5' gat ctc gat ccc gcg aaa tta ata cga ctc act ata … · tuesday, november 8, 2011 2:58 pm...
TRANSCRIPT
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
Tuesday, November 8, 2011 2:58 PM Page 2 of 8MBP165.PPYF.T203V.pRSET
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
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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
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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
Tuesday, November 8, 2011 2:58 PM Page 5 of 8MBP165.PPYF.T203V.pRSET
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
Tuesday, November 8, 2011 2:58 PM Page 6 of 8MBP165.PPYF.T203V.pRSET
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
Tuesday, November 8, 2011 2:58 PM Page 7 of 8MBP165.PPYF.T203V.pRSET
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
Tuesday, November 8, 2011 2:58 PM Page 8 of 8MBP165.PPYF.T203V.pRSET
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