https://www.alphaknockout.com

Mouse Tpm2 Knockout Project (CRISPR/Cas9)

Objective: To create a Tpm2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Tpm2 (NCBI Reference Sequence: NM_009416 ; Ensembl: ENSMUSG00000028464 ) is located on Mouse 4. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000107913). Exon 3~8 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 3 starts from about 28.29% of the coding region. Exon 3~8 covers 62.44% of the coding region. The size of effective KO region: ~1515 bp. The KO 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 3 4 5 6 7 8 9

Legends Exon of mouse Tpm2 Knockout region

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Overview of the Dot Plot (up) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp

Forward Reverse Complement

Sequence 12

Note: The 2000 bp section downstream of Exon 8 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.

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Overview of the GC Content Distribution (up) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(24.55% 491) | C(25.3% 506) | T(25.7% 514) | G(24.45% 489)

Note: The 2000 bp section upstream of Exon 3 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.

Overview of the GC Content Distribution (down) Window size: 300 bp

Sequence 12

Summary: Full Length(2000bp) | A(28.8% 576) | C(24.1% 482) | T(26.95% 539) | G(20.15% 403)

Note: The 2000 bp section downstream of Exon 8 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 2000 1 2000 2000 100.0% chr4 - 43519745 43521744 2000 browser details YourSeq 79 900 1073 2000 89.8% chr1 - 91807890 91808267 378 browser details YourSeq 72 901 1048 2000 90.0% chr5 + 100366830 100367005 176 browser details YourSeq 71 900 1054 2000 85.2% chr17 - 24790091 24790281 191 browser details YourSeq 65 762 1069 2000 69.7% chr13 + 97158087 97158201 115 browser details YourSeq 63 899 1070 2000 87.9% chr12 + 78892575 78892789 215 browser details YourSeq 61 900 1071 2000 89.7% chr1 + 192839431 192839616 186 browser details YourSeq 57 969 1056 2000 92.6% chr6 - 88177275 88177386 112 browser details YourSeq 56 739 1050 2000 73.1% chr9 - 32026631 32026872 242 browser details YourSeq 56 969 1111 2000 89.9% chr11 + 79261667 79262081 415 browser details YourSeq 55 900 1055 2000 93.7% chr11 - 75380696 75380879 184 browser details YourSeq 55 902 1048 2000 95.1% chr5 + 93017466 93017673 208 browser details YourSeq 53 881 1043 2000 93.6% chr1 - 119550116 119550348 233 browser details YourSeq 49 931 1068 2000 87.4% chr10 + 127971542 127971720 179 browser details YourSeq 48 908 990 2000 94.5% chr7 - 137290835 137290928 94 browser details YourSeq 48 900 1053 2000 85.3% chr17 - 24191359 24191776 418 browser details YourSeq 48 992 1068 2000 78.0% chr12 - 87366519 87366585 67 browser details YourSeq 48 899 985 2000 91.4% chr10 - 111410625 111410722 98 browser details YourSeq 47 899 1056 2000 92.8% chr1 - 84956403 84956606 204 browser details YourSeq 47 899 1056 2000 92.8% chr1 - 85205350 85205553 204

Note: The 2000 bp section upstream of Exon 3 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 2000 1 2000 2000 100.0% chr4 - 43516230 43518229 2000 browser details YourSeq 163 1422 1606 2000 95.5% chr11 + 75316567 75316750 184 browser details YourSeq 158 1424 1610 2000 95.0% chr10 + 117086224 117086537 314 browser details YourSeq 157 1422 1611 2000 93.5% chr1 - 55206798 55206998 201 browser details YourSeq 156 1423 1594 2000 97.1% chr9 - 21965149 21965691 543 browser details YourSeq 156 1416 1599 2000 94.3% chr18 + 82655801 82655996 196 browser details YourSeq 155 1422 1610 2000 93.9% chr15 + 79355081 79355283 203 browser details YourSeq 155 1422 1608 2000 96.0% chr11 + 113580260 113580466 207 browser details YourSeq 155 1423 1610 2000 93.9% chr1 + 88039796 88168290 128495 browser details YourSeq 154 1436 1607 2000 95.9% chr12 + 83515240 83515426 187 browser details YourSeq 153 1415 1602 2000 94.8% chr8 - 109949360 109949558 199 browser details YourSeq 153 1422 1612 2000 92.9% chr5 - 149105484 149105800 317 browser details YourSeq 153 1422 1596 2000 94.3% chr4 - 127136803 127136988 186 browser details YourSeq 153 1422 1595 2000 95.9% chr10 + 117658236 117658420 185 browser details YourSeq 152 1422 1594 2000 95.8% chr9 - 63192294 63192481 188 browser details YourSeq 152 1422 1602 2000 94.8% chr19 - 3331371 3331589 219 browser details YourSeq 152 1422 1607 2000 91.8% chr1 - 52178120 52178307 188 browser details YourSeq 152 1415 1611 2000 92.3% chr13 + 38190116 38190316 201 browser details YourSeq 151 1422 1595 2000 93.7% chr5 - 130125456 130125639 184 browser details YourSeq 150 1422 1613 2000 91.8% chr19 - 41782963 41783179 217

Note: The 2000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Tpm2 2, beta [ Mus musculus (house mouse) ] Gene ID: 22004, updated on 1-Oct-2019

Gene summary

Official Symbol Tpm2 provided by MGI Official Full Name tropomyosin 2, beta provided by MGI Primary source MGI:MGI:98810 See related Ensembl:ENSMUSG00000028464 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 Tpm-2; Trop-2 Summary This gene belongs to the tropomyosin family which encodes that bind to filaments and stabilize them by Expression regulating access to actin modifying proteins. The encoded protein is a high molecular weight tropomyosin expressed in slow . In humans, mutations in this gene are associated with , cap disease and distal syndromes. Alternative splicing of this gene results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Apr 2013] Orthologs Biased expression in bladder adult (RPKM 555.0), mammary gland adult (RPKM 100.5) and 4 other tissues See more human all

Genomic context

Location: 4; 4 A5 See Tpm2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (43513726..43523583, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (43527584..43536260, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 6 transcripts

Gene: Tpm2 ENSMUSG00000028464

Description tropomyosin 2, beta [Source:MGI Symbol;Acc:MGI:98810] Gene Synonyms Tpm-2, Trop-2 Location Chromosome 4: 43,514,711-43,523,765 reverse strand. GRCm38:CM000997.2 About this gene This gene has 6 transcripts (splice variants), 110 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Tpm2-202 ENSMUST00000107913.9 2098 284aa ENSMUSP00000103546.3 Protein coding CCDS18100 P58774 TSL:1 GENCODE basic APPRIS P3

Tpm2-203 ENSMUST00000107914.9 1164 284aa ENSMUSP00000103547.3 Protein coding CCDS71374 P58774 Q6PJ18 TSL:1 GENCODE basic APPRIS ALT1

Tpm2-201 ENSMUST00000030184.11 2175 284aa ENSMUSP00000030184.5 Protein coding - A2AIM4 TSL:5 GENCODE basic APPRIS ALT1

Tpm2-206 ENSMUST00000150592.1 401 105aa ENSMUSP00000119908.1 Protein coding - A2AIM5 CDS 3' incomplete TSL:3

Tpm2-205 ENSMUST00000150262.7 1545 No protein - lncRNA - - TSL:2

Tpm2-204 ENSMUST00000133355.1 337 No protein - lncRNA - - TSL:5

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29.05 kb Forward strand 43.51Mb 43.52Mb 43.53Mb Car9-201 >protein coding (Comprehensive set...

Car9-202 >lncRNA

Car9-206 >protein coding

Car9-204 >lncRNA Car9-207 >lncRNA

Car9-203 >lncRNA

Car9-205 >lncRNA

Contigs AL732506.10 > Genes (Comprehensive set... < Gm12454-202lncRNA < Tpm2-203protein coding < Tln1-201protein coding

< Gm12454-201lncRNA < Tpm2-201protein coding

< Tpm2-205lncRNA

< Tpm2-202protein coding

< Tpm2-204lncRNA

< Tpm2-206protein coding

Regulatory Build

43.51Mb 43.52Mb 43.53Mb Reverse strand 29.05 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

RNA gene

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Transcript: ENSMUST00000107913

< Tpm2-202protein coding

Reverse strand 8.68 kb

ENSMUSP00000103... MobiDB lite Low complexity (Seg) Coiled-coils (Ncoils) Superfamily SSF57997 Prints Tropomyosin Pfam Tropomyosin PROSITE patterns Tropomyosin PANTHER PTHR19269

PTHR19269:SF46 Gene3D 1.20.5.340

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

Variant Legend missense variant synonymous variant

Scale bar 0 40 80 120 160 200 240 284

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

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