https://www.alphaknockout.com

Mouse Itgb3bp Knockout Project (CRISPR/Cas9)

Objective: To create a Itgb3bp knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Itgb3bp (NCBI Reference Sequence: NM_026348 ; Ensembl: ENSMUSG00000028549 ) is located on Mouse 4. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000146258). Exon 3~4 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 9.28% of the coding region. Exon 3~4 covers 38.45% of the coding region. The size of effective KO region: ~3537 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 9

Legends Exon of mouse Itgb3bp 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 4 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(27.25% 545) | C(18.95% 379) | T(32.85% 657) | G(20.95% 419)

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(26.65% 533) | C(21.3% 426) | T(33.55% 671) | G(18.5% 370)

Note: The 2000 bp section downstream of Exon 4 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 - 99802233 99804232 2000 browser details YourSeq 91 522 1180 2000 88.7% chr17 + 47100469 47460025 359557 browser details YourSeq 61 491 634 2000 81.6% chr2 - 172391457 172391593 137 browser details YourSeq 60 498 655 2000 79.5% chr9 + 65166103 65166238 136 browser details YourSeq 58 506 631 2000 94.1% chr8 - 4191752 4191878 127 browser details YourSeq 57 526 631 2000 88.0% chr7 - 131259210 131259316 107 browser details YourSeq 54 451 539 2000 75.4% chr1 + 120031083 120031159 77 browser details YourSeq 53 491 631 2000 90.5% chr14 - 55942741 55942883 143 browser details YourSeq 49 491 631 2000 93.0% chr11 + 23184283 23184423 141 browser details YourSeq 47 499 665 2000 92.8% chr8 + 127609349 127609522 174 browser details YourSeq 46 494 648 2000 81.7% chr10 - 66906847 66907001 155 browser details YourSeq 46 491 631 2000 96.2% chrX + 151259601 151259741 141 browser details YourSeq 45 595 655 2000 87.3% chr11 - 112803013 112803072 60 browser details YourSeq 45 490 626 2000 91.0% chr7 + 19121464 19121601 138 browser details YourSeq 45 491 649 2000 86.6% chr12 + 102194106 102194262 157 browser details YourSeq 44 609 673 2000 88.0% chr4 - 55180724 55180786 63 browser details YourSeq 44 501 629 2000 96.0% chr14 - 67617875 67618004 130 browser details YourSeq 43 980 1028 2000 96.0% chr5 + 27217823 27217876 54 browser details YourSeq 42 527 626 2000 68.7% chr11 - 104023178 104023276 99 browser details YourSeq 42 590 657 2000 79.6% chr10 + 3175198 3175259 62

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 - 99796696 99798695 2000 browser details YourSeq 344 1450 1938 2000 90.8% chr18 - 4459648 4460280 633 browser details YourSeq 320 1411 2000 2000 90.7% chr10 + 38571409 38747602 176194 browser details YourSeq 298 1450 2000 2000 86.7% chr12 + 38999668 39000253 586 browser details YourSeq 297 1446 2000 2000 86.1% chr10 + 97817965 97818509 545 browser details YourSeq 293 1451 2000 2000 88.9% chr1 + 117434975 117435534 560 browser details YourSeq 285 1450 1938 2000 89.2% chr4 + 102936009 102936545 537 browser details YourSeq 280 1485 2000 2000 86.9% chr10 + 47238279 47238789 511 browser details YourSeq 278 1453 1995 2000 86.4% chr8 - 78937324 78937852 529 browser details YourSeq 273 1451 2000 2000 89.7% chr15 + 50546920 50547492 573 browser details YourSeq 256 1451 1984 2000 84.4% chr16 - 20320100 20320599 500 browser details YourSeq 256 1450 1933 2000 87.1% chr2 + 137927150 137927605 456 browser details YourSeq 245 1450 1986 2000 85.5% chr19 + 33984906 33985417 512 browser details YourSeq 231 1446 2000 2000 87.0% chr1 - 159072319 159072884 566 browser details YourSeq 230 1451 2000 2000 90.5% chr1 + 21742869 21743519 651 browser details YourSeq 226 1451 2000 2000 90.7% chr14 - 11228697 11229303 607 browser details YourSeq 225 1698 2000 2000 88.9% chr18 + 15429617 15429981 365 browser details YourSeq 221 1383 2000 2000 86.2% chr8 - 93157042 93157945 904 browser details YourSeq 219 1451 1886 2000 91.3% chr13 + 25709834 25710277 444 browser details YourSeq 218 1522 2000 2000 85.6% chr1 - 67016228 67016692 465

Note: The 2000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found.

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Gene and information: Itgb3bp binding protein (beta3-endonexin) [ Mus musculus (house mouse) ] Gene ID: 67733, updated on 12-Aug-2019

Gene summary

Official Symbol Itgb3bp provided by MGI Official Full Name integrin beta 3 binding protein (beta3-endonexin) provided by MGI Primary source MGI:MGI:1914983 See related Ensembl:ENSMUSG00000028549 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 CENP-R; AU022583; 4930471O16Rik Expression Broad expression in testis adult (RPKM 2.2), CNS E11.5 (RPKM 1.9) and 22 other tissues See more Orthologs human all

Genomic context

Location: 4; 4 C6 See Itgb3bp in Genome Data Viewer Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 4 NC_000070.6 (99767406..99829215, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 4 NC_000070.5 (99432093..99495809, complement)

Chromosome 4 - NC_000070.6

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Transcript information: This gene has 5 transcripts

Gene: Itgb3bp ENSMUSG00000028549

Description integrin beta 3 binding protein (beta3-endonexin) [Source:MGI Symbol;Acc:MGI:1914983] Gene Synonyms 4930471O16Rik Location Chromosome 4: 99,765,402-99,929,813 reverse strand. GRCm38:CM000997.2 View alleles of this gene on alternative sequences About this gene This gene has 5 transcripts (splice variants), 1 gene allele, 123 orthologues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Itgb3bp-204 ENSMUST00000146258.1 2806 176aa ENSMUSP00000117153.1 Protein coding CCDS18387 Q9CQ82 TSL:1 GENCODE basic APPRIS P1

Itgb3bp-203 ENSMUST00000123830.7 3387 No protein - lncRNA - - TSL:2

Itgb3bp-202 ENSMUST00000123045.7 720 No protein - lncRNA - - TSL:3

Itgb3bp-201 ENSMUST00000102786.10 716 No protein - lncRNA - - TSL:5

Itgb3bp-205 ENSMUST00000146739.1 346 No protein - lncRNA - - TSL:5

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184.41 kb Forward strand 99.80Mb 99.85Mb 99.90Mb Alg6-201 >protein coding Efcab7-205 >protein coding Efcab7-204 >protein coding Pgm1-201 >protein coding (Comprehensive set...

Alg6-207 >retained intron Efcab7-203 >protein coding Gm26425-201 >snRNA

Efcab7-202 >protein coding

Efcab7-201 >protein coding

Contigs < BX005053.5 BX324127.8 > CR536609.3 > Genes < Itgb3bp-204protein coding (Comprehensive set...

< Itgb3bp-203lncRNA

< Itgb3bp-201lncRNA

< Itgb3bp-202lncRNA

< Itgb3bp-205lncRNA

Regulatory Build

99.80Mb 99.85Mb 99.90Mb Reverse strand 184.41 kb

Regulation Legend

CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site

Gene Legend Protein Coding

Ensembl protein coding merged Ensembl/Havana

Non-Protein Coding

processed transcript RNA gene

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Transcript: ENSMUST00000146258

< Itgb3bp-204protein coding

Reverse strand 63.78 kb

ENSMUSP00000117... MobiDB lite Pfam Centromere protein R PIRSF Centromere protein R PANTHER Centromere protein R

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

Variant Legend missense variant synonymous variant

Scale bar 0 20 40 60 80 100 120 140 176

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

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