Team:Edinburgh/crtI

= crtI =

AMINO ACID SEQUENCE
MKPTTVIGAGFGGLALAIRLQAAGIPVLLLEQRDKPGGRAYVYE

DQGFTFDAGPTVITDPSAIEELFALAGKQLKEYVELLPVTPFYRLCWESGKVFNYDND

QTRLEAQIQQFNPRDVEGYRQFLDYSRAVFKEGYLKLGTVPFLSFRDMLRAAPQLAKL

QAWRSVYSKVASYIEDEHLRQAFSFHSLLVGGNPFATSSIYTLIHALEREWGVWFPRG

GTGALVQGMIKLFQDLGGEVVLNARVSHMETTGNKIEAVHLEDGRRFLTQAVASNADV

VHTYRDLLSQHPAAVKQSNKLQTKRMSNSLFVLYFGLNHHHDQLAHHTVCFGPRYREL

IDEIFNHDGLAEDFSLYLHAPCVTDSSLAPEGCGSYYVLAPVPHLGTANLDWTVEGPK

LRDRIFAYLEQHYMPGLRSQLVTHRMFTPFDFRDQLNAYHGSAFSVEPVLTQSAWFRP

HNRDKTITNLYLVGAGTHPGAGIPGVIGSAKATAGLMLEDLI*

DNA SEQUENCE
3541 ttgcaagcca ttatgacgac tcatcgttaa agagcgacta catgaaacca actacggtaa

3601 ttggtgcagg cttcggtggc ctggcactgg caattcgtct acaagctgcg gggatccccg

3661 tcttactgct tgaacaacgt gataaacccg gcggtcgggc ttatgtctac gaggatcagg

3721 ggtttacctt tgatgcaggc ccgacggtta tcaccgatcc cagtgccatt gaagaactgt

3781 ttgcactggc aggaaaacag ttaaaagagt atgtcgaact gctgccggtt acgccgtttt

3841 accgcctgtg ttgggagtca gggaaggtct ttaattacga taacgatcaa acccggctcg

3901 aagcgcagat tcagcagttt aatccccgcg atgtcgaagg ttatcgtcag tttctggact

3961 attcacgcgc ggtgtttaaa gaaggctatc taaagctcgg tactgtccct tttttatcgt

4021 tcagagacat gcttcgcgcc gcacctcaac tggcgaaa ct gcag gcatgg agaagcgttt

4081 acagtaaggt tgccagttac atcgaagatg aacatctgcg ccaggcgttt tctttccact

4141 cgctgttggt gggcggcaat cccttcgcca cctcatccat ttatacgttg atacacgcgc

4201 tggagcgtga gtggggcgtc tggtttccgc gtggcggcac cggcgcatta gttcagggga

4261 tgataaagct gtttcaggat ctgggtggcg aagtcgtgtt aaacgccaga gtcagccata

4321 tggaaacgac aggaaacaag attgaagccg tgcatttaga ggacggtcgc aggttcctga

4381 cgcaagccgt cgcgtcaaat gcagatgtgg ttcataccta tcgcgacctg ttaagccagc

4441 accctgccgc ggttaagcag tccaacaaa c tgcag actaa gcgcatgagt aactctctgt

4501 ttgtgctcta ttttggtttg aatcaccatc atgatcagct cgcgcatcac acggtttgtt

4561 tcggcccgcg ttaccgcgag ctgattgacg aaatttttaa tcatgatggc ctcgcagagg

4621 acttctcact ttatctgcac gcgccctgtg tcacggattc gtcactggcg cctgaaggtt

4681 gcggcagtta ctatgtgttg gcgccggtgc cgcatttagg caccgcgaac ctcgactgga

4741 cggttgaggg gccaaaacta cgcgaccgta tttttgcgta ccttgagcag cattacatgc

4801 ctggcttacg gagtcagctg gtcacgcacc ggatgtttac gccgtttgat tttcgcgacc

4861 agcttaatgc ctatcatggc tcagcctttt ctgtggagcc cgttcttacc cagagcgcct

4921 ggtttcggcc gcataaccgc gataaaacca ttactaatct ctacctggtc ggcgcaggca

4981 cgcatcccgg cgcaggcatt cctggcgtca tcggctcggc aaaagcgaca gcaggtttga

5041 tgctggagga tctgatatga ataatccgtc gttactcaat catgcggtcg aaacgatggc

BIOBRICKTM PRIMERS
Forward: gat gaattc gcggccgc t tctag atg aaa cca act acg gta att g

Reverse: gct actagt a tta tt a tat cag atc ctc cag cat c

MUTAGENIC PRIMERS
The native crtI sequence contains two internal PstI sites. These were removed by MABEL site directed mutagenesis by Douglas Leslie (Honours project, 2008). Initially mutagenesis by megaprimer PCR was attempted (see iGEM 2007 lab book page 57, also file 'mutagenicPrimersForcrtI20aug07.doc'), using primers crtImut1 and crtImut2, but this was not successful, so primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used instead.The mutagenic primers were as follows:

* crtImut1: gct tct cca tgc ttg cag ttt cgc cag * crtImut2: cat gcg ctt agt ttg cag ttt gtt gg   * crtImutf1: gtt tac agt aag gtt gcc : none, no, 51.81 C.    * crtImutf2: agt aac tct ctg ttt gtg c : very weak, no, 51.46 C

In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.