https://www.alphaknockout.com
Mouse Cntnap4 Knockout Project (CRISPR/Cas9)
Objective: To create a Cntnap4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.
Strategy summary: The Cntnap4 gene (NCBI Reference Sequence: NM_130457 ; Ensembl: ENSMUSG00000031772 ) is located on Mouse chromosome 8. 24 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 24 (Transcript: ENSMUST00000034225). Exon 3 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 knock-out mice show increased midbrain dopaminergic release in the nucleus accumbens, synaptic defects, impaired sensory-motor gating, and increased grooming behavior.
Exon 3 starts from about 5.17% of the coding region. Exon 3 covers 4.94% of the coding region. The size of effective KO region: ~194 bp. The KO region does not have any other known gene.
Page 1 of 8 https://www.alphaknockout.com
Overview of the Targeting Strategy
Wildtype allele 5' gRNA region gRNA region 3'
1 3 24
Legends Exon of mouse Cntnap4 Knockout region
Page 2 of 8 https://www.alphaknockout.com
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. 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 3 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.
Page 3 of 8 https://www.alphaknockout.com
Overview of the GC Content Distribution (up) Window size: 300 bp
Sequence 12
Summary: Full Length(2000bp) | A(27.15% 543) | C(20.45% 409) | T(32.8% 656) | G(19.6% 392)
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(28.9% 578) | C(17.55% 351) | T(33.3% 666) | G(20.25% 405)
Note: The 2000 bp section downstream 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.
Page 4 of 8 https://www.alphaknockout.com
BLAT Search Results (up)
QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 2000 1 2000 2000 100.0% chr8 + 112663108 112665107 2000 browser details YourSeq 57 1407 1463 2000 100.0% chr8 + 112662820 112662876 57 browser details YourSeq 56 691 749 2000 98.4% chr10 - 113239033 113239096 64 browser details YourSeq 55 691 749 2000 100.0% chr16 + 8566845 8566910 66 browser details YourSeq 53 695 749 2000 98.2% chr2 - 34191498 34191552 55 browser details YourSeq 42 123 200 2000 90.4% chr16 - 7310716 7310794 79 browser details YourSeq 41 194 244 2000 90.2% chr4 - 128520855 128520905 51 browser details YourSeq 40 155 232 2000 93.5% chr9 + 64213108 64213229 122 browser details YourSeq 34 202 247 2000 86.9% chr6 - 134528664 134528707 44 browser details YourSeq 34 207 248 2000 87.5% chr2 - 59856291 59856331 41 browser details YourSeq 34 481 523 2000 90.3% chr14 + 117129152 117129197 46 browser details YourSeq 32 207 248 2000 88.1% chr2 - 132614460 132614501 42 browser details YourSeq 32 483 525 2000 88.9% chr16 - 89561232 89561273 42 browser details YourSeq 31 194 240 2000 83.0% chr5 + 141878180 141878226 47 browser details YourSeq 29 215 286 2000 91.5% chr8 - 81610775 81610847 73 browser details YourSeq 28 209 240 2000 93.8% chr6 + 101076498 101076529 32 browser details YourSeq 26 509 537 2000 85.2% chr2 + 4050136 4050162 27 browser details YourSeq 26 503 535 2000 93.4% chr11 + 88725591 88725639 49 browser details YourSeq 25 693 717 2000 100.0% chr10 + 56247498 56247522 25 browser details YourSeq 24 210 237 2000 92.9% chr4 - 130000156 130000183 28
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% chr8 + 112665302 112667301 2000 browser details YourSeq 59 424 513 2000 93.0% chr16 - 81219233 81219334 102 browser details YourSeq 51 408 466 2000 96.5% chr6 + 144568089 144568171 83 browser details YourSeq 49 405 514 2000 94.8% chr18 - 81476732 81476842 111 browser details YourSeq 43 612 662 2000 94.2% chr11 - 54848669 54848721 53 browser details YourSeq 42 627 737 2000 91.7% chr2 - 166492250 166492361 112 browser details YourSeq 38 611 648 2000 100.0% chr7 - 74965835 74965872 38 browser details YourSeq 36 1935 1970 2000 100.0% chr6 + 28064408 28064443 36 browser details YourSeq 33 430 465 2000 97.3% chr8 + 68795280 68795361 82 browser details YourSeq 32 632 667 2000 97.3% chr11 - 107450595 107450636 42 browser details YourSeq 31 612 644 2000 90.7% chr4 - 38967290 38967321 32 browser details YourSeq 31 1938 1968 2000 100.0% chr5 + 21143291 21143321 31 browser details YourSeq 27 1276 1304 2000 89.3% chr14 - 113631779 113631806 28 browser details YourSeq 27 595 633 2000 78.2% chr11 - 107379915 107379949 35 browser details YourSeq 26 612 637 2000 100.0% chr9 - 96372196 96372221 26 browser details YourSeq 26 1272 1304 2000 96.5% chr7 + 16953161 16953195 35 browser details YourSeq 24 1270 1303 2000 85.3% chr17 - 36019498 36019531 34 browser details YourSeq 23 1271 1293 2000 100.0% chr6 + 147700640 147700662 23 browser details YourSeq 22 1261 1282 2000 100.0% chr9 + 36286565 36286586 22 browser details YourSeq 22 1283 1304 2000 100.0% chr12 + 38066375 38066396 22
Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.
Page 5 of 8 https://www.alphaknockout.com
Gene and protein information: Cntnap4 contactin associated protein-like 4 [ Mus musculus (house mouse) ] Gene ID: 170571, updated on 12-Aug-2019
Gene summary
Official Symbol Cntnap4 provided by MGI Official Full Name contactin associated protein-like 4 provided by MGI Primary source MGI:MGI:2183572 See related Ensembl:ENSMUSG00000031772 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Caspr4; E130114F09Rik Expression Biased expression in cerebellum adult (RPKM 7.4), CNS E18 (RPKM 5.5) and 5 other tissues See more Orthologs human all
Genomic context
Location: 8; 8 E1 See Cntnap4 in Genome Data Viewer Exon count: 24
Annotation release Status Assembly Chr Location
108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (112569798..112882707)
Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (115093943..115406607)
Chromosome 8 - NC_000074.6
Page 6 of 8 https://www.alphaknockout.com
Transcript information: This gene has 6 transcripts
Gene: Cntnap4 ENSMUSG00000031772
Description contactin associated protein-like 4 [Source:MGI Symbol;Acc:MGI:2183572] Gene Synonyms Caspr4, E130114F09Rik Location Chromosome 8: 112,570,043-112,882,717 forward strand. GRCm38:CM001001.2 About this gene This gene has 6 transcripts (splice variants), 130 orthologues, 35 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts
Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags
Cntnap4-201 ENSMUST00000034225.6 4848 1310aa ENSMUSP00000034225.6 Protein coding CCDS40482 Q99P47 TSL:1 GENCODE basic APPRIS P2
Cntnap4-202 ENSMUST00000118171.7 4860 1310aa ENSMUSP00000112511.1 Protein coding - D3YWB9 TSL:5 GENCODE basic APPRIS ALT1
Cntnap4-204 ENSMUST00000125976.7 3862 No protein - Retained intron - - TSL:5
Cntnap4-203 ENSMUST00000125196.7 2651 No protein - Retained intron - - TSL:5
Cntnap4-206 ENSMUST00000140753.1 553 No protein - lncRNA - - TSL:3
Cntnap4-205 ENSMUST00000127636.1 278 No protein - lncRNA - - TSL:3
332.68 kb Forward strand 112.6Mb 112.7Mb 112.8Mb Genes (Comprehensive set... Cntnap4-201 >protein coding
Cntnap4-202 >protein coding
Cntnap4-204 >retained intron
Cntnap4-203 >retained intron
Gm22664-201 >misc RNA Cntnap4-205 >lncRNA Cntnap4-206 >lncRNA
Contigs < AC123843.4 < AC165305.4 AC132107.3 > Genes < Gm26994-201lncRNA < Gm45904-201lncRNA (Comprehensive set...
Regulatory Build
112.6Mb 112.7Mb 112.8Mb Reverse strand 332.68 kb
Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank
Gene Legend Protein Coding
Ensembl protein coding
Non-Protein Coding
processed transcript RNA gene
Page 7 of 8 https://www.alphaknockout.com
Transcript: ENSMUST00000034225
312.66 kb Forward strand
Cntnap4-201 >protein coding
ENSMUSP00000034... Transmembrane heli... Low complexity (Seg) Cleavage site (Sign... Superfamily Galactose-binding-like domain superfamily Fibrinogen-like, C-terminal
Concanavalin A-like lectin/glucanase domain superfamily
SSF57196 SMART Laminin G domain
Coagulation factor 5/8 C-terminal domain EGF-like domain Pfam Coagulation factor 5/8 C-terminal domain
Laminin G domain PROSITE profiles Coagulation factor 5/8 C-terminal domain Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain
Laminin G domain
EGF-like domain PROSITE patterns Coagulation factor 5/8 C-terminal domain
Coagulation factor 5/8 C-terminal domain PANTHER PTHR15036
Contactin-associated protein-like 4 Gene3D Galactose-binding-like domain superfamily 2.60.120.1000
2.60.120.200
2.10.25.10 CDD Coagulation factor 5/8 C-terminal domain cd00054
cd00110
All sequence SNPs/i... Sequence variants (dbSNP and all other sources)
Variant Legend missense variant splice region variant synonymous variant
Scale bar 0 200 400 600 800 1000 1310
We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC.
Page 8 of 8