...'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to improve restriction enzyme.... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a...final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Preparing the PCR product for...

...reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR ...

... different PAM sites (NGG, NGAN, NGNG, NGAG, and NGCG). Since then, the Osamu Nureki Lab described a rationally...

...SpCas9 variants with alternative PAMs like NGAG and NGCG and SaCas9 with an NNGRRN PAM by mutagenizing the...

...VRER variant D1135V, G1218R, R1335E and T1337R NGCG The D1135E variant is far more selective for...

...KKH TadA-TadA* NNNRRT 8 SpCas9-VRER TadA-TadA* NGCG 8  Tips for designing base editing experiments...

...activity than BE3  85173  pBK-VRER-BE3  VRER-Cas9 (NGCG)    85174  pBK-YE1-BE3  SpCas9 (NGG)  Editing ...

...abcg2a TAL3400 & TAL3401 TGTCCTCATCCCCGTCCCgccgcggcgccaccgtCAGCTTCCACAACATCAA adam15 TAL3016 & TAL3017...TAL3017 TGTTTCTGGCGGACTCTCgtcaccattcttctgtTTGAAGGCACACCTTTGA ahcyl1 TAL3018 & TAL3019 TGACAGACTGTGGCGAGGatgccaagctggaggcGACGAAACAGGCCAGCAA...TGACAGACTGTGGCGAGGatgccaagctggaggcGACGAAACAGGCCAGCAA amh TAL3210 & TAL3211 TCCAGGCAAGATTTGGGCtgatgctgatgatgacCGTGGCGATTGGGTCGTA Aminopeptidase-o TAL3020...TAL3020 & TAL3021 TGATCTGGATCCTAACAAggatgacttgcccctgtGTGCGAACACCAACCATA arf6 TAL3212 & TAL3213 TGGGGAAGATGCTTTCCAagatcttcggcaacaaGGAGATGAGAATATTGA...ARSA TAL 3002 & TAL 3003 TCAAGAGGCGGTCTCCCTctcaacgagaccaccaTAGCCGAGGTGCTGAAGA asun TAL3404 & TAL3405 TAACTCAAGCCAACTCCAggaaacatcaggagtttGCAGGAAGATTGAGTTCA... atg12 TAL3216 & TAL3217 TTAATCAGTCGTTTGCGCcatcaccggaccaagaAGTTGGTGTGCTTTTTGA avpl TAL3220 & TAL3221 TGCTGTGTGTGCTCGCGCtctccacgctctcgtcTGCCTGCTACATCCAGAA... TGCTGTGTGTGCTCGCGCtctccacgctctcgtcTGCCTGCTACATCCAGAA avpr1b TAL3028 & TAL3029 TCACGGCGAACAACACTAccgatccgttcgggagGAACGAAGATGTGGCCAA...

...rerio GGAGCGAGCGGAGCGGTACA 42244 cut S. pyogenes 23360964 Joung fh D. rerio GGAGCGGTACATGGCGACCG 42243...synthetic GCGTATAGTACACATTATACG 66585 cut S. pyogenes 26018130 Xue LRTM2 H. sapiens GCTGGCGGAAGACAGAGTGC 69237... sapiens GTTCCGCGTTACATAACTTA 50927 S. pyogenes 24346702 Wolfe Neomycin TCATGGCTGATGCAATGCGG 67594 cut...gondii ATTGGTCTGACAGCGATGTG 59855 cut S. pyogenes 25480939 Sibley th D. rerio GGATGCGCGTAAGGAGCGCG 42247...sapiens GGTGAGTGAGTGTGTGCGTG 47507 cut S. pyogenes 23792628 Joung VR synthetic TGCGCTCGGTCGTTCGGCTG 62286...GFP Synthetic gRNA1: GAGCTGGACGGCGACGTAAA; gRNA2: CAGAACACCCCCATCGGCGA 72619 cut S. pyogenes 26493208 ... Malat1 H. sapiens gRNA1: GAACCGGTGGGGCTGCGTCA; gRNA2: GGCAGGAGAGGCCAGTTGCG 72620 cut S. pyogenes 26493208...

...OPEN OPEN gCGTGACCTCgctgatAAGAAGAGg xylt1 OZ509 and OZ510 OPEN OPEN cACCCTCTACaggacaGTAGCGGGTg zgc:153086...OPEN OPEN gCGCCACGGAcaataaaGAGGACTGCa GUSB OZ551 and OZ552 OPEN OPEN gGTCATCTGCcattcGAGGCGGACa REST corepressor...CODA CODA gTCCAGCAGCGGTGAGGATGAAGACg unc119b OZ581 and OZ582 CODA CODA tTGCTGCCGCGGATGCTGTGGCGGTTa cx52.9...cTGCTACTTCtggatGGAGAAACGa pitx2 OZ533 and OZ534 OPEN OPEN cCGCTTCAGCggtctGTGGACTCGa zebrafish similar to grp151 (or gpcr-2037... or cav1.3b OZ547 and OZ548 OPEN OPEN gAGCTCCGTCtcgctgGCGGCCGAAg histamine receptor H3 (hrh3) OZ549 and...corepressor 2 OZ553 and OZ554 OPEN OPEN gCTCACCATCtgttggaTGCGATGGAa cdkn1a (p21) OZ555 and OZ556 OPEN OPEN...tGTCTCCGGCagaggGAGGAGGAGa esrrga OZ563 and OZ564 CODA CODA tAGCTACCGCTGGCGTCTGAGGAGc HACE1 OZ565 and OZ566 CODA CODA gCTCATCTCCACCGAGCTGGAGAAg...

...(RFC 10) Prefix: GAATTC GCGGCCGC T TCTAGA G Alternate Prefix: GAATTC GCGGCCGC T TCTAGA TG (contains start...Prefix: GAATTC GCGGCCGC T ACTAGT G Prefix Enzymes: EcoR1 , NotI , Spel Suffix: GCTAGC GCGGCCG CTGCAG Suffix...Prefix: GAATTC GCGGCCGC T TCTAGA G Prefix Enzymes: EcoR1 , NotI , XbaI Suffix: T ACTAGT A GCGGCCG CTGCAG Suffix... 25) Prefix: GAATTC GCGGCCGC T TCTAGA TG GCCGGC Alternate Prefix: GAATTC GCGGCCGC T TCTAG (for "N-parts...Enzymes: EcoR1 , NotI , XbaI Suffix: T ACTAGT A GCGGCCG CTGCAG Suffix Enzymes: SpeI , NotI , PstI Scar:... , XbaI, (NgoMIV) Suffix: ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG Suffix Enzymes: Agel , Spel , Notl, Pstl...

...Letter Code Codons (RNA) Alanine Ala A GCU, GCC, GCA, GCG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Asparagine...

...CATATG TTAATTAA GGCGCGCC CAATTG - 3' = 28 bp Bottom oligo: 3' - GTATAC AATTAATT CCGCGCGG GTTAAC - 5' = ... CATATG TTAATTAA GGCGCGCC CAATTG G - 3' Bottom oligo: 3' - G GTATAC AATTAATT CCGCGCGG GTTAAC CAGCT - 5...' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG - 3' Bottom oligo: 5' - TCGACCAATTGGGCGCGCCTTAATTAACATATGG - 3'...tandem ( NdeI - CATATG , PacI - TTAATTAA , AscI - GGCGCGCC , MfeI - CAATTG ). The bottom oligo will be the...

...'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to improve restriction enzyme.... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a...final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR...

...galanin receptor 3 - GAST gastrin GAS GCG glucagon GLP1, GLP2, GRPP GCGR glucagon receptor GGR, MGC138246 ...

...probe targeting ITR: ITR Forward Primer: 5’-CGGCCTCAGTGAGCGA ITR Reverse Primer: 5’-GGAACCCCTAGTGATGGAGTT...GGAACCCCTAGTGATGGAGTT ITR Probe: -FAM-CACTCCCTCTCTGCGCGCTCG-BBQ- Reagent Preparation 1X PCR Buffer containing 0.05% Pluronic...

...order for the enzyme to cleave efficiently (e.g. GCGGCG-restriction site-your sequence). Protocol Video...

...NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant 3'...

...pUC Reverse AGCGGATAACAATTTCACACAGG (Invitrogen) In lacZ gene MBP-F GATGAAGCCCTGAAAGACGCGCAG (Waugh lab...MSCV-rev CAGCGGGGCTGCTAAAGCGCATGC Murine stem cell virus, reverse primer MT1-F GCTGTCCTCTAAGCGTCACC Mouse...NOS-F GCGTTCAAAAGTCGCCTAAG Nopaline synthase promoter, forward primer Nmt1-F GCAATGTGCAGCGAAACTAA S. pombe...Terminal GCTAGTTATTGCTCAGCGG T7 terminator, reverse primer Tac promoter GAGCGGATAACAATTTCACACAGG (Waugh ...to 3′. Commonly Used Primers CMV Forward CGCAAATGGGCGGTAGGCGTG (Invitrogen) Human CMV immediate early promoter...factor signal sequence, forward primer Amp-R ATAATACCGCGCCACATAGC 5' end of ampicillin resistance gene, reverse...resistance gene, reverse primer CMV Forward CGCAAATGGGCGGTAGGCGTG (Invitrogen) Human CMV immediate early promoter...

...'-GGAACCCCTAGTGATGGAGTT rev ITR primer, 5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve ...