https://www.alphaknockout.com
Mouse Esam Knockout Project (CRISPR/Cas9)
Objective: To create a Esam knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Esam gene (NCBI Reference Sequence: NM_027102 ; Ensembl: ENSMUSG00000001946 ) is located on Mouse chromosome 9. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000002011). Exon 2 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: Mice homozygous for a null allele exhbit a decrease in body weight, impaired neutrophil transmigration and decreased immune and VEGF-stimulated vascular permeability. Tumor growth is inhibited due to decreased pathological angiogenesis in homozygous mutant mice.
Exon 2 starts from about 6.01% of the coding region. Exon 2 covers 15.91% of the coding region. The size of effective KO region: ~188 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 2 7
Legends Exon of mouse Esam 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 2 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 2 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(20.95% 419) | C(27.35% 547) | T(23.5% 470) | G(28.2% 564)
Note: The 2000 bp section upstream of Exon 2 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(22.8% 456) | C(21.05% 421) | T(33.2% 664) | G(22.95% 459)
Note: The 2000 bp section downstream of Exon 2 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% chr9 + 37529489 37531488 2000 browser details YourSeq 23 590 612 2000 100.0% chr7 + 136446450 136446472 23 browser details YourSeq 21 769 791 2000 95.7% chr1 + 90734300 90734322 23 browser details YourSeq 20 1476 1495 2000 100.0% chr1 - 93688617 93688636 20
Note: The 2000 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 2000 1 2000 2000 100.0% chr9 + 37531677 37533676 2000 browser details YourSeq 168 1604 1906 2000 87.0% chr4 - 69925196 69925496 301 browser details YourSeq 163 1604 1901 2000 88.9% chr4 + 134473537 134473832 296 browser details YourSeq 153 1604 1901 2000 87.1% chr17 - 70392604 70392887 284 browser details YourSeq 107 553 1087 2000 77.6% chr5 + 122929005 122929413 409 browser details YourSeq 90 1604 1738 2000 93.4% chr9 - 115281252 115281388 137 browser details YourSeq 81 1787 1910 2000 86.8% chr6 - 27433340 27433474 135 browser details YourSeq 77 988 1087 2000 85.3% chr15 + 85956829 85956920 92 browser details YourSeq 76 1474 1578 2000 88.3% chr17 + 70392910 70393013 104 browser details YourSeq 73 1483 1578 2000 83.9% chr4 - 134473413 134473505 93 browser details YourSeq 73 1483 1578 2000 83.9% chr4 + 69925528 69925620 93 browser details YourSeq 70 988 1087 2000 83.0% chr18 - 30917462 30917552 91 browser details YourSeq 63 28 323 2000 92.0% chr8 + 33790839 33791277 439 browser details YourSeq 63 1479 1551 2000 90.3% chr6 + 79930188 79930259 72 browser details YourSeq 60 1605 1691 2000 90.7% chr6 - 79930074 79930161 88 browser details YourSeq 55 993 1087 2000 74.7% chr1 - 105751187 105751262 76 browser details YourSeq 54 257 324 2000 86.6% chr9 + 65847412 65847478 67 browser details YourSeq 52 262 324 2000 92.8% chr1 - 137047992 137048053 62 browser details YourSeq 51 262 322 2000 88.4% chrX - 69256030 69256089 60 browser details YourSeq 49 259 324 2000 87.8% chr15 + 57475049 57475112 64
Note: The 2000 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: Esam endothelial cell-specific adhesion molecule [ Mus musculus (house mouse) ] Gene ID: 69524, updated on 10-Oct-2019
Gene summary
Official Symbol Esam provided by MGI Official Full Name endothelial cell-specific adhesion molecule provided by MGI Primary source MGI:MGI:1916774 See related Ensembl:ENSMUSG00000001946 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 Esam1; W117m; 2310008D05Rik Expression Broad expression in lung adult (RPKM 97.8), subcutaneous fat pad adult (RPKM 50.9) and 22 other tissuesS ee more Orthologs human all
Genomic context
Location: 9; 9 A4 See Esam in Genome Data Viewer Exon count: 7
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (37528089..37538319)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (37335674..37345904)
Chromosome 9 - NC_000075.6
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Transcript information: This gene has 6 transcripts
Gene: Esam ENSMUSG00000001946
Description endothelial cell-specific adhesion molecule [Source:MGI Symbol;Acc:MGI:1916774] Gene Synonyms 2310008D05Rik, W117m Location Chromosome 9: 37,528,078-37,538,319 forward strand. GRCm38:CM001002.2 About this gene This gene has 6 transcripts (splice variants), 261 orthologues, 17 paralogues, is a member of 1 Ensembl protein family and is associated with 6 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Esam- ENSMUST00000002011.13 1865 394aa ENSMUSP00000002011.7 Protein coding CCDS22979 Q3U102 TSL:1 201 Q925F2 GENCODE basic APPRIS P1
Esam- ENSMUST00000146860.7 859 252aa ENSMUSP00000122473.1 Protein coding - D3Z5Y0 CDS 3' 205 incomplete TSL:5
Esam- ENSMUST00000123198.1 545 71aa ENSMUSP00000116300.1 Protein coding - D3Z5S5 CDS 3' 202 incomplete TSL:3
Esam- ENSMUST00000144596.1 415 44aa ENSMUSP00000114632.1 Protein coding - D3YV70 CDS 3' 204 incomplete TSL:3
Esam- ENSMUST00000214142.1 216 28aa ENSMUSP00000149283.1 Nonsense mediated - A0A1L1SR18 CDS 5' 206 decay incomplete TSL:5
Esam- ENSMUST00000131832.1 627 No - Retained intron - - TSL:2 203 protein
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30.24 kb Forward strand 37.52Mb 37.53Mb 37.54Mb Genes (Comprehensive set... Msantd2-204 >protein coding Esam-201 >protein coding Vsig2-201 >protein coding
Msantd2-201 >protein coding Esam-205 >protein coding Vsig2-206 >protein coding
Msantd2-208 >protein coding Esam-204 >protein codingEsam-206 >nonsense mediated decay Vsig2-202 >retained intron
Msantd2-206 >lncRNA Esam-202 >protein coding Esam-203 >retained intron Vsig2-203 >protein coding
Msantd2-207 >retained intron Vsig2-204 >protein coding
Vsig2-205 >retained intron
Contigs AC105958.11 > Genes < Nrgn-201protein coding (Comprehensive set...
< Nrgn-202lncRNA
Regulatory Build
37.52Mb 37.53Mb 37.54Mb Reverse strand 30.24 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: ENSMUST00000002011
10.24 kb Forward strand
Esam-201 >protein coding
ENSMUSP00000002... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily Immunoglobulin-like domain superfamily SMART Immunoglobulin subtype
Immunoglobulin subtype 2 Pfam Immunoglobulin V-set domain PF13927
PROSITE profiles Immunoglobulin-like domain PANTHER PTHR44549 Gene3D Immunoglobulin-like fold CDD cd00096
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 40 80 120 160 200 240 280 320 394
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
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