https://www.alphaknockout.com

Mouse Stxbp1 Conditional Knockout Project (CRISPR/Cas9)

Objective: To create a Stxbp1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering.

Strategy summary: The Stxbp1 gene (NCBI Reference Sequence: NM_001113569 ; Ensembl: ENSMUSG00000026797 ) is located on Mouse chromosome 2. 20 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 19 (Transcript: ENSMUST00000077458). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Stxbp1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-252F13 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: Mice homozygous for a null allele exhibit total loss of neurotransmitter secretion from synaptic vesicles throughout development and massive neuron apoptosis after initial synaptogenesis, leading to widespread neurodegeneration and complete neonatal lethality.

Exon 3 starts from about 4.86% of the coding region. The knockout of Exon 3 will result in frameshift of the gene. The size of intron 2 for 5'-loxP site insertion: 1619 bp, and the size of intron 3 for 3'-loxP site insertion: 1924 bp. The size of effective cKO region: ~582 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 gRNA region 5' gRNA region 3'

1 2 3 4 20 Targeting vector

Targeted allele

Constitutive KO allele (After Cre recombination)

Legends Exon of mouse Stxbp1 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(7082bp) | A(23.91% 1693) | C(23.5% 1664) | T(27.37% 1938) | G(25.23% 1787)

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% chr2 - 32822167 32825166 3000 browser details YourSeq 129 2815 3000 3000 88.7% chr10 - 78089018 78089223 206 browser details YourSeq 128 2813 3000 3000 89.1% chr19 - 46297449 46297637 189 browser details YourSeq 123 2817 3000 3000 91.3% chr9 + 109110872 109111056 185 browser details YourSeq 119 2804 3000 3000 91.7% chr19 + 25227869 25228072 204 browser details YourSeq 118 2826 3000 3000 85.9% chr4 - 118299125 118299295 171 browser details YourSeq 118 2812 3000 3000 82.4% chr10 + 82721406 82721583 178 browser details YourSeq 117 2813 3000 3000 88.8% chr17 - 28465793 28465992 200 browser details YourSeq 117 2814 2976 3000 85.9% chr7 + 130185691 130185849 159 browser details YourSeq 117 2813 3000 3000 84.5% chr12 + 102327968 102328146 179 browser details YourSeq 117 2810 3000 3000 88.8% chr12 + 76036675 76036865 191 browser details YourSeq 116 2815 3000 3000 85.7% chr4 - 149978537 149978709 173 browser details YourSeq 114 2814 2988 3000 91.4% chr10 - 76869505 76869684 180 browser details YourSeq 113 2814 2996 3000 87.5% chrX + 94730510 94730700 191 browser details YourSeq 112 2812 3000 3000 87.9% chr3 + 66409480 66409668 189 browser details YourSeq 112 2812 3000 3000 85.5% chr2 + 170047217 170047400 184 browser details YourSeq 111 2844 2993 3000 93.1% chr18 - 39017379 39017529 151 browser details YourSeq 111 2832 3000 3000 94.5% chr12 - 17441083 17441479 397 browser details YourSeq 110 2848 3000 3000 90.5% chr9 - 108218656 108218809 154 browser details YourSeq 110 2817 2995 3000 92.4% chr7 + 16937142 16937339 198

Note: The 3000 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 3000 1 3000 3000 100.0% chr2 - 32818585 32821584 3000 browser details YourSeq 194 2142 2710 3000 91.1% chr3 - 36524301 36581690 57390 browser details YourSeq 177 2130 2738 3000 86.9% chr8 - 84221009 84221456 448 browser details YourSeq 176 2127 2335 3000 90.2% chr13 + 93681803 93682006 204 browser details YourSeq 173 2127 2337 3000 91.8% chr2 - 154388506 154388735 230 browser details YourSeq 172 2134 2330 3000 94.4% chr17 - 17383042 17383243 202 browser details YourSeq 172 2127 2322 3000 95.4% chr16 - 20669422 20669750 329 browser details YourSeq 169 2134 2331 3000 95.3% chr2 - 18002640 18002841 202 browser details YourSeq 169 2126 2338 3000 92.1% chr13 + 98787586 98787811 226 browser details YourSeq 168 2134 2323 3000 92.6% chr19 - 46200521 46200707 187 browser details YourSeq 167 2128 2335 3000 90.0% chr2 + 32171565 32171770 206 browser details YourSeq 166 2134 2330 3000 93.7% chr7 - 29977935 29978146 212 browser details YourSeq 166 2134 2331 3000 93.3% chr14 - 26039506 26039848 343 browser details YourSeq 166 2134 2331 3000 93.3% chr14 - 25899736 25900078 343 browser details YourSeq 166 2127 2324 3000 90.2% chr19 + 43713133 43713326 194 browser details YourSeq 165 2128 2322 3000 90.1% chr7 - 112492862 112493052 191 browser details YourSeq 165 2134 2322 3000 92.9% chr9 + 116548007 116548192 186 browser details YourSeq 165 2127 2321 3000 92.4% chr7 + 45484894 45485085 192 browser details YourSeq 165 2134 2322 3000 92.0% chr19 + 29217220 29217405 186 browser details YourSeq 165 2134 2331 3000 92.8% chr11 + 106851071 106851276 206

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: Stxbp1 syntaxin binding protein 1 [ Mus musculus (house mouse) ] Gene ID: 20910, updated on 3-Sep-2019

Gene summary

Official Symbol Stxbp1 provided by MGI Official Full Name syntaxin binding protein 1 provided by MGI Primary source MGI:MGI:107363 See related Ensembl:ENSMUSG00000026797 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 Ms10g; nsec1; N-sec1; Sxtbp1; Unc18h; MMS10-G; Rb-sec1; Unc18-1; AI317162; AI326233; Munc-18a; Munc18-1 Expression Biased expression in cerebellum adult (RPKM 216.7), cortex adult (RPKM 165.3) and 6 other tissues See more Orthologs human all

Genomic context

Location: 2 B; 2 22.09 cM See Stxbp1 in Genome Data Viewer

Exon count: 20

Annotation release Status Assembly Chr Location

108 current GRCm38.p6 (GCF_000001635.26) 2 NC_000068.7 (32787607..32847237, complement)

Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 2 NC_000068.6 (32643127..32702757, complement)

Chromosome 2 - NC_000068.7

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

Transcript information: This gene has 5 transcripts

Gene: Stxbp1 ENSMUSG00000026797

Description syntaxin binding protein 1 [Source:MGI Symbol;Acc:MGI:107363] Gene Synonyms Munc-18a, Munc18-1, N-sec1, Rb-sec1, Sxtbp1, Unc18h, nsec1 Location Chromosome 2: 32,787,602-32,847,245 reverse strand. GRCm38:CM000995.2 About this gene This gene has 5 transcripts (splice variants), 264 orthologues, 7 paralogues, is a member of 1 Ensembl protein family and is associated with 11 phenotypes. Transcripts

Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags

Stxbp1-201 ENSMUST00000050000.15 3892 594aa ENSMUSP00000052440.9 Protein coding CCDS15933 O08599 TSL:1 GENCODE basic APPRIS P3

Stxbp1-202 ENSMUST00000077458.6 3616 603aa ENSMUSP00000089051.3 Protein coding CCDS50572 O08599 TSL:1 GENCODE basic APPRIS ALT1

Stxbp1-205 ENSMUST00000208840.1 526 100aa ENSMUSP00000146437.1 Protein coding - A0A140LHJ4 CDS 3' incomplete TSL:5

Stxbp1-204 ENSMUST00000192333.1 1083 No protein - Retained intron - - TSL:NA

Stxbp1-203 ENSMUST00000113222.3 3598 No protein - lncRNA - - TSL:1

79.64 kb Forward strand 32.78Mb 32.80Mb 32.82Mb 32.84Mb Genes Gm24165-201 >snoRNA Gm13524-201 >lncRNA (Comprehensive set...

Contigs AL772271.11 > AL845471.7 > Genes (Comprehensive set... < Cfap157-201protein coding < Stxbp1-204retained intron

< Cfap157-202retained intron < Stxbp1-205protein coding

< Stxbp1-201protein coding

< Stxbp1-203lncRNA

< Stxbp1-202protein coding

Regulatory Build

32.78Mb 32.80Mb 32.82Mb 32.84Mb Reverse strand 79.64 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

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

Transcript: ENSMUST00000077458

< Stxbp1-202protein coding

Reverse strand 59.24 kb

ENSMUSP00000089... Low complexity (Seg) Superfamily Sec1-like superfamily

Pfam Sec1-like protein

PIRSF Sec1-like protein PANTHER PTHR11679:SF35

Sec1-like protein Gene3D 3.40.50.2060 3.90.830.10 1.25.40.60

Sec1-like, domain 2

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

Variant Legend

missense variant splice region variant synonymous variant

Scale bar 0 60 120 180 240 300 360 420 480 540 603

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

Page 7 of 7