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Team:Edinburgh/glgC
From 2008.igem.org
(New page: <div id="header">{{Template:Team:Edinburgh/Templates/Header}}</div> = ''glgC'' = === Amino acid sequence == MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPA<br /> VHFGGK FRIIDFALSN CINS...) |
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= ''glgC'' = | = ''glgC'' = | ||
| - | === Amino acid sequence == | + | === Amino acid sequence === |
MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPA<br /> | MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPA<br /> | ||
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GMVIGENA EEDARRFYRS EEGIVLVTRE MLRKLGHKQE R<br /> | GMVIGENA EEDARRFYRS EEGIVLVTRE MLRKLGHKQE R<br /> | ||
| - | === Nucleotide sequence == | + | === Nucleotide sequence === |
3565981 atgccccaat aacatcagcc agaccgccgg ttttaagcag cgggaacatc tctgaacata<br /> | 3565981 atgccccaat aacatcagcc agaccgccgg ttttaagcag cgggaacatc tctgaacata<br /> | ||
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Thus a digest with EcoRI and PstI should yield a vector fragment plus fragments of 570, 567, and 228 bp.<br /> | Thus a digest with EcoRI and PstI should yield a vector fragment plus fragments of 570, 567, and 228 bp.<br /> | ||
| - | == BioBrick<sup>TM</sup> primers == | + | === BioBrick<sup>TM</sup> primers === |
F primer: atg gtt agt tta gag aag aac gat c 25mer<br /> | F primer: atg gtt agt tta gag aag aac gat c 25mer<br /> | ||
R primer: tta tta tcg ctc ctg ttt atg ccc taa c<br /> | R primer: tta tta tcg ctc ctg ttt atg ccc taa c<br /> | ||
| - | == Mutagenesis == | + | === Mutagenesis === |
Site 1: codons 191 and 192, gaa-ttc, EF. Possible silent changes gag-ttc or gaa-ttt. We opted for gaa-ttt.<br /> | Site 1: codons 191 and 192, gaa-ttc, EF. Possible silent changes gag-ttc or gaa-ttt. We opted for gaa-ttt.<br /> | ||
Latest revision as of 23:09, 29 October 2008
Contents |
glgC
Amino acid sequence
MVSLEKNDHL MLARQLPLKS VALILAGGRG TRLKDLTNKR AKPA
VHFGGK FRIIDFALSN CINSGIRRMG VITQYQSHTL VQHIQRGWSF FNEEMNEFVD LL
PAQQRMKG ENWYRGTADA VTQNLDIIRR YKAEYVVILA GDHIYKQDYS RMLIDHVEKG
ARCTVACMPV PIEEASAFGV MAVDENDKII EFVEKPANPP SMPNDPSKSL ASMGIYVF
DA DYLYELLEED DRDENSSHDF GKDLIPKITE AGLAYAHPFP LSCVQSDPDA EPYWRD
VGTL EAYWKANLDL ASVVPELDMY DRNWPIRTYN ESLPPAKFVQ DRSGSHGMTL NSLV
SGGCVI SGSVVVQSVL FSRVRVNSFC NIDSAVLLPE VWVGRSCRLR RCVIDRACVI PE
GMVIGENA EEDARRFYRS EEGIVLVTRE MLRKLGHKQE R
Nucleotide sequence
3565981 atgccccaat aacatcagcc agaccgccgg ttttaagcag cgggaacatc tctgaacata
3566041 catgtaaaac ctgcattatc gctcctgttt atgccctaac ttccgtagca tttcgcgcgt
3566101 taccagcacg atgccttctt ctgaacgata gaaacgacgt gcatcttcct ctgcgttttc
3566161 accaatcacc atgccttccg gaataacaca agcacgatcg atgacgcagc ggcgcagacg
3566221 gcacgagcga cctacccata cttccggtaa caatacggcg gaatcaatgt tgcagaatga
3566281 attcacgcga acgcgcgaga acagaacgga ctgcaccacc accgaaccgg agatcacaca
3566341 accgccggaa accagtgagt taagggtcat cccgtggcta ccggagcgat cctgcacgaa
3566401 tttcgctggc ggtaatgatt cattgtaggt gcgaattggc caattgcgat cgtacatatc
3566461 cagttccggc accacagagg ccagatcgag gttcgctttc cagtaagctt ccagcgtacc
3566521 cacatcgcgc cagtacggct cggcatccgg gtcggattgt acgcaagaga gcgggaacgg
3566581 gtgcgcatag gccagaccgg cttcggtgat cttgggaatc aaatctttgc caaagtcgtg
3566641 gctggagttc tcatcgcgat cgtcttcttc cagcagttca tacagatagt cggcgtcaaa
3566701 gacgtagata cccatactcg ccagagattt gctcggatcg ttcggcattg acggcgggtt
3566761 agcaggtttt tcaacgaatt cgataatttt atcgttctca tcaaccgcca taacgccaaa
3566821 tgcggaggct tcttcaatcg gtactggcat acaagcaacg gtgcaacgtg cgcctttttc
3566881 gacgtgatcg ataagcatac gcgagtagtc ttgcttgtag atatggtcgc ccgccaggat
3566941 caccacgtat tccgctttat aacggcggat aatgtcgagg ttttgggtga ccgcatctgc
3567001 ggtgccgcga taccagtttt cccctttcat tctctgctgt gctggcagca gatcgacaaa
3567061 ctcgttcatt tcttcattga agaatgacca gccgcgctga atgtgctgca ccagagtgtg
3567121 ggactggtac tgggtgatca cgcccatacg acggatcccg gagttgatgc agttagacag
3567181 cgcaaagtcg ataatgcgga acttaccgcc gaagtgtacg gccggttttg ctcgcttatt
3567241 ggttaaatcc ttcaggcggg taccacgtcc tcccgccagt atcagggcaa cagatttcaa
3567301 tggcagctgg cgcgccaaca ttaagtgatc gttcttctct aaactaacca tgactaactc
3567361 cttttttatc atctctggaa cacacacaat ccgtgtgcag gtccctgcca gacagccgta
Complement
tacggctgtctggcagggacctgcacacggattgtgtgtgttccagagat 50
gataaaaaaggagttagtcatggttagtttagagaagaacgatcacttaa 100
tgttggcgcgccagctgccattgaaatctgttgccctgatactggcggga 150
ggacgtggtacccgcctgaaggatttaaccaataagcgagcaaaaccggc 200
cgtacacttcggcggtaagttccgcattatcgactttgcgctgtctaact 250
gcatcaactccgggatccgtcgtatgggcgtgatcacccagtaccagtcc 300
cacactctggtgcagcacattcagcgcggctggtcattcttcaatgaaga 350
aatgaacgagtttgtcgatctgctgccagcacagcagagaatgaaagggg 400
aaaactggtatcgcggcaccgcagatgcggtcacccaaaacctcgacatt 450
atccgccgttataaagcggaatacgtggtgatcctggcgggcgaccatat 500
ctacaagcaagactactcgcgtatgcttatcgatcacgtcgaaaaaggcg 550
cacgttgcaccgttgcttgtatgccagtaccgattgaagaagcctccgca 600
tttggcgttatggcggttgatgagaacgataaaattatcgaattcgttga 650
aaaacctgctaacccgccgtcaatgccgaacgatccgagcaaatctctgg 700
cgagtatgggtatctacgtctttgacgccgactatctgtatgaactgctg 750
gaagaagacgatcgcgatgagaactccagccacgactttggcaaagattt 800
gattcccaagatcaccgaagccggtctggcctatgcgcacccgttcccgc 850
tctcttgcgtacaatccgacccggatgccgagccgtactggcgcgatgtg 900
ggtacgctggaagcttactggaaagcgaacctcgatctggcctctgtggt 950
gccggaactggatatgtacgatcgcaattggccaattcgcacctacaatg 1000
aatcattaccgccagcgaaattcgtgcaggatcgctccggtagccacggg 1050
atgacccttaactcactggtttccggcggttgtgtgatctccggttcggt 1100
ggtggtgcagtccgttctgttctcgcgcgttcgcgtgaattcattctgca 1150
acattgattccgccgtattgttaccggaagtatgggtaggtcgctcgtgc 1200
cgtctgcgccgctgcgtcatcgatcgtgcttgtgttattccggaaggcat 1250
ggtgattggtgaaaacgcagaggaagatgcacgtcgtttctatcgttcag 1300
aagaaggcatcgtgctggtaacgcgcgaaatgctacggaagttagggcat 1350
aaacaggagcgataatgcaggttttacatgtatgttcagagatgttcccg 1400
ctgcttaaaaccggcggtctggctgatgttattggggcat 1440
Sequence map
tacggctgtctggcagggacctgcacacggattgtgtgtgttccagagat 50
atgccgacagaccgtccctggacgtgtgcctaacacacacaaggtctcta
1 2 3 4 5 6 7 8 9 10
gataaaaaaggagttagtc atg gtt agt tta gag aag aac gat cac tta a 100
ctattttttcctcaatcag tac caa tca aat ctc ttc ttg cta gtg aat t
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
tg ttg gcg cgc cag ctg cca ttg aaa tct gtt gcc ctg ata ctg gcg gga 150
ac aac cgc gcg gtc gac ggt aac ttt aga caa cgg gac tat gac cgc cct
28 29 KpnI 32 33 34 35 36 37 38 39 40 41 42 43 4
gga cgt ggt acc cgc ctg aag gat tta acc aat aag cga gca aaa ccg gc 200
cct gca cca tgg gcg gac ttc cta aat tgg tta ttc gct cgt ttt ggc cg
4 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
c gta cac ttc ggc ggt aag ttc cgc att atc gac ttt gcg ctg tct aac t 250
g cat gtg aag ccg cca ttc aag gcg taa tag ctg aaa cgc gac aga ttg a
61 62 63 64 BamHI 67 68 69 70 71 72 73 74 75 76 77
gc atc aac tcc ggg atc cgt cgt atg ggc gtg atc acc cag tac cag tcc 300
cg tag ttg agg ccc tag gca gca tac ccg cac tag tgg gtc atg gtc agg
78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 9
cac act ctg gtg cag cac att cag cgc ggc tgg tca ttc ttc aat gaa ga 350
gtg tga gac cac gtc gtg taa gtc gcg ccg acc agt aag aag tta ctt ct
4 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 1
a atg aac gag ttt gtc gat ctg ctg cca gca cag cag aga atg aaa ggg g 400
t tac ttg ctc aaa cag cta gac gac ggt cgt gtc gtc tct tac ttt ccc c
11 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
aa aac tgg tat cgc ggc acc gca gat gcg gtc acc caa aac ctc gac att 450
tt ttg acc ata gcg ccg tgg cgt cta cgc cag tgg gtt ttg gag ctg taa
128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 14
atc cgc cgt tat aaa gcg gaa tac gtg gtg atc ctg gcg ggc gac cat at 500
tag gcg gca ata ttt cgc ctt atg cac cac tag gac cgc ccg ctg gta ta
4 145 146 147 148 149 150 151 152 153 ClaI 156 157 158 159 160 1
c tac aag caa gac tac tcg cgt atg ctt atc gat cac gtc gaa aaa ggc g 550
g atg ttc gtt ctg atg agc gca tac gaa tag cta gtg cag ctt ttt ccg c
61 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177
ca cgt tgc acc gtt gct tgt atg cca gta ccg att gaa gaa gcc tcc gca 600
gt gca acg tgg caa cga aca tac ggt cat ggc taa ctt ctt cgg agg cgt
178 179 180 181 182 183 184 185 186 187 188 189 190 EcoRI 193 19
ttt ggc gtt atg gcg gtt gat gag aac gat aaa att atc gaa ttc gtt ga 650
aaa ccg caa tac cgc caa cta ctc ttg cta ttt taa tag ctt aag caa ct
4 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 2
a aaa cct gct aac ccg ccg tca atg ccg aac gat ccg agc aaa tct ctg g 700
t ttt gga cga ttg ggc ggc agt tac ggc ttg cta ggc tcg ttt aga gac c
11 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227
cg agt atg ggt atc tac gtc ttt gac gcc gac tat ctg tat gaa ctg ctg 750
gc tca tac cca tag atg cag aaa ctg cgg ctg ata gac ata ctt gac gac
228 229 230 231 PvuI 234 235 236 237 238 239 241 241 242 243 24
gaa gaa gac gat cgc gat gag aac tcc agc cac gac ttt ggc aaa gat tt 800
ctt ctt ctg cta gcg cta ctc ttg agg tcg gtg ctg aaa ccg ttt cta aa
4 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 2
g att ccc aag atc acc gaa gcc ggt ctg gcc tat gcg cac ccg ttc ccg c 850
c taa ggg ttc tag tgg ctt cgg cca gac cgg ata cgc gtg ggc aag ggc g
61 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277
tc tct tgc gta caa tcc gac ccg gat gcc gag ccg tac tgg cgc gat gtg 900
ag aga acg cat gtt agg ctg ggc cta cgg ctc ggc atg acc gcg cta cac
278 279 280 HindIII 284 285 286 287 288 289 290 291 292 293 29
ggt acg ctg gaa gct tac tgg aaa gcg aac ctc gat ctg gcc tct gtg gt 950
cca tgc gac ctt cga atg acc ttt cgc ttg gag cta gac cgg aga cac ca
4 295 296 297 298 299 300 PvuI 304 305 306 307 308 309 310 3
g ccg gaa ctg gat atg tac gat cgc aat tgg cca att cgc acc tac aat g 1000
c ggc ctt gac cta tac atg cta gcg tta acc ggt taa gcg tgg atg tta c
11 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327
aa tca tta ccg cca gcg aaa ttc gtg cag gat cgc tcc ggt agc cac ggg 1050
tt agt aat ggc ggt cgc ttt aag cac gtc cta gcg agg cca tcg gtg ccc
328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 34
atg acc ctt aac tca ctg gtt tcc ggc ggt tgt gtg atc tcc ggt tcg gt 1100
tac tgg gaa ttg agt gac caa agg ccg cca aca cac tag agg cca agc ca
4 345 346 347 348 349 350 351 352 353 354 355 356 EcoRI 359 360 3
g gtg gtg cag tcc gtt ctg ttc tcg cgc gtt cgc gtg aat tca ttc tgc a 1150
c cac cac gtc agg caa gac aag agc gcg caa gcg cac tta agt aag acg t
61 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
ac att gat tcc gcc gta ttg tta ccg gaa gta tgg gta ggt cgc tcg tgc 1200
tg taa cta agg cgg cat aac aat ggc ctt cat acc cat cca gcg agc acg
378 379 380 381 382 383 ClaIPvuI 387 388 389 390 391 392 393 39
cgt ctg cgc cgc tgc gtc atc gat cgt gct tgt gtt att ccg gaa ggc at 1250
gca gac gcg gcg acg cag tag cta gca cga aca caa taa ggc ctt ccg ta
4 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 4
g gtg att ggt gaa aac gca gag gaa gat gca cgt cgt ttc tat cgt tca g 1300
c cac taa cca ctt ttg cgt ctc ctt cta cgt gca gca aag ata gca agt c
11 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427
aa gaa ggc atc gtg ctg gta acg cgc gaa atg cta cgg aag tta ggg cat 1350
tt ctt ccg tag cac gac cat tgc gcg ctt tac gat gcc ttc aat ccc gta
428 429 430 431 *
aaa cag gag cga taa tgcaggttttacatgtatgttcagagatgttcccg 1400
ttt gtc ctc gct att acgtccaaaatgtacatacaagtctctacaagggc
ctgcttaaaaccggcggtctggctgatgttattggggcat 1440
gacgaattttggccgccagaccgactacaataaccccgta
EcoRI FOUND 2.
PstI not found.
SpeI not found.
XbaI not found.
Two EcoRI sites are present at +570 and +1137.
Thus a digest with EcoRI and PstI should yield a vector fragment plus fragments of 570, 567, and 228 bp.
BioBrickTM primers
F primer: atg gtt agt tta gag aag aac gat c 25mer
R primer: tta tta tcg ctc ctg ttt atg ccc taa c
Mutagenesis
Site 1: codons 191 and 192, gaa-ttc, EF. Possible silent changes gag-ttc or gaa-ttt. We opted for gaa-ttt.
Forward: tgttgaaaaacctgctaacc - 54°C
Reverse: aattcgataattttatcgttc -52°C
Site 2: codons 356 to 358, gtg-aat-tca, VNS. Possible silent changes gtc-aat-tca, or gtg-aac-tca, no good possibility changing base 1 of the serine codon only. We went for g-aac-tca.
Forward: atcattctgcaacattgattcc - 54°C
Reverse: ttcacgcgaacgcgcg - 54°C
The mutation required to produce glgC16 is G336D, GGC changed to GAC.
Forward: acggttgtgtgatctccg - 54°C
Reverse: cggaaaccagtgagttaag - 56°C
