https://www.alphaknockout.com

Mouse Nktr Knockout Project (CRISPR/Cas9)

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

Strategy summary: The Nktr gene (NCBI Reference Sequence: NM_010918 ; Ensembl: ENSMUSG00000032525 ) is located on Mouse chromosome 9. 17 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 17 (Transcript: ENSMUST00000035112). Exon 9~15 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 9 starts from about 12.64% of the coding region. Exon 9~15 covers 83.09% of the coding region. The size of effective KO region: ~9899 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 9 10 11 12 13 14 15 17

Legends Exon of mouse Nktr Knockout region

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

Forward Reverse Complement

Sequence 12

Note: The 821 bp section upstream of Exon 9 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 527 bp section downstream of Exon 15 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(821bp) | A(20.83% 171) | C(24.48% 201) | T(34.59% 284) | G(20.1% 165)

Note: The 821 bp section upstream of Exon 9 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(527bp) | A(28.65% 151) | C(19.92% 105) | T(32.64% 172) | G(18.79% 99)

Note: The 527 bp section downstream of Exon 15 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 821 1 821 821 100.0% chr9 + 121741745 121742565 821 browser details YourSeq 26 785 810 821 100.0% chr3 + 7867266 7867291 26 browser details YourSeq 25 344 372 821 85.8% chr1 - 108485435 108485462 28 browser details YourSeq 25 344 372 821 85.8% chr1 - 100061170 100061197 28 browser details YourSeq 24 479 505 821 96.2% chr10 + 118309561 118309587 27 browser details YourSeq 21 206 226 821 100.0% chr5 - 129362461 129362481 21 browser details YourSeq 21 549 569 821 100.0% chr10 - 24759377 24759397 21 browser details YourSeq 21 351 377 821 88.9% chr11 + 36303117 36303143 27 browser details YourSeq 21 351 373 821 95.7% chr1 + 186648779 186648801 23

Note: The 821 bp section upstream of Exon 9 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 527 1 527 527 100.0% chr9 + 121752465 121752991 527 browser details YourSeq 26 47 76 527 93.4% chr19 - 4093665 4093694 30 browser details YourSeq 25 175 207 527 96.3% chr4 + 114851320 114851353 34 browser details YourSeq 25 49 73 527 100.0% chr11 + 70876626 70876650 25 browser details YourSeq 24 49 76 527 92.9% chr9 - 61007210 61007237 28 browser details YourSeq 24 49 76 527 92.9% chrX + 53001169 53001196 28 browser details YourSeq 24 49 74 527 96.2% chr9 + 108020516 108020541 26 browser details YourSeq 24 49 76 527 92.9% chr5 + 122363909 122363936 28 browser details YourSeq 24 52 75 527 100.0% chr2 + 61724739 61724762 24 browser details YourSeq 24 49 76 527 92.9% chr16 + 4589365 4589392 28 browser details YourSeq 24 49 76 527 92.9% chr1 + 42043750 42043777 28 browser details YourSeq 23 280 302 527 100.0% chr3 + 61453374 61453396 23 browser details YourSeq 22 51 72 527 100.0% chr3 + 41812885 41812906 22 browser details YourSeq 21 170 190 527 100.0% chr7 - 109237641 109237661 21 browser details YourSeq 21 49 69 527 100.0% chr14 - 80490871 80490891 21 browser details YourSeq 21 52 72 527 100.0% chr12 - 78886796 78886816 21 browser details YourSeq 21 239 261 527 95.7% chr14 + 53149254 53149276 23 browser details YourSeq 21 307 327 527 100.0% chr13 + 106189873 106189893 21 browser details YourSeq 20 163 184 527 95.5% chr12 - 89575595 89575616 22 browser details YourSeq 20 165 184 527 100.0% chr13 + 90955439 90955458 20

Note: The 527 bp section downstream of Exon 15 is BLAT searched against the genome. No significant similarity is found.

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Gene and protein information: Nktr natural killer tumor recognition sequence [ Mus musculus (house mouse) ] Gene ID: 18087, updated on 10-Oct-2019

Gene summary

Official Symbol Nktr provided by MGI Official Full Name natural killer tumor recognition sequence provided by MGI Primary source MGI:MGI:97346 See related Ensembl:ENSMUSG00000032525 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 D9Wsu172e; 5330401F18Rik Expression Ubiquitous expression in limb E14.5 (RPKM 21.4), CNS E14 (RPKM 18.6) and 28 other tissues See more Orthologs human all

Genomic context

Location: 9 F4; 9 72.57 cM See Nktr in Genome Data Viewer Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (121713881..121756842)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (121628299..121665959)

Chromosome 9 - NC_000075.6

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Transcript information: This gene has 18 transcripts

Gene: Nktr ENSMUSG00000032525

Description natural killer tumor recognition sequence [Source:MGI Symbol;Acc:MGI:97346] Gene Synonyms 5330401F18Rik, D9Wsu172e Location Chromosome 9: 121,719,169-121,756,843 forward strand. GRCm38:CM001002.2 About this gene This gene has 18 transcripts (splice variants), 202 orthologues, 17 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Nktr- ENSMUST00000035112.12 7022 1453aa ENSMUSP00000035112.5 Protein coding CCDS23636 P30415 TSL:1 201 GENCODE basic APPRIS P1

Nktr- ENSMUST00000182225.7 2942 966aa ENSMUSP00000138168.1 Protein coding - Q8CBP6 CDS 3' 205 incomplete TSL:1

Nktr- ENSMUST00000182713.1 811 95aa ENSMUSP00000138587.1 Protein coding - S4R2C2 CDS 5' 211 incomplete TSL:3

Nktr- ENSMUST00000182337.1 452 101aa ENSMUSP00000138164.1 Protein coding - S4R1C3 CDS 3' 207 incomplete TSL:3

Nktr- ENSMUST00000182311.7 407 99aa ENSMUSP00000138690.1 Protein coding - S4R2L3 CDS 3' 206 incomplete TSL:5

Nktr- ENSMUST00000183070.7 2576 105aa ENSMUSP00000138611.1 Nonsense mediated - Q3TQ51 TSL:1 214 decay

Nktr- ENSMUST00000182179.7 1460 105aa ENSMUSP00000138437.1 Nonsense mediated - Q3TQ51 TSL:1 204 decay

Nktr- ENSMUST00000182763.7 717 69aa ENSMUSP00000138272.1 Nonsense mediated - S4R1L7 TSL:5 212 decay

Nktr- ENSMUST00000182503.7 539 100aa ENSMUSP00000138463.1 Nonsense mediated - S4R220 CDS 5' 209 decay incomplete TSL:3

Nktr- ENSMUST00000182904.8 8950 No - Retained intron - - TSL:1 213 protein

Nktr- ENSMUST00000183311.7 2548 No - Retained intron - - TSL:2 216 protein

Nktr- ENSMUST00000216573.1 1065 No - Retained intron - - TSL:2 218 protein

Nktr- ENSMUST00000182607.1 789 No - Retained intron - - TSL:3 210 protein

Nktr- ENSMUST00000213351.1 741 No - Retained intron - - TSL:2 217 protein

Nktr- ENSMUST00000182086.1 703 No - Retained intron - - TSL:3 203 protein

Nktr- ENSMUST00000182076.1 678 No - Retained intron - - TSL:2 202 protein

Nktr- ENSMUST00000182395.1 417 No - Retained intron - - TSL:3 208 protein

Nktr- ENSMUST00000183207.1 268 No - lncRNA - - TSL:5 215 protein

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57.67 kb Forward strand 121.71Mb 121.72Mb 121.73Mb 121.74Mb 121.75Mb 121.76Mb Genes (Comprehensive set... Ss18l2-201 >protein coding Nktr-214 >nonsense mediated decay Nktr-217 >retained intron Zfp651-204 >retained intron

Ss18l2-206 >protein coding Nktr-201 >protein coding Zfp651-201 >protein coding

Ss18l2-203 >retained intronNktr-212 >nonsense mediated decay Nktr-209 >nonsense mediated decay

Ss18l2-205 >protein coding Nktr-213 >retained intron Nktr-218 >retained intron Nktr-210 >retained intron

Ss18l2-202 >retained intronNktr-206 >protein coding Nktr-211 >protein coding

Ss18l2-204 >protein coding Nktr-216 >retained intron Nktr-203 >retained intron Nktr-202 >retained intron

Ss18l2-207 >retained intronNktr-215 >lncRNA Nktr-208 >retained intron Zfp651-203 >nonsense mediated decay

Nktr-204 >nonsense mediated decay

Nktr-205 >protein coding

Nktr-207 >protein coding

Contigs < AC159810.2 < AC165080.4 Genes < E530011L22Rik-201lncRNA (Comprehensive set...

< E530011L22Rik-202lncRNA

Regulatory Build

121.71Mb 121.72Mb 121.73Mb 121.74Mb 121.75Mb 121.76Mb Reverse strand 57.67 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

processed transcript RNA gene

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Transcript: ENSMUST00000035112

37.67 kb Forward strand

Nktr-201 >protein coding

ENSMUSP00000035... MobiDB lite Low complexity (Seg) Superfamily Cyclophilin-like domain superfamily Prints Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain Pfam Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain

PROSITE profiles Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain PROSITE patterns Cyclophilin-type peptidyl-prolyl cis-trans isomerase, conserved site PANTHER PTHR11071:SF257

PTHR11071 Gene3D Cyclophilin-like domain superfamily

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

Variant Legend

inframe deletion missense variant splice region variant synonymous variant

Scale bar 0 200 400 600 800 1000 1200 1453

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

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