https://www.alphaknockout.com
Mouse Pram1 Knockout Project (CRISPR/Cas9)
Objective: To create a Pram1 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Pram1 gene (NCBI Reference Sequence: NM_001002842 ; Ensembl: ENSMUSG00000032739 ) is located on Mouse chromosome 17. 10 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 10 (Transcript: ENSMUST00000052079). Exon 2~4 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: Homozygous null mice have neutrophils that exhibit decreased adhesion-dependent reactive oxygen intermediate production and degranulation.
Exon 2 starts from about 0.2% of the coding region. Exon 2~4 covers 78.86% of the coding region. The size of effective KO region: ~1743 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 3 4 10
Legends Exon of mouse Pram1 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. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats.
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. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis.
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Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(32.75% 655) | C(19.15% 383) | T(21.65% 433) | G(26.45% 529)
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(25.85% 517) | C(23.15% 463) | T(26.85% 537) | G(24.15% 483)
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% chr17 + 33638465 33640464 2000 browser details YourSeq 67 819 1060 2000 74.7% chr3 + 37976270 37976389 120 browser details YourSeq 65 769 888 2000 85.4% chr1 - 121271824 121271929 106 browser details YourSeq 63 673 808 2000 91.9% chr17 - 66646973 66647175 203 browser details YourSeq 63 666 819 2000 94.6% chr5 + 128891993 128892486 494 browser details YourSeq 63 824 1064 2000 75.7% chr19 + 48065253 48065411 159 browser details YourSeq 62 925 1014 2000 86.3% chr1 + 75350090 75350193 104 browser details YourSeq 61 912 1064 2000 95.6% chr3 - 150630864 150631069 206 browser details YourSeq 60 769 890 2000 97.0% chr2 - 19517176 19517309 134 browser details YourSeq 58 826 928 2000 92.9% chr4 - 149975916 149976388 473 browser details YourSeq 57 719 820 2000 79.2% chr1 + 181697597 181697680 84 browser details YourSeq 55 766 825 2000 98.4% chr14 + 88782364 88782454 91 browser details YourSeq 54 912 995 2000 81.7% chr7 - 101319624 101319694 71 browser details YourSeq 54 760 832 2000 93.9% chr19 + 19007597 19007676 80 browser details YourSeq 53 767 824 2000 98.3% chr10 + 74082194 74082292 99 browser details YourSeq 52 912 995 2000 82.8% chr9 - 110126946 110127018 73 browser details YourSeq 52 911 995 2000 78.4% chr13 - 13320552 13320621 70 browser details YourSeq 51 824 890 2000 96.5% chr7 + 26578498 26578810 313 browser details YourSeq 51 767 956 2000 96.5% chr1 + 37867018 37867487 470 browser details YourSeq 50 912 995 2000 80.4% chr6 - 11676969 11677039 71
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% chr17 + 33642208 33644207 2000 browser details YourSeq 69 952 1049 2000 86.4% chr10 + 32945876 32945975 100 browser details YourSeq 68 958 1049 2000 88.8% chr9 + 10560049 10560142 94 browser details YourSeq 67 959 1049 2000 88.7% chr2 + 23325255 23325347 93 browser details YourSeq 63 959 1049 2000 86.4% chr11 + 25963395 25963487 93 browser details YourSeq 61 887 1046 2000 70.7% chr1 - 67899769 67899880 112 browser details YourSeq 61 956 1047 2000 89.9% chr15 + 89448345 89448436 92 browser details YourSeq 61 948 1049 2000 85.3% chr1 + 60746776 60746889 114 browser details YourSeq 60 956 1049 2000 87.7% chr19 - 10178091 10178186 96 browser details YourSeq 60 948 1049 2000 85.3% chr14 + 119050845 119050954 110 browser details YourSeq 59 956 1049 2000 87.7% chr2 - 29719357 29719451 95 browser details YourSeq 59 945 1025 2000 95.6% chr3 + 52456779 52456864 86 browser details YourSeq 58 956 1049 2000 86.5% chr1 + 49054664 49054759 96 browser details YourSeq 57 956 1045 2000 88.4% chrX - 93558292 93558381 90 browser details YourSeq 57 952 1049 2000 80.9% chr19 - 42495919 42496019 101 browser details YourSeq 57 956 1049 2000 88.2% chr10 - 113377136 113377231 96 browser details YourSeq 56 958 1050 2000 88.0% chr9 - 19457134 19457228 95 browser details YourSeq 56 958 1049 2000 88.2% chr10 - 62768836 62768929 94 browser details YourSeq 56 952 1048 2000 84.4% chr13 + 109779644 109779741 98 browser details YourSeq 55 956 1049 2000 86.7% chrX - 97799600 97799695 96
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 protein information: Pram1 PML-RAR alpha-regulated adaptor molecule 1 [ Mus musculus (house mouse) ] Gene ID: 378460, updated on 12-Aug-2019
Gene summary
Official Symbol Pram1 provided by MGI Official Full Name PML-RAR alpha-regulated adaptor molecule 1 provided by MGI Primary source MGI:MGI:3576625 See related Ensembl:ENSMUSG00000032739 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 AY665714 Expression Ubiquitous expression in liver E18 (RPKM 5.7), spleen adult (RPKM 2.1) and 24 other tissues See more Orthologs human all
Genomic context
Location: 17; 17 B1 See Pram1 in Genome Data Viewer Exon count: 10
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (33638056..33645706)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (33775001..33782651)
Chromosome 17 - NC_000083.6
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Transcript information: This gene has 3 transcripts
Gene: Pram1 ENSMUSG00000032739
Description PML-RAR alpha-regulated adaptor molecule 1 [Source:MGI Symbol;Acc:MGI:3576625] Location Chromosome 17: 33,638,056-33,645,706 forward strand. GRCm38:CM001010.2 About this gene This gene has 3 transcripts (splice variants), 141 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Pram1- ENSMUST00000052079.7 2278 675aa ENSMUSP00000057065.7 Protein coding CCDS37569 Q6BCL1 TSL:1 201 GENCODE basic APPRIS P1
Pram1- ENSMUST00000116619.8 2268 44aa ENSMUSP00000112318.2 Nonsense mediated - E9PYJ5 TSL:1 202 decay
Pram1- ENSMUST00000166215.1 582 No - Retained intron - - TSL:2 203 protein
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27.65 kb Forward strand 33.63Mb 33.64Mb 33.65Mb Genes (Comprehensive set... Zfp414-201 >protein coding Pram1-201 >protein coding
Zfp414-203 >protein coding Pram1-202 >nonsense mediated decay
Zfp414-206 >retained intron Pram1-203 >retained intron
Zfp414-205 >protein coding
Zfp414-207 >lncRNA
Zfp414-202 >protein coding
Zfp414-204 >retained intron
Gm20507-201 >lncRNA
Contigs CT033847.5 > Genes < Hnrnpm-206protein coding (Comprehensive set...
< Hnrnpm-202protein coding
< Hnrnpm-205protein coding
< Hnrnpm-213retained intro < Hnrnpm-201protein coding < Hnrnpm-204protein coding < Hnrnpm-210lncRNA < Hnrnpm-214retained intron < Hnrnpm-211protein coding Regulatory Build 33.63Mb 33.64Mb 33.65Mb Reverse strand 27.65 kb Regulation Legend Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000052079 7.65 kb Forward strand Pram1-201 >protein coding ENSMUSP00000057... MobiDB lite Low complexity (Seg) Superfamily SH3-like domain superfamily Pfam Helically-extended SH3 domain PROSITE profiles SH3 domain PANTHER PTHR16830 PTHR16830:SF11 Gene3D 2.30.30.40 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend frameshift variant inframe deletion missense variant synonymous variant Scale bar 0 60 120 180 240 300 360 420 480 540 600 675 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9