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Mouse Tubgcp6 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Tubgcp6 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tubgcp6 gene (NCBI Reference Sequence: NM_001163319.1 ; Ensembl: ENSMUSG00000051786 ) is located on Mouse chromosome 15. 25 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 25 (Transcript: ENSMUST00000109353). 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 Tubgcp6 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-65G19 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 13.98% 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: 1403 bp, and the size of intron 2 for 3'-loxP site insertion: 4254 bp. The size of effective cKO region: ~664 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 25 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Tubgcp6 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(7164bp) | A(24.25% 1737) | C(23.63% 1693) | T(26.93% 1929) | G(25.2% 1805)

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.

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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% chr15 - 89120924 89123923 3000 browser details YourSeq 178 2131 2599 3000 90.5% chr16 + 17587908 17588392 485 browser details YourSeq 166 2144 2622 3000 90.3% chr3 + 32434608 32535050 100443 browser details YourSeq 163 2113 2600 3000 84.3% chr6 + 47923277 47923685 409 browser details YourSeq 135 2126 2277 3000 94.7% chr10 - 60093592 60093743 152 browser details YourSeq 135 2127 2386 3000 83.0% chr5 + 122133338 122133509 172 browser details YourSeq 135 2127 2546 3000 93.6% chr15 + 10730470 10731018 549 browser details YourSeq 134 2127 2301 3000 92.4% chr13 - 58895873 58896047 175 browser details YourSeq 134 2127 2282 3000 91.5% chr10 - 112069907 112070059 153 browser details YourSeq 133 2127 2278 3000 94.1% chr2 - 31444105 31444257 153 browser details YourSeq 132 2127 2278 3000 94.1% chr11 - 23461359 23461512 154 browser details YourSeq 132 2126 2273 3000 94.6% chr9 + 118038316 118038463 148 browser details YourSeq 130 2126 2282 3000 92.2% chrX - 102647927 102648102 176 browser details YourSeq 130 2126 2280 3000 90.1% chr10 - 75383275 75383427 153 browser details YourSeq 130 2122 2282 3000 91.2% chr18 + 5668733 5668904 172 browser details YourSeq 129 2126 2282 3000 91.8% chr3 - 109394945 109395103 159 browser details YourSeq 129 2127 2280 3000 94.6% chr14 - 76063407 76063564 158 browser details YourSeq 129 2126 2277 3000 91.1% chr5 + 85242119 85242267 149 browser details YourSeq 129 2127 2268 3000 95.8% chr19 + 8960049 8960191 143 browser details YourSeq 129 2126 2280 3000 92.3% chr18 + 87088969 87089139 171

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% chr15 - 89117260 89120259 3000 browser details YourSeq 257 1310 2105 3000 91.1% chrX + 157316041 157664181 348141 browser details YourSeq 197 1319 1820 3000 87.3% chr16 + 38511635 38512110 476 browser details YourSeq 169 1475 1833 3000 87.3% chr4 - 32803586 33080817 277232 browser details YourSeq 161 1331 1875 3000 92.6% chr17 - 24683614 24684234 621 browser details YourSeq 157 1323 1534 3000 89.6% chr11 - 80562201 80562411 211 browser details YourSeq 152 1323 1800 3000 94.2% chr4 + 133736255 133736741 487 browser details YourSeq 149 1328 1820 3000 94.7% chr10 - 124035268 124035861 594 browser details YourSeq 143 1327 1920 3000 84.1% chrX - 7979918 7980278 361 browser details YourSeq 142 1323 1889 3000 83.9% chr5 + 101355255 101355458 204 browser details YourSeq 141 1310 1466 3000 94.0% chr2 - 157961881 157962033 153 browser details YourSeq 138 1393 1820 3000 78.1% chr11 - 87187053 87187297 245 browser details YourSeq 138 1310 1472 3000 89.9% chr5 + 65356005 65356162 158 browser details YourSeq 137 1323 1471 3000 96.6% chr8 - 84825115 84825277 163 browser details YourSeq 137 1325 1467 3000 98.0% chr13 - 104149770 104149912 143 browser details YourSeq 136 1323 1877 3000 82.8% chr4 + 139334381 139334555 175 browser details YourSeq 134 1323 1467 3000 94.5% chr7 - 118295185 118295327 143 browser details YourSeq 134 1331 1876 3000 81.5% chr19 - 5025034 5025197 164 browser details YourSeq 134 1324 1472 3000 93.2% chr9 + 85543100 85543245 146 browser details YourSeq 134 1323 1467 3000 94.5% chr1 + 85099265 85099407 143

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: Tubgcp6 tubulin, gamma complex associated protein 6 [ Mus musculus (house mouse) ] Gene ID: 328580, updated on 26-Jun-2020

Gene summary

Official Symbol Tubgcp6 provided by MGI Official Full Name tubulin, gamma complex associated protein 6 provided by MGI Primary source MGI:MGI:2146071 See related Ensembl:ENSMUSG00000051786 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 GCP-6; AI843275; mKIAA1669; D430023H11 Expression Ubiquitous expression in thymus adult (RPKM 11.9), genital fat pad adult (RPKM 11.8) and 28 other tissues See more Orthologs human all

Genomic context

Location: 15; 15 E3 See Tubgcp6 in Genome Data Viewer

Exon count: 25

Annotation release Status Assembly Chr Location

108.20200622 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (89099097..89123208, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (88929528..88953580, complement)

Chromosome 15 - NC_000081.6

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Transcript information: This gene has 11 transcripts

Gene: Tubgcp6 ENSMUSG00000051786

Description tubulin, gamma complex associated protein 6 [Source:MGI Symbol;Acc:MGI:2146071] Location Chromosome 15: 89,098,357-89,123,112 reverse strand. GRCm38:CM001008.2 About this gene This gene has 11 transcripts (splice variants), 256 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tubgcp6- ENSMUST00000109353.8 7635 1769aa ENSMUSP00000104977.2 Protein coding CCDS49697 G5E8P0 TSL:5 202 GENCODE basic APPRIS P2

Tubgcp6- ENSMUST00000041656.13 5357 1761aa ENSMUSP00000040132.7 Protein coding - E9Q6P4 TSL:5 201 GENCODE basic APPRIS ALT2

Tubgcp6- ENSMUST00000163290.7 1328 293aa ENSMUSP00000131359.1 Nonsense mediated - F6ZAT3 CDS 5' 203 decay incomplete TSL:5

Tubgcp6- ENSMUST00000166480.1 759 128aa ENSMUSP00000132108.1 Nonsense mediated - F6X8A1 CDS 5' 205 decay incomplete TSL:5

Tubgcp6- ENSMUST00000169069.1 586 139aa ENSMUSP00000132786.1 Nonsense mediated - F6W5U8 CDS 5' 208 decay incomplete TSL:5

Tubgcp6- ENSMUST00000170877.1 508 40aa ENSMUSP00000129291.1 Nonsense mediated - F6YBF3 CDS 5' 210 decay incomplete TSL:3

Tubgcp6- ENSMUST00000231098.1 4674 No - Retained intron - - - 211 protein

Tubgcp6- ENSMUST00000164717.1 1920 No - Retained intron - - TSL:1 204 protein

Tubgcp6- ENSMUST00000166994.1 953 No - Retained intron - - TSL:5 206 protein

Tubgcp6- ENSMUST00000168256.1 802 No - Retained intron - - TSL:2 207 protein

Tubgcp6- ENSMUST00000169208.1 514 No - Retained intron - - TSL:2 209 protein

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44.76 kb Forward strand

89.09Mb 89.10Mb 89.11Mb 89.12Mb 89.13Mb Genes Selenoo-201 >protein coding (Comprehensive set...

Selenoo-202 >nonsense mediated decay

Contigs AC113069.10 > Genes (Comprehensive set... < Tubgcp6-202protein coding < Hdac10-201protein coding

< Tubgcp6-204retained intron < Tubgcp6-211retained intron < Hdac10-208processed transcript

< Tubgcp6-201protein coding < Hdac10-204processed transcript

< Tubgcp6-203nonsense mediated decay < Hdac10-203retained intron

< Tubgcp6-206retained intron < Hdac10-207retained intron

< Tubgcp6-209retained intron < Hdac10-205retained intron

< Tubgcp6-205nonsense mediated decay < Hdac10-202protein coding

< Tubgcp6-207retained intron < Hdac10-206processed transcript

< Tubgcp6-210nonsense mediated decay < Mapk12-205processed transcript

< Tubgcp6-208nonsense mediated decay < Mapk12-206processed transcript

< Mapk12-207processed transcript

< Mapk12-204retained intron

< Mapk12-203retained intron

< Mapk12-201protein coding

Regulatory Build

89.09Mb 89.10Mb 89.11Mb 89.12Mb 89.13Mb Reverse strand 44.76 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana Ensembl protein coding

Non-Protein Coding

processed transcript

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Transcript: ENSMUST00000109353

< Tubgcp6-202protein coding

Reverse strand 24.73 kb

ENSMUSP00000104... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Pfam Gamma tubulin complex component protein, N-terminal Gamma tubulin complex component, C-terminal

PANTHER Gamma-tubulin complex component protein

PTHR19302:SF55 Gene3D Gamma-tubulin complex, C-terminal domain superfamily

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

Variant Legend

inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1400 1769

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

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