 77028368 281 browser details YourSeq 156 1280 1459 3000 93.4% chr2 - 31868120 31868299 180

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Vps29 VPS29 retromer complex component [ Mus musculus (house mouse) ] Gene ID: 56433, updated on 14-Aug-2019

Gene summary

Official Symbol Vps29 provided by MGI Official Full Name VPS29 retromer complex component provided by MGI Primary source MGI:MGI:1928344 See related Ensembl:ENSMUSG00000029462 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 PEP11; AW049835; 2010015D08Rik Expression Ubiquitous expression in CNS E18 (RPKM 27.9), placenta adult (RPKM 22.1) and 28 other tissues See more Orthologs human all

Genomic context

Location: 5; 5 F See Vps29 in Genome Data Viewer

Exon count: 6

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 5 NC_000071.6 (122354328..122364984)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 5 NC_000071.5 (122804422..122813296)

Chromosome 5 - NC_000071.6

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Transcript information: This gene has 9 transcripts

Gene: Vps29 ENSMUSG00000029462

Description VPS29 retromer complex component [Source:MGI Symbol;Acc:MGI:1928344] Gene Synonyms 2010015D08Rik, PEP11 Location Chromosome 5: 122,354,369-122,364,984 forward strand. GRCm38:CM000998.2 About this gene This gene has 9 transcripts (splice variants), 234 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Vps29- ENSMUST00000155671.7 2716 182aa ENSMUSP00000121020.1 Protein coding CCDS39256 Q9QZ88 TSL:1 208 GENCODE basic APPRIS P3

Vps29- ENSMUST00000118830.7 1014 186aa ENSMUSP00000113525.1 Protein coding CCDS84954 Q9QZ88 TSL:1 204 GENCODE basic APPRIS ALT1

Vps29- ENSMUST00000117868.7 720 144aa ENSMUSP00000113345.1 Protein coding - D3Z645 TSL:2 202 GENCODE basic

Vps29- ENSMUST00000118765.7 713 91aa ENSMUSP00000112579.1 Protein coding - D3YW98 TSL:3 203 GENCODE basic

Vps29- ENSMUST00000145821.1 407 124aa ENSMUSP00000123593.1 Protein coding - D3YYD5 CDS 3' 206 incomplete TSL:3

Vps29- ENSMUST00000111729.9 874 53aa ENSMUSP00000107358.3 Nonsense mediated - H7BXA2 TSL:2 201 decay

Vps29- ENSMUST00000154686.1 488 37aa ENSMUSP00000118991.1 Nonsense mediated - D6RH43 TSL:5 207 decay

Vps29- ENSMUST00000199086.1 3364 No - Retained intron - - TSL:NA 209 protein

Vps29- ENSMUST00000132785.1 529 No - lncRNA - - TSL:5 205 protein

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30.62 kb Forward strand 122.35Mb 122.36Mb 122.37Mb Genes (Comprehensive set... Vps29-208 >protein coding Gpn3-203 >nonsense mediated decay

Vps29-204 >protein coding Gpn3-202 >nonsense mediated decay

Vps29-202 >protein coding Gpn3-206 >retained intron

Vps29-205 >lncRNA Gpn3-201 >protein coding

Vps29-203 >protein coding

Vps29-201 >nonsense mediated decay

Vps29-206 >protein coding

Vps29-207 >nonsense mediated decay

Vps29-209 >retained intron

Contigs < AC093473.6 < AC113285.9 Genes < Rad9b-202protein coding < Fam216a-201protein coding (Comprehensive set...

< Rad9b-204protein coding < Rad9b-205retained intron

< Rad9b-201protein coding

< Rad9b-203protein coding

Regulatory Build

122.35Mb 122.36Mb 122.37Mb Reverse strand 30.62 kb

Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

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Transcript: ENSMUST00000155671

10.62 kb Forward strand

Vps29-208 >protein coding

ENSMUSP00000121... PDB-ENSP mappings TIGRFAM Phosphodiesterase MJ0936/Vps29

Superfamily SSF56300

Pfam Calcineurin-like phosphoesterase domain, lpxH-type PANTHER Phosphodiesterase MJ0936/Vps29

Vacuolar protein sorting-associated protein 29 Gene3D Metallo-dependent phosphatase-like CDD Vacuolar protein sorting-associated protein 29

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

Variant Legend synonymous variant

Scale bar 0 20 40 60 80 100 120 140 160 182

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

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