5' GAT CTC GAT CCC GCG AAA TTA ATA CGA CTC ACT ATA … · Tuesday, November 8, 2011 2:58 PM Page 1 of 8 MBP165.PPYF.T203V.pRSET T7 promoter 5' GAT CTC GAT CCC GCG AAA TTA ATA CGA

Tuesday, November 8, 2011 2:58 PM Page 1 of 8MBP165.PPYF.T203V.pRSET

T7 promoter

GAT CTC GAT CCC GCG AAA TTA ATA CGA CTC ACT ATA GGG AGA CCA CAA CGG TTT5' 54

NdeI

M R G

1 2 3

CCC TCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG CGG GGT5' 108

His6Gly T7 gene10 leader

S H H H H H H G M A S M T G G Q Q M

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG5' 162

BamHI

Xpress epitope MBP

G R D L Y D D D D K D R W

22 23 24 25 26 27 28 29 30 31 32 33 34

G S K I E

-2 -1 1 2 3

GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC AAA ATC GAA5' 216

MBP

E G K L V I W I N G D K G Y N G L A

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

GAA GGT AAA CTG GTA ATC TGG ATT AAC GGC GAT AAA GGC TAT AAC GGT CTC GCT5' 270

MBP

E V G K K F E K D T G I K V T V E H

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

GAA GTC GGT AAG AAA TTC GAG AAA GAT ACC GGA ATT AAA GTC ACC GTT GAG CAT5' 324

BspEI

MBP

P D K L E E K F P Q V A A T G D G P

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

CCG GAT AAA CTG GAA GAG AAA TTC CCA CAG GTT GCG GCA ACT GGC GAT GGC CCT5' 378

MBP

D I I F W A H D R F G G Y A Q S G L

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

GAC ATT ATC TTC TGG GCA CAC GAC CGC TTT GGT GGC TAC GCT CAA TCT GGC CTG5' 432


MBP

L A E I T P D K A F Q D K L Y P F T

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93

TTG GCT GAA ATC ACC CCG GAC AAA GCG TTC CAG GAC AAG CTG TAT CCG TTT ACC5' 486

MBP

W D A V R Y N G K L I A Y P I A V E

94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111

TGG GAT GCC GTA CGT TAC AAC GGC AAG CTG ATT GCT TAC CCG ATC GCT GTT GAA5' 540

BglII

MBP

A L S L I Y N K D L L P N P P K T W

112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129

GCG TTA TCG CTG ATT TAT AAC AAA GAT CTG CTG CCG AAC CCG CCA AAA ACC TGG5' 594

MBP

E E I P A L D K E L K A K G K S A L

130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147

GAA GAG ATC CCG GCG CTG GAT AAA GAA CTG AAA GCG AAA GGT AAG AGC GCG CTG5' 648

MBP

M F N L Q E P Y F T W P L I A A D P

148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165

ATG TTC AAC CTG CAA GAA CCG TAC TTC ACC TGG CCG CTG ATT GCT GCT GAC CCC5' 702

GFP C-term163

P S Y N V F I M A D K Q K N G I K A

146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163

CCC AGC TAC AAC GTC TTC ATC ATG GCC GAC AAG CAG AAG AAC GGC ATC AAG GCG5' 756

GFP C-term175

180

N F K I R H N I E D G G V Q L A Y H

164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181

AAC TTC AAG ATC CGC CAC AAC ATC GAG GAC GGC GGC GTG CAG CTC GCC TAC CAC5' 810

GFP C-term

Y Q Q N T P I G D G P V L L P D N H

182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199

TAC CAG CAG AAC ACC CCC ATC GGC GAC GGC CCC GTG CTG CTG CCC GAC AAC CAC5' 864


GFP C-term206

Y L S V Q S K L S K D P N E K R D H

200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217

TAC CTG AGC gtC CAG TCC AAA CTT TCG AAA GAC CCC AAC GAG AAG CGC GAT CAC5' 918

GFP C-term

M V L L E F V T A A G I T L G M D E

218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235

ATG GTC CTG CTG GAG TTC GTG ACC GCC GCC GGG ATC ACT CTC GGC ATG GAC GAG5' 972

eGFP N-HalfGFP C-term

Linker

L Y K

236 237 238

G G T G G S

1 2 3 4 5 6

M V S K G E E L F

0 1 2 3 4 5 6 7 8

CTG TAC AAG GGC GGT ACC GGA GGG AGC ATG GTG AGC AAG GGC GAG GAG CTG TTC5' 1026

eGFP N-Half

T G V V P I L V E L D G D V N G H K

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

ACC GGG GTG GTG CCC ATC CTG GTC GAG CTG GAC GGC GAC GTA AAC GGC CAC AAG5' 1080

eGFP N-Half

F S V S G E G E G D A T Y G K L T L

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

TTC AGC GTG TCC GGC GAG GGC GAG GGC GAT GCC ACC TAC GGC AAG CTG ACC CTG5' 1134

eGFP N-Half

K F I C T T G K L P V P W P T L V T

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

AAG TTC ATC TGC ACC ACC GGC AAG CTG CCC GTG CCC TGG CCC ACC CTC GTG ACC5' 1188

eGFP N-Half

T L T Y G V Q C F S R Y P D H M K Q

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

ACC CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC TAC CCC GAC CAC ATG AAG CAG5' 1242

eGFP N-Half93

H D F F K S A M P E G Y I Q E R T I

81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

CAC GAC TTC TTC AAG TCC GCC ATG CCC GAA GGC TAC ATC CAG GAG CGC ACC ATC5' 1296


eGFP N-Half

F F K D D G N Y K T R A E V K F E G

99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116

TTC TTC AAG GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG GTG AAG TTC GAG GGC5' 1350

eGFP N-Half

D T L V N R I E L K G I D F K E D G

117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134

GAC ACC CTG GTG AAC CGC ATC GAG CTG AAG GGC ATC GAC TTC AAG GAG GAC GGC5' 1404

MBP C-HalfeGFP N-Half

N I L G H K L E Y N F N

135 136 137 138 139 140 141 142 143 144 145 146

G G Y A F K

165 166 167 168 169 170

AAC ATC CTG GGG CAC AAG CTG GAG TAC AAC TTT AAC ggg ggt TAT GCG TTC AAG5' 1458

MBP C-Half

Y E N G K Y D I K D V G V D N A G A

171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188

TAT GAA AAC GGC AAG TAC GAC ATT AAA GAC GTG GGC GTG GAT AAC GCT GGC GCG5' 1512

MBP C-Half

K A G L T F L V D L I K N K H M N A

189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206

AAA GCG GGT CTG ACC TTC CTG GTT GAC CTG ATT AAA AAC AAA CAC ATG AAT GCA5' 1566

MBP C-Half

D T D Y S I A E A A F N K G E T A M

207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224

GAC ACC GAT TAC TCC ATC GCA GAA GCT GCC TTT AAT AAA GGC GAA ACA GCG ATG5' 1620

MBP C-Half

T I N G P W A W S N I D T S K V N Y

225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242

ACC ATC AAC GGC CCG TGG GCA TGG TCC AAC ATC GAC ACC AGC AAA GTG AAT TAT5' 1674

MBP C-Half

G V T V L P T F K G Q P S K P F V G

243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

GGT GTA ACG GTA CTG CCG ACC TTC AAG GGT CAA CCA TCC AAA CCG TTC GTT GGC5' 1728


MBP C-Half

V L S A G I N A A S P N K E L A K E

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278

GTG CTG AGC GCA GGT ATT AAC GCC GCC AGT CCG AAC AAA GAG CTG GCA AAA GAG5' 1782

MBP C-Half

F L E N Y L L T D E G L E A V N K D

279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296

TTC CTC GAA AAC TAT CTG CTG ACT GAT GAA GGT CTG GAA GCG GTT AAT AAA GAC5' 1836

MBP C-Half

K P L G A V A L K S Y E E E L V K D

297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314

AAA CCG CTG GGT GCC GTA GCG CTG AAG TCT TAC GAG GAA GAG TTG GtG AAA GAT5' 1890

MBP C-Half

P R I A A T M E N A Q K G E I M P N

315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332

CCG CGG ATT GCC GCC ACT ATG GAA AAC GCC CAG AAA GGT GAA ATC ATG CCG AAC5' 1944

MBP C-Half

I P Q M S A F W Y A V R T A V I N A

333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350

ATC CCG CAG ATG TCC GCT TTC TGG TAT GCC GTG CGT ACT GCG GTG ATC AAC GCC5' 1998

MBP C-Half

A S G R Q T V D E D L K D A Q T R I

351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368

GCC AGC GGT CGT CAG ACT GTC GAT GAA GaC CTG AAA GAC GCG CAG ACT cgg atc5' 2052

HindIIIEcoRI

MBP C-Half

T K G S H H H H H H G .

369 370 371 372 373 374 375 376 377 378 379 380

acc aaa GGA AGT CAC CAT CAT CAC CAC CAT GGC taa GAA TTC GAA GCT TGA TCC5' 2106

GGC TGC TAA CAA AGC CCG AAA GGA AGC TGA GTT GGC TGC TGC CAC CGC TGA GCA5' 2160

ATA ACT AGC ATA ACC CCT TGG GGC CTC TAA ACG GGT CTT GAG GGG TTT TTT GCT5' 2214


BspEI

GAA AGG AGG AAC TAT ATC CGG ATC TGG CGT AAT AGC GAA GAG GCC CGC ACC GAT5' 2268

f1 origin

CGC CCT TCC CAA CAG TTG CGC AGC CTG AAT GGC GAA TGG GAC GCG CCC TGT AGC5' 2322

f1 origin

GGC GCA TTA AGC GCG GCG GGT GTG GTG GTT ACG CGC AGC GTG ACC GCT ACA CTT5' 2376

f1 origin

GCC AGC GCC CTA GCG CCC GCT CCT TTC GCT TTC TTC CCT TCC TTT CTC GCC ACG5' 2430

f1 origin

TTC GCC GGC TTT CCC CGT CAA GCT CTA AAT CGG GGG CTC CCT TTA GGG TTC CGA5' 2484

f1 origin

TTT AGT GCT TTA CGG CAC CTC GAC CCC AAA AAA CTT GAT TAG GGT GAT GGT TCA5' 2538

f1 origin

CGT AGT GGG CCA TCG CCC TGA TAG ACG GTT TTT CGC CCT TTG ACG TTG GAG TCC5' 2592

f1 origin

ACG TTC TTT AAT AGT GGA CTC TTG TTC CAA ACT GGA ACA ACA CTC AAC CCT ATC5' 2646

f1 origin

TCG GTC TAT TCT TTT GAT TTA TAA GGG ATT TTG CCG ATT TCG GCC TAT TGG TTA5' 2700

f1 origin

AAA AAT GAG CTG ATT TAA CAA AAA TTT AAC GCG AAT TTT AAC AAA ATA TTA ACG5' 2754

f1 origin

CTT ACA ATT TAG GTG GCA CTT TTC GGG GAA ATG TGC GCG GAA CCC CTA TTT GTT5' 2808

TAT TTT TCT AAA TAC ATT CAA ATA TGT ATC CGC TCA TGA GAC AAT AAC CCT GAT5' 2862

AAA TGC TTC AAT AAT ATT GAA AAA GGA AGA GTA TGA GTA TTC AAC ATT TCC GTG5' 2916

TCG CCC TTA TTC CCT TTT TTG CGG CAT TTT GCC TTC CTG TTT TTG CTC ACC CAG5' 2970

AAA CGC TGG TGA AAG TAA AAG ATG CTG AAG ATC AGT TGG GTG CAC GAG TGG GTT5' 3024


ACA TCG AAC TGG ATC TCA ACA GCG GTA AGA TCC TTG AGA GTT TTC GCC CCG AAG5' 3078

AAC GTT TTC CAA TGA TGA GCA CTT TTA AAG TTC TGC TAT GTG GCG CGG TAT TAT5' 3132

CCC GTA TTG ACG CCG GGC AAG AGC AAC TCG GTC GCC GCA TAC ACT ATT CTC AGA5' 3186

ATG ACT TGG TTG AGT ACT CAC CAG TCA CAG AAA AGC ATC TTA CGG ATG GCA TGA5' 3240

CAG TAA GAG AAT TAT GCA GTG CTG CCA TAA CCA TGA GTG ATA ACA CTG CGG CCA5' 3294

ACT TAC TTC TGA CAA CGA TCG GAG GAC CGA AGG AGC TAA CCG CTT TTT TGC ACA5' 3348

ACA TGG GGG ATC ATG TAA CTC GCC TTG ATC GTT GGG AAC CGG AGC TGA ATG AAG5' 3402

CCA TAC CAA ACG ACG AGC GTG ACA CCA CGA TGC CTG TAG CAA TGG CAA CAA CGT5' 3456

TGC GCA AAC TAT TAA CTG GCG AAC TAC TTA CTC TAG CTT CCC GGC AAC AAT TAA5' 3510

TAG ACT GGA TGG AGG CGG ATA AAG TTG CAG GAC CAC TTC TGC GCT CGG CCC TTC5' 3564

CGG CTG GCT GGT TTA TTG CTG ATA AAT CTG GAG CCG GTG AGC GTG GGT CTC GCG5' 3618

GTA TCA TTG CAG CAC TGG GGC CAG ATG GTA AGC CCT CCC GTA TCG TAG TTA TCT5' 3672

ACA CGA CGG GGA GTC AGG CAA CTA TGG ATG AAC GAA ATA GAC AGA TCG CTG AGA5' 3726

TAG GTG CCT CAC TGA TTA AGC ATT GGT AAC TGT CAG ACC AAG TTT ACT CAT ATA5' 3780

TAC TTT AGA TTG ATT TAA AAC TTC ATT TTT AAT TTA AAA GGA TCT AGG TGA AGA5' 3834

TCC TTT TTG ATA ATC TCA TGA CCA AAA TCC CTT AAC GTG AGT TTT CGT TCC ACT5' 3888

GAG CGT CAG ACC CCG TAG AAA AGA TCA AAG GAT CTT CTT GAG ATC CTT TTT TTC5' 3942

TGC GCG TAA TCT GCT GCT TGC AAA CAA AAA AAC CAC CGC TAC CAG CGG TGG TTT5' 3996

GTT TGC CGG ATC AAG AGC TAC CAA CTC TTT TTC CGA AGG TAA CTG GCT TCA GCA5' 4050


GAG CGC AGA TAC CAA ATA CTG TTC TTC TAG TGT AGC CGT AGT TAG GCC ACC ACT5' 4104

TCA AGA ACT CTG TAG CAC CGC CTA CAT ACC TCG CTC TGC TAA TCC TGT TAC CAG5' 4158

TGG CTG CTG CCA GTG GCG ATA AGT CGT GTC TTA CCG GGT TGG ACT CAA GAC GAT5' 4212

AGT TAC CGG ATA AGG CGC AGC GGT CGG GCT GAA CGG GGG GTT CGT GCA CAC AGC5' 4266

CCA GCT TGG AGC GAA CGA CCT ACA CCG AAC TGA GAT ACC TAC AGC GTG AGC TAT5' 4320

GAG AAA GCG CCA CGC TTC CCG AAG GGA GAA AGG CGG ACA GGT ATC CGG TAA GCG5' 4374

GCA GGG TCG GAA CAG GAG AGC GCA CGA GGG AGC TTC CAG GGG GAA ACG CCT GGT5' 4428

ATC TTT ATA GTC CTG TCG GGT TTC GCC ACC TCT GAC TTG AGC GTC GAT TTT TGT5' 4482

GAT GCT CGT CAG GGG GGC GGA GCC TAT GGA AAA ACG CCA GCA ACG CGG CCT TTT5' 4536

TAC GGT TCC TGG CCT TTT GCT GGC CTT TTG CTC ACA TGT TCT TTC CTG CGT TAT5' 4590

CCC CTG ATT CTG TGG ATA ACC GTA TTA CCG CCT TTG AGT GAG CTG ATA CCG CTC5' 4644

GCC GCA GCC GAA CGA CCG AGC GCA GCG AGT CAG TGA GCG AGG AAG CGG AAG AGC5' 4698

GCC CAA TAC GCA AAC CGC CTC TCC CCG CGC GTT GGC CGA TTC ATT AAT GCA G5' 4750

Documents

5' GAT CTC GAT CCC GCG AAA TTA ATA CGA CTC ACT ATA … · Tuesday, November 8, 2011 2:58 PM Page 1 of 8 MBP165.PPYF.T203V.pRSET T7 promoter 5' GAT CTC GAT CCC GCG AAA TTA ATA CGA