RAS Pathway 2.0 Clone Collection Ras Reference Reagents Program Cancer Research Technology Program, Leidos Biomedical Research Inc. Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA Version 1.0, September 2015

Overview The RAS Pathway 2.0 Clone Collection is a set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) to permit construction of RAS pathway gene expression vectors for in vitro and in vivo research. While these clones can be used for standard Gateway cloning, their utility is greatly enhanced with the use of the FNLCR Combinatorial Cloning Platform (CCP) which allows simplified construction of vectors with different elements (see Wall VE et al, Methods Mol. Biol. (2014), 1116: 193-208 for more information). Some uses of this system are: - Construction of protein expression constructs with various fusion tags - Generation of expression constructs with different promoters for in vivo expression - Production of clones with fluorescent tags for localization experiments - Generation of constructs for making mutant cell lines or transgenic animals - Production of vectors for shRNA or miRNA delivery The advantage of the CCP is in the exquisite specificity of the Multisite Gateway reactions, which permit linkage of multiple elements in a directional fashion, and which involve no additional DNA amplification, thus ensuring the accuracy of the DNA sequence of the final products without the need for additional sequencing. There is also no need for restriction-based cloning processes which have a high rate of failure and may require optimization depending on the sites available in a given clone. This collection contains 180 genes in the “RAS pathway version 2.0”, a crowdsourced pathway map of the RAS signaling pathway generated on the RASCentral website of the Frederick National Laboratory RAS Initiative (www.cancer.gov/RAS) . In each case, the primary splice variant was chosen to represent the most commonly found transcript by RNAseq data across all TCGA tumor samples. Many of these transcript variants are not available in cDNA or ORF libraries, or are available only in mutated forms. The sequences in this collection encode wildtype proteins, and match the RefSeq data for the particular splice variants with the exception of known SNPs or cases in which the RefSeq data is not consistent with genome sequence data (see below). All clones in the collection are in two separate formats. The “closed” format odd-numbered clones contain a Kozak translational initiation sequence and ATG start site at the 5’ end, and a stop codon at the 3’ end. These constructs can be used to make native or aminoterminally tagged constructs using Gateway vectors or the CCP. The “open” format even-numbered clones have the same 5’ sequence, but lack a stop codon and are in frame with the Gateway attB2 site at their 3’ ends. This allows production of C-terminal fusions using appropriate Gateway vectors or the CCP. These Entry clones have been completely sequence validated and functionally tested in Gateway reactions to ensure proper performance.

Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

Vectors included: Except where noted, all clones are in a standard spectinomycin resistant attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon. Odd numbered clones contain a stop codon at the 3’ end, while even numbered clones lack the 3’ stop codon and are in frame with the Gateway attL2 site for C-terminal fusion.

Clone # R777-E001 R777-E002 R777-E003 R777-E004 R777-E005 R777-E006 R777-E007 R777-E008 R777-E009 R777-E010 R777-E011 R777-E012 R777-E013 R777-E014 R777-E015 R777-E016 R777-E017 R777-E018 R777-E019 R777-E020 R777-E021 R777-E022 R777-E023 R777-E024 R777-E025 R777-E026 R777-E027 R777-E028 R777-E029 R777-E030 R777-E031 R777-E032 R777-E033 R777-E034 R777-E035 R777-E036

Gene Hs.AKT1 Hs.AKT1-nostop Hs.AKT2 Hs.AKT2-nostop Hs.AKT3 Hs.AKT3-nostop Hs.ALK Hs.ALK-nostop Hs.ARAF Hs.ARAF-nostop Hs.ARHGAP35 Hs.ARHGAP35-nostop Hs.ARHGEF2 Hs.ARHGEF2-nostop Hs.BRAF Hs.BRAF-nostop Hs.CCND1 Hs.CCND1-nostop Hs.CCND2 Hs.CCND2-nostop Hs.CCND3 Hs.CCND3-nostop Hs.CDK4 Hs.CDK4-nostop Hs.CDK6 Hs.CDK6-nostop Hs.CNKSR1 Hs.CNKSR1-nostop Hs.CNKSR2 Hs.CNKSR2-nostop Hs.CYTH2 Hs.CYTH2-nostop Hs.DUSP1 Hs.DUSP1-nostop Hs.DUSP2 Hs.DUSP2-nostop

RefSeq NM_005163.2 NM_005163.2 NM_001626.5 NM_001626.5 NM_005465.4 NM_005465.4 NM_004304.4 NM_004304.4 NM_001256196.1 NM_001256196.1 NM_004491.4 NM_004491.4 NM_001162383.1 NM_001162383.1 NM_004333.4 NM_004333.4 NM_053056.2 NM_053056.2 NM_001759.1 NM_001759.1 NM_001760.4 NM_001760.4 NM_000075.3 NM_000075.3 NM_001259.6 NM_001259.6 NM_001297647.1 NM_001297647.1 NM_001168647.1 NM_001168647.1 NM_017457.5 NM_017457.5 NM_004417.3 NM_004417.3 NM_004418.3 NM_004418.3

Notes

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E037 R777-E038 R777-E039 R777-E040 R777-E041 R777-E042 R777-E043 R777-E044 R777-E045 R777-E046 R777-E047 R777-E048 R777-E049 R777-E050 R777-E051 R777-E052 R777-E053 R777-E054 R777-E055 R777-E056 R777-E057 R777-E058 R777-E059 R777-E060 R777-E061 R777-E062 R777-E063 R777-E064 R777-E065 R777-E066 R777-E067 R777-E068 R777-E069 R777-E070 R777-E071 R777-E072 R777-E073 R777-E074 R777-E075 R777-E076 R777-E077 R777-E078 R777-E079 R777-E080

Hs.DUSP3 Hs.DUSP3-nostop Hs.DUSP4 Hs.DUSP4-nostop Hs.DUSP5 Hs.DUSP5-nostop Hs.DUSP6 Hs.DUSP6-nostop Hs.E2F1 Hs.E2F1-nostop Hs.E2F2 Hs.E2F2-nostop Hs.E2F3 Hs.E2F3-nostop Hs.ECT2 Hs.ECT2-nostop Hs.EGFR Hs.EGFR-nostop Hs.EIF4EBP1 Hs.EIF4EBP1-nostop Hs.EIF4EBP2 Hs.EIF4EBP2-nostop Hs.EIF4EBP3 Hs.EIF4EBP3-nostop Hs.ERBB2 Hs.ERBB2-nostop Hs.ETS1 Hs.ETS1-nostop Hs.ETS2 Hs.ETS2-nostop Hs.EXOC1 Hs.EXOC1-nostop Hs.EXOC2 Hs.EXOC2-nostop Hs.EXOC3 Hs.EXOC3-nostop Hs.EXOC4 Hs.EXOC4-nostop Hs.EXOC5 Hs.EXOC5-nostop Hs.EXOC6 Hs.EXOC6-nostop Hs.EXOC7 Hs.EXOC7-nostop

NM_004090.3 NM_004090.3 NM_001394.6 NM_001394.6 NM_004419.3 NM_004419.3 NM_001946.3 NM_001946.3 NM_005225.2 NM_005225.2 NM_004091.3 NM_004091.3 NM_001949.4 NM_001949.4 NM_018098.5 NM_018098.5 NM_005228.3 NM_005228.3 NM_004095.3 NM_004095.3 NM_004096.4 NM_004096.4 NM_003732.2 NM_003732.2 NM_004448.3 NM_004448.3 NM_005238.3 NM_005238.3 NM_005239.5 NM_005239.5 NM_001024924.1 NM_001024924.1 NM_018303.5 NM_018303.5 NM_007277.4 NM_007277.4 NM_021807.3 NM_021807.3 NM_006544.3 NM_006544.3 NM_019053.4 NM_019053.4 NM_001013839.3 NM_001013839.3

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E081 R777-E082 R777-E083 R777-E084 R777-E085 R777-E086 R777-E087 R777-E088 R777-E089 R777-E090 R777-E091 R777-E092 R777-E093 R777-E094 R777-E095 R777-E096 R777-E097 R777-E098 R777-E099 R777-E100 R777-E101 R777-E102 R777-E103 R777-E104 R777-E105 R777-E106 R777-E107 R777-E108 R777-E109 R777-E110 R777-E111 R777-E112 R777-E113 R777-E114 R777-E115 R777-E116 R777-E117 R777-E118 R777-E119 R777-E120 R777-E121 R777-E122 R777-E123 R777-E124

Hs.EXOC8 Hs.EXOC8-nostop Hs.FGFR1 Hs.FGFR1-nostop Hs.FGFR2 Hs.FGFR2-nostop Hs.FGFR3 Hs.FGFR3-nostop Hs.FGFR4 Hs.FGFR4-nostop Hs.FLT3 Hs.FLT3-nostop Hs.FNTA Hs.FNTA-nostop Hs.FNTB Hs.FNTB-nostop Hs.FOS Hs.FOS-nostop Hs.GRB2 Hs.GRB2-nostop Hs.HRAS Hs.HRAS-nostop Hs.ICMT Hs.ICMT-nostop Hs.INSR Hs.INSR-nostop Hs.INSRR Hs.INSRR-nostop Hs.IRS1 Hs.IRS1-nostop Hs.IRS2 Hs.IRS2-nostop Hs.JUN Hs.JUN-nostop Hs.KRAS Hs.KRAS-nostop Hs.KSR1 Hs.KSR1-nostop Hs.KSR2 Hs.KSR2-nostop Hs.MAP2K1 Hs.MAP2K1-nostop Hs.MAP2K2 Hs.MAP2K2-nostop

NM_175876.4 NM_175876.4 NM_001174063.1 NM_001174063.1 NM_000141.4 NM_000141.4 NM_000142.4 NM_000142.4 NM_002011.4 NM_002011.4 NM_004119.2 NM_004119.2 NM_002027.2 NM_002027.2 NM_002028.3 NM_002028.3 NM_005252.3 NM_005252.3 NM_002086.4 NM_002086.4 NM_005343.2 NM_005343.2 NM_012405.3 NM_012405.3 NM_001079817.1 NM_001079817.1 NM_014215.2 NM_014215.2 NM_005544.2 NM_005544.2 NM_003749 NM_003749 NM_002228.3 NM_002228.3 NM_004985.4 NM_004985.4 NM_014238.1 NM_014238.1 XM_011538224.1 XM_011538224.1 NM_002755.3 NM_002755.3 NM_030662.3 NM_030662.3

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E125 R777-E126 R777-E127 R777-E128 R777-E129 R777-E130 R777-E131 R777-E132 R777-E133 R777-E134 R777-E135 R777-E136 R777-E137 R777-E138 R777-E139 R777-E140 R777-E141 R777-E142 R777-E143 R777-E144 R777-E145 R777-E146 R777-E147 R777-E148 R777-E149 R777-E150 R777-E151 R777-E152 R777-E153 R777-E154 R777-E155 R777-E156 R777-E157 R777-E158 R777-E159 R777-E160 R777-E161 R777-E162 R777-E163 R777-E164 R777-E165 R777-E166 R777-E167 R777-E168

Hs.MAPK1 Hs.MAPK1-nostop Hs.MAPK3 Hs.MAPK3-nostop Hs.MDM2 Hs.MDM2-nostop Hs.MET Hs.MET-nostop Hs.MLST8 Hs.MLST8-nostop Hs.MRAS Hs.MRAS-nostop Hs.MTOR Hs.MTOR-nostop Hs.NF1 Hs.NF1-nostop Hs.NFE2L2 Hs.NFE2L2-nostop Hs.NFKB1 Hs.NFKB1-nostop Hs.NRAS Hs.NRAS-nostop Hs.PAK1 Hs.PAK1-nostop Hs.PAK2 Hs.PAK2-nostop Hs.PAK3 Hs.PAK3-nostop Hs.PAK4 Hs.PAK4-nostop Hs.PDGFRA Hs.PDGFRA-nostop Hs.PDGFRB Hs.PDGFRB-nostop Hs.PDPK1 Hs.PDPK1-nostop Hs.PEBP1 Hs.PEBP1-nostop Hs.PIK3CA Hs.PIK3CA-nostop Hs.PIK3CB Hs.PIK3CB-nostop Hs.PIK3CD Hs.PIK3CD-nostop

NM_002745.4 NM_002745.4 NM_002746.2 NM_002746.2 NM_002392.5 NM_002392.5 NM_000245.2 NM_000245.2 NM_022372.4 NM_022372.4 NM_001085049.2 NM_001085049.2 NM_004958.3 NM_004958.3 NM_001042492.2 NM_001042492.2 NM_006164.4 NM_006164.4 NM_003998.3 NM_003998.3 NM_002524.4 NM_002524.4 NM_002576.4 NM_002576.4 NM_002577.4 NM_002577.4 NM_002578.3 NM_002578.3 NM_005884.3 NM_005884.3 NM_006206.4 NM_006206.4 NM_002609.3 NM_002609.3 NM_002613.4 NM_002613.4 NM_002567.2 NM_002567.2 NM_006218.2 NM_006218.2 NM_006219.2 NM_006219.2 NM_005026.3 NM_005026.3

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E169 R777-E170 R777-E171 R777-E172 R777-E173 R777-E174 R777-E175 R777-E176 R777-E177 R777-E178 R777-E179 R777-E180 R777-E181 R777-E182 R777-E183 R777-E184 R777-E185 R777-E186 R777-E187 R777-E188 R777-E189 R777-E190 R777-E191 R777-E192 R777-E193 R777-E194 R777-E195 R777-E196 R777-E197 R777-E198 R777-E199 R777-E200 R777-E201 R777-E202 R777-E203 R777-E204 R777-E205 R777-E206 R777-E207 R777-E208 R777-E209 R777-E210 R777-E211 R777-E212

Hs.PIK3CG Hs.PIK3CG-nostop Hs.PIK3R1 Hs.PIK3R1-nostop Hs.PIK3R2 Hs.PIK3R2-nostop Hs.PIK3R3 Hs.PIK3R3-nostop Hs.PIK3R4 Hs.PIK3R4-nostop Hs.PIK3R5 Hs.PIK3R5-nostop Hs.PIK3R6 Hs.PIK3R6-nostop Hs.PIN1 Hs.PIN1-nostop Hs.PLXNB1 Hs.PLXNB1-nostop Hs.PPP1CA Hs.PPP1CA-nostop Hs.PREX2 Hs.PREX2-nostop Hs.PRKAA1 Hs.PRKAA1-nostop Hs.PRKAA2 Hs.PRKAA2-nostop Hs.PRKAB1 Hs.PRKAB1-nostop Hs.PRKAB2 Hs.PRKAB2-nostop Hs.PRKAG1 Hs.PRKAG1-nostop Hs.PRKAG2 Hs.PRKAG2-nostop Hs.PRKAG3 Hs.PRKAG3-nostop Hs.PTEN Hs.PTEN-nostop Hs.RAC1 Hs.RAC1-nostop Hs.RAC2 Hs.RAC2-nostop Hs.RAC3 Hs.RAC3-nostop

NM_002649.3 NM_002649.3 NM_181523.2 NM_181523.2 NM_005027.3 NM_005027.3 NM_003629.3 NM_003629.3 NM_014602.2 NM_014602.2 NM_014308.3 NM_014308.3 NM_001010855.3 NM_001010855.3 NM_006221.3 NM_006221.3 NM_001130082.2 NM_001130082.2 NM_002708.3 NM_002708.3 NM_024870.2 NM_024870.2 NM_006251.5 NM_006251.5 NM_006252.3 NM_006252.3 NM_006253.4 NM_006253.4 NM_005399.4 NM_005399.4 NM_002733.4 NM_002733.4 NM_024429.1 NM_024429.1 NM_017431.2 NM_017431.2 NM_000314.5 NM_000314.5 NM_006908.4 NM_006908.4 NM_002872.4 NM_002872.4 NM_005052.2 NM_005052.2

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E213 R777-E214 R777-E215 R777-E216 R777-E217 R777-E218 R777-E219 R777-E220 R777-E221 R777-E222 R777-E223 R777-E224 R777-E225 R777-E226 R777-E227 R777-E228 R777-E229 R777-E230 R777-E231 R777-E232 R777-E233 R777-E234 R777-E235 R777-E236 R777-E237 R777-E238 R777-E239 R777-E240 R777-E241 R777-E242 R777-E243 R777-E244 R777-E245 R777-E246 R777-E247 R777-E248 R777-E249 R777-E250 R777-E251 R777-E252 R777-E253 R777-E254 R777-E255 R777-E256

Hs.RAF1 Hs.RAF1-nostop Hs.RALA Hs.RALA-nostop Hs.RALB Hs.RALB-nostop Hs.RALBP1 Hs.RALBP1-nostop Hs.RALGDS Hs.RALGDS-nostop Hs.RAPGEF1 Hs.RAPGEF1-nostop Hs.RAPGEF2 Hs.RAPGEF2-nostop Hs.RASA1 Hs.RASA1-nostop Hs.RASA2 Hs.RASA2-nostop Hs.RASA3 Hs.RASA3-nostop Hs.RASAL1 Hs.RASAL1-nostop Hs.RASAL2 Hs.RASAL2-nostop Hs.RASAL3 Hs.RASAL3-nostop Hs.RASGRF1 Hs.RASGRF1-nostop Hs.RASGRF2 Hs.RASGRF2-nostop Hs.RASGRP1 Hs.RASGRP1-nostop Hs.RASGRP2 Hs.RASGRP2-nostop Hs.RASGRP3 Hs.RASGRP3-nostop Hs.RASGRP4 Hs.RASGRP4-nostop Hs.RASSF1 Hs.RASSF1-nostop Hs.RASSF10 Hs.RASSF10-nostop Hs.RASSF2 Hs.RASSF2-nostop

NM_002880.3 NM_002880.3 NM_005402.3 NM_005402.3 NM_002881.2 NM_002881.2 NM_006788.3 NM_006788.3 NM_006266.3 NM_006266.3 NM_198679.1 NM_198679.1 NM_014247.2 NM_014247.2 NM_002890.2 NM_002890.2 NM_006506.3 NM_006506.3 NM_007368.2 NM_007368.2 NM_001193521.1 NM_001193521.1 NM_170692.2 NM_170692.2 NM_022904.1 NM_022904.1 NM_001145648.1 NM_001145648.1 NM_006909.2 NM_006909.2 NM_005739.3 NM_005739.3 NM_153819.1 NM_153819.1 NM_001139488.1 NM_001139488.1 NM_001146202.1 NM_001146202.1 NM_007182.4 NM_007182.4 NM_001080521.2 NM_001080521.2 NM_170774.1 NM_170774.1

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E257 R777-E258 R777-E259 R777-E260 R777-E261 R777-E262 R777-E263 R777-E264 R777-E265 R777-E266 R777-E267 R777-E268 R777-E269 R777-E270 R777-E271 R777-E272 R777-E273 R777-E274 R777-E275 R777-E276 R777-E277 R777-E278 R777-E279 R777-E280 R777-E281 R777-E282 R777-E283 R777-E284 R777-E285 R777-E286 R777-E287 R777-E288 R777-E289 R777-E290 R777-E291 R777-E292 R777-E293 R777-E294 R777-E295 R777-E296 R777-E297 R777-E298 R777-E299 R777-E300

Hs.RASSF3 Hs.RASSF3-nostop Hs.RASSF4 Hs.RASSF4-nostop Hs.RASSF5 Hs.RASSF5-nostop Hs.RASSF6 Hs.RASSF6-nostop Hs.RASSF7 Hs.RASSF7-nostop Hs.RASSF8 Hs.RASSF8-nostop Hs.RASSF9 Hs.RASSF9-nostop Hs.RB1 Hs.RB1-nostop Hs.RCE1 Hs.RCE1-nostop Hs.RHEB Hs.RHEB-nostop Hs.RHOA Hs.RHOA-nostop Hs.RHOB Hs.RHOB-nostop Hs.RHOC Hs.RHOC-nostop Hs.ROCK1 Hs.ROCK1-nostop Hs.ROCK2 Hs.ROCK2-nostop Hs.ROS1 Hs.ROS1-nostop Hs.RPS6KA1 Hs.RPS6KA1-nostop Hs.RPS6KA2 Hs.RPS6KA2-nostop Hs.RPS6KA3 Hs.RPS6KA3-nostop Hs.RPS6KA6 Hs.RPS6KA6-nostop Hs.RPS6KB1 Hs.RPS6KB1-nostop Hs.RPS6KB2 Hs.RPS6KB2-nostop

NM_178169.3 NM_178169.3 NM_032023.3 NM_032023.3 NM_182665.3 NM_182665.3 NM_177532.4 NM_177532.4 NM_001143993.1 NM_001143993.1 NM_007211.4 NM_007211.4 NM_005447.3 NM_005447.3 NM_000321.2 NM_000321.2 NM_005133.2 NM_005133.2 NM_005614.3 NM_005614.3 NM_001664.2 NM_001664.2 NM_004040.3 NM_004040.3 NM_175744.4 NM_175744.4 NM_005406.2 NM_005406.2 NM_004850.3 NM_004850.3 NM_002944.2 NM_002944.2 NM_002953.3 NM_002953.3 NM_021135.4 NM_021135.4 NM_004586.2 NM_004586.2 NM_014496.4 NM_014496.4 NM_003161.3 NM_003161.3 NM_003952.2 NM_003952.2

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E301 R777-E302 R777-E303 R777-E304 R777-E305 R777-E306 R777-E307 R777-E308 R777-E309 R777-E310 R777-E311 R777-E312 R777-E313 R777-E314 R777-E315 R777-E316 R777-E317 R777-E318 R777-E319 R777-E320 R777-E321 R777-E322 R777-E323 R777-E324 R777-E325 R777-E326 R777-E327 R777-E328 R777-E329 R777-E330 R777-E331 R777-E332 R777-E333 R777-E334 R777-E335 R777-E336 R777-E337 R777-E338 R777-E339 R777-E340 R777-E341 R777-E342 R777-E343 R777-E344

Hs.RPTOR Hs.RPTOR-nostop Hs.SAV1 Hs.SAV1-nostop Hs.SCRIB Hs.SCRIB-nostop Hs.SHC1 Hs.SHC1-nostop Hs.SHC2 Hs.SHC2-nostop Hs.SHC3 Hs.SHC3-nostop Hs.SHC4 Hs.SHC4-nostop Hs.SHOC2 Hs.SHOC2-nostop Hs.SOS1 Hs.SOS1-nostop Hs.SOS2 Hs.SOS2-nostop Hs.SPRED1 Hs.SPRED1-nostop Hs.SPRED2 Hs.SPRED2-nostop Hs.SPRED3 Hs.SPRED3-nostop Hs.SPRY1 Hs.SPRY1-nostop Hs.SPRY2 Hs.SPRY2-nostop Hs.SPRY3 Hs.SPRY3-nostop Hs.SPRY4 Hs.SPRY4-nostop Hs.STK11 Hs.STK11-nostop Hs.STK3 Hs.STK3-nostop Hs.STK4 Hs.STK4-nostop Hs.TBK1 Hs.TBK1-nostop Hs.TFDP1 Hs.TFDP1-nostop

NM_020761.2 NM_020761.2 NM_021818.3 NM_021818.3 NM_015356.4 NM_015356.4 NM_003029.4 NM_003029.4 NM_012435.2 NM_012435.2 NM_016848.5 NM_016848.5 NM_203349.3 NM_203349.3 NM_007373.3 NM_007373.3 NM_005633.3 NM_005633.3 NM_006939.2 NM_006939.2 NM_152594.2 NM_152594.2 NM_181784.2 NM_181784.2 NM_001042522.2 NM_001042522.2 NM_001258038.1 NM_001258038.1 NM_005842.2 NM_005842.2 NM_005840.2 NM_005840.2 NM_001127496.1 NM_001127496.1 NM_000455.4 NM_000455.4 NM_006281.3 NM_006281.3 NM_006282.2 NM_006282.2 NM_013254.3 NM_013254.3 NM_007111.4 NM_007111.4

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Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

R777-E345 R777-E346 R777-E347 R777-E348 R777-E349 R777-E350 R777-E351 R777-E352 R777-E353 R777-E354 R777-E355 R777-E356 R777-E357 R777-E358 R777-E359 R777-E360

Hs.TFDP2 Hs.TFDP2-nostop Hs.TIAM1 Hs.TIAM1-nostop Hs.TIAM2 Hs.TIAM2-nostop Hs.TP53 Hs.TP53-nostop Hs.TSC1 Hs.TSC1-nostop Hs.TSC2 Hs.TSC2-nostop Hs.VAV1 Hs.VAV1-nostop Hs.YAP1 Hs.YAP1-nostop

NM_006286.4 NM_006286.4 NM_003253.2 NM_003253.2 NM_001010927.2 NM_001010927.2 NM_000546.5 NM_000546.5 NM_000368.4 NM_000368.4 NM_001114382.1 NM_001114382.1 NM_005428.3 NM_005428.3 NM_001130145.2 NM_001130145.2

1 1

1) clone contains silent mutation(s) from the RefSeq sequence which correspond to known SNPs from genomic DNA sequence data 2) clone contains an A220M mutation not in the RefSeq sequence data, but supported by chimpanzee genomic data and alternate human genome sequence data, suggesting it is a non-synonymous SNP 3) clone contains a T227M mutation not in the RefSeq sequence data, but supported by alternate human genome sequence data, suggesting it is a non-synonymous SNP 4) clone contains additional 30 bp 5’ UTR sequence due to GC content interference with PCR amplification and cloning stability. This sequence is in frame with the attL1 site allowing for N-terminal fusions. 5) clone sequence has been synonymously codon optimized in full or in part to avoid toxicity in E. coli or cloning difficulties due to high GC content. 6) clone contains additional N-terminal sequence not found in the RefSeq sequence, but supported by other genomic and experimental data. 7) clone is in pDonor-225, a low copy number, kanamycin resistant donor vector due to instability in higher copy number Entry vectors. Toxicity associated with this clone could lead to issues in downstream expression clone production.

Materials Available: Glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Unless otherwise noted above, all Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 ug/ml spectinomycin to ensure plasmid stability. Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

Contact Information For additional information on the system or to get more information on the CCP, please contact Dr. Dominic Esposito, Director, RAS Reference Reagents Program, Frederick National Laboratory at 301-846-7376 or [email protected] For technical questions about the system, please contact: Jennifer Mehalko, FNLCR Protein Expression Laboratory, 301-228-4074; [email protected] Vanessa Wall, FNLCR Protein Expression Laboratory, 301-846-1661; [email protected]

Gateway Recombinational Cloning For more information on the Gateway system, please see Hartley JL, Temple GF, and Brasch MA. (2000) “DNA cloning using in vitro site-specific recombination.” Genome Res. 10, 1788-1795. For more information on the FNLCR Combinatorial Cloning Platform (CCP), please see Wall VA, et. al. (2014) “Combinatorial assembly of clone libraries using site-specific recombination.” Meth. Mol. Biol. 1116, 193-208.

Frederick National Laboratory for Cancer Research P.O. Box B, Frederick, MD 21702-1201

RAS pathway v2.0 EGFR, ERBB2, FLT3, PDGFRA,B, MET, ROS1, ALK, FGFR1-4, INSR, INSRR

SOS1, SOS2.GRB2.SHC1-4

ARHGEF2, VAV1, ECT2

RHOA, B, C

HRAS, NRAS, KRAS

FNTA.B RCE1 ICMT

RASGRP1-4, RAPGEF1,2, RASGRF1,2

MRAS

PLXNB1

U

ROCK1,2

ARHGAP35 RASA1-3 RASAL1-3 NF1.SPRED1-3

IRS1, 2

RASSF1-10

RALGDS

STK3,4.SAV1

RAF1, ARAF, BRAF

PEBP1

KSR1,2 CNKSR1,2

RALA,B

MAP2K1,2

SHOC2.PPP1CA PIN1 SCRIB

PIK3CA,D,G, PIK3R1-3,5,6 PTEN

PIP3

MAPK1,3

YAP1 RALBP1

TIAM1,2, PREX2

AKT1-3

PDPK1 EXOC1-8

TSC1.2

RPS6KA1-3, RPS6KA6

ETS1,2, FOS, JUN

RAC1-3

STK11 CYTH2

TBK1

RHEB DUSP1-6

MDM2

NFKB1

CCND1-3

SPRY1-4

CDK4, CDK6 TP53

PRKAA1,2 PRKAB1,2 PRKAG1-3

MTOR.RPTOR.MLST8

PAK1-4

RB1 EIF4EBP1-3

E2F1,2,3.TFDP1,TFDP2

RPS6KB1,2

NFE2L2