https://www.alphaknockout.com

Mouse Got2 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Got2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Got2 (NCBI Reference Sequence: NM_010325 ; Ensembl: ENSMUSG00000031672 ) is located on Mouse 8. 10 are identified, with the ATG start codon in 1 and the TAA stop codon in exon 10 (Transcript: ENSMUST00000034097). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Got2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-264J18 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note:

Exon 2 starts from about 6.98% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 10347 bp, and the size of intron 3 for 3'-loxP site insertion: 1282 bp. The size of effective cKO region: ~2624 bp. The cKO region does not have any other known gene.

Page 1 of 7 https://www.alphaknockout.com

Overview of the Targeting Strategy

Wildtype allele 5' gRNA region gRNA region 3'

1 2 3 4 10 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Got2 Homology arm cKO region loxP site

Page 2 of 7 https://www.alphaknockout.com

Overview of the Dot Plot Window size: 10 bp

Forward Reverse Complement

Sequence 12

Note: The sequence of homologous arms and cKO region 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 GC Content Distribution Window size: 300 bp

Sequence 12

Summary: Full Length(9124bp) | A(28.89% 2636) | C(22.27% 2032) | T(27.22% 2484) | G(21.61% 1972)

Note: The sequence of homologous arms and cKO region 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 3 of 7 https://www.alphaknockout.com

BLAT Search Results (up)

QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ------browser details YourSeq 3000 1 3000 3000 100.0% chr8 - 95878120 95881119 3000 browser details YourSeq 495 459 2027 3000 92.1% chr14 - 48269783 48765015 495233 browser details YourSeq 377 1295 2033 3000 90.6% chr2 + 124734658 125237839 503182 browser details YourSeq 342 1295 2081 3000 90.8% chr1 - 170851089 171266290 415202 browser details YourSeq 335 460 1872 3000 95.0% chr4 - 140669455 141216982 547528 browser details YourSeq 268 485 2030 3000 88.8% chr4 - 34416023 34510220 94198 browser details YourSeq 266 486 1453 3000 96.0% chr11 + 61844241 61939327 95087 browser details YourSeq 231 1417 2025 3000 81.8% chr2 + 118683752 118684205 454 browser details YourSeq 229 1494 2082 3000 81.5% chr3 - 127654144 127654602 459 browser details YourSeq 228 1415 1790 3000 90.7% chr11 - 63306491 63307265 775 browser details YourSeq 211 479 763 3000 95.8% chr14 - 99291682 99292138 457 browser details YourSeq 202 1509 1797 3000 93.6% chr11 + 4841850 4842148 299 browser details YourSeq 187 1471 1727 3000 91.2% chr2 + 26263196 26263475 280 browser details YourSeq 174 1333 2123 3000 82.4% chr15 - 89482146 89482550 405 browser details YourSeq 171 1376 2031 3000 82.0% chr14 - 51017054 51017369 316 browser details YourSeq 161 1304 1951 3000 78.2% chr7 + 29011004 29011290 287 browser details YourSeq 160 1476 1713 3000 88.9% chr7 + 15891674 15892320 647 browser details YourSeq 156 1298 1798 3000 90.6% chr15 + 81548149 81548797 649 browser details YourSeq 156 1283 1623 3000 87.1% chr10 + 80633063 80633481 419 browser details YourSeq 155 1304 1946 3000 78.2% chr1 - 158433577 158433862 286

Note: The 3000 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 3000 1 3000 3000 100.0% chr8 - 95872496 95875495 3000 browser details YourSeq 506 2097 2884 3000 87.0% chr3 - 98485192 98488332 3141 browser details YourSeq 480 1734 2840 3000 88.8% chr13 - 108032865 108034173 1309 browser details YourSeq 447 2109 2893 3000 86.6% chr1 + 141345781 141346652 872 browser details YourSeq 439 1882 2715 3000 85.7% chr12 + 66341799 66342594 796 browser details YourSeq 438 2099 2716 3000 88.9% chr18 + 79179231 79179845 615 browser details YourSeq 436 1621 2714 3000 87.1% chr7 + 4880896 4882001 1106 browser details YourSeq 432 1642 2714 3000 85.5% chr9 - 16521662 16522587 926 browser details YourSeq 430 2097 2714 3000 86.0% chr8 - 25938643 25939245 603 browser details YourSeq 430 2097 2735 3000 87.2% chr4 + 101948022 101948655 634 browser details YourSeq 428 2144 2880 3000 89.1% chr17 - 66104008 66104851 844 browser details YourSeq 428 2097 2714 3000 86.6% chr16 - 10153068 10153695 628 browser details YourSeq 426 2093 2715 3000 87.6% chr14 + 67834670 67835262 593 browser details YourSeq 422 2099 2821 3000 85.4% chr7 - 105658744 105659373 630 browser details YourSeq 422 2097 2714 3000 86.8% chr6 - 29096742 29097363 622 browser details YourSeq 421 2097 2714 3000 87.5% chr3 - 98541167 98541793 627 browser details YourSeq 421 2097 2714 3000 87.5% chr3 - 98489844 98490470 627 browser details YourSeq 417 2109 2714 3000 85.9% chr3 - 129057075 129057690 616 browser details YourSeq 417 2097 2714 3000 88.0% chr13 - 54484005 54484630 626 browser details YourSeq 416 2097 2714 3000 87.3% chrX - 153327042 153327667 626

Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found.

Page 4 of 7 https://www.alphaknockout.com

Gene and protein information: Got2 glutamatic-oxaloacetic 2, mitochondrial [ Mus musculus (house mouse) ] Gene ID: 14719, updated on 12-Aug-2019

Gene summary

Official Symbol Got2 provided by MGI Official Full Name glutamatic-oxaloacetic transaminase 2, mitochondrial provided by MGI Primary source MGI:MGI:95792 See related Ensembl:ENSMUSG00000031672 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 Got-2; Kyat4; mAspAT; FABP-pm; AL022787 Expression Ubiquitous expression in heart adult (RPKM 277.4), liver adult (RPKM 151.9) and 26 other tissues See more Orthologs human all

Genomic context

Location: 8 D1; 8 47.79 cM See Got2 in Genome Data Viewer

Exon count: 10

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (95864133..95888401, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (98388037..98412265, complement)

Chromosome 8 - NC_000074.6

Page 5 of 7 https://www.alphaknockout.com

Transcript information: This gene has 4 transcripts

Gene: Got2 ENSMUSG00000031672

Description glutamatic-oxaloacetic transaminase 2, mitochondrial [Source:MGI Symbol;Acc:MGI:95792] Gene Synonyms FABP-pm, Kyat4, mAspAT, mitochondrial aspartate aminotransferase, plasma membrane fatty acid binding protein Location Chromosome 8: 95,864,134-95,888,547 reverse strand. GRCm38:CM001001.2 About this gene This gene has 4 transcripts (splice variants), 231 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Got2-201 ENSMUST00000034097.7 2530 430aa ENSMUSP00000034097.7 Protein coding CCDS22568 P05202 TSL:1 GENCODE basic APPRIS P1

Got2-204 ENSMUST00000212861.1 2057 No protein - Retained intron - - TSL:NA

Got2-202 ENSMUST00000147040.2 639 No protein - Retained intron - - TSL:2

Got2-203 ENSMUST00000211836.1 704 No protein - lncRNA - - TSL:3

44.41 kb Forward strand

95.86Mb 95.87Mb 95.88Mb 95.89Mb Gm45760-201 >TEC (Comprehensive set...

Contigs < AC127300.3 Genes (Comprehensive set... < Got2-201protein coding

< Got2-203lncRNA< Got2-202retained intron < Got2-204retained intron

Regulatory Build

95.86Mb 95.87Mb 95.88Mb 95.89Mb Reverse strand 44.41 kb

Regulation Legend CTCF Open Chromatin Promoter Promoter Flank

Gene Legend Protein Coding

merged Ensembl/Havana

Non-Protein Coding

RNA gene processed transcript

Page 6 of 7 https://www.alphaknockout.com

Transcript: ENSMUST00000034097

< Got2-201protein coding

Reverse strand 24.41 kb

ENSMUSP00000034... PDB-ENSP mappings Low complexity (Seg) Superfamily -dependent

Prints Aspartate/other aminotransferase Pfam Aminotransferase, class I/classII

PROSITE patterns Aminotransferases, class-I, pyridoxal-phosphate-binding site

PANTHER Aspartate/other aminotransferase

PTHR11879:SF39 Gene3D Pyridoxal phosphate-dependent transferase, major domain

Pyridoxal phosphate-dependent transferase domain 1 CDD cd00609

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 40 80 120 160 200 240 280 320 360 430

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

Page 7 of 7