113011 hw4-sol

113011 hw4-sol - ’9 6‘3 Héw‘bw‘mleA 17 7 firva...

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Unformatted text preview: ’9 6‘3 Héw‘bw‘mleA 17:: 7% firva @_ Mac/eafif/g (57.24374 6? WM: SQM/ fefvit‘de (amt/bxjfl‘efi 70mm W I ' - / 4 “"962 2/ 13438:") [Ha/MK ‘V/ Skiflfi 03* Hawk (9, QCC C) 7a” jku%3 (0 10606 [330 gee % Vic/xvi? W32 fer axe/cue. Q 4 I I w ,, 0 age: #8 fath 4‘ 77/16 fir)le [AA/LLva La beam/fed 469 DWHFE‘MC fltcflt’WOL/me/ML 01,3 >48 QJ/Dfaém \ X J lbw/M 44% Q dt‘ficd 6 ooan C,“ Flam/LA (5 Padluci‘uj (/LL’LUWCMLUtu/LF 5 M . \ / L P0 [3 P [‘CGLLL VOOLU ago/k (3m 'LLLOLLLLLLLQL/I Ekfl @9115 03576 a AF ' Cami/58 Jigsaw MUEM PM addrfl‘ow 7163 0W/ @7166 @OXiLQéZ/EF’Q‘JCLJ ‘ ‘ ‘ ‘ Q n I I ‘ #:5 Cuff/Oéf( MK5 3 /CLH—'014_ Cdl{ [email protected] Vt acéfizr I M £3 (.0 /," 61 121 ccgccctctc QECCCngtg tctgggcctc gaggtacctc cagcttgaat gatggaggtc tgtcctgcgg gcatgtggat _ agcccagaag cactgctgac _ gaagctgtac ggcatatcca L gaaccccgtc gcaatgacat , tcacagggcc ggacagagcc _ aggacacgct tggagaactt _ ggtggcaaga gcctctggct aaaccaccaa ctccaggccc tggtcacccg cacgaatgtc ccagtcctgg ttggaggcca gagaatatca gggcagcagg ggccaggccc aaagccgtca gaaqccatct actttccgca acaggggagg ccacctccct gaggggCtCt ctcaggggcc aacttgaggg atgctgggaa ttggaggcga aggtggcaag gcccccttga ctcatggggt gtggg gcgc cccca ctg ctggct gc ccacc aggaggccga ctgtcccaga ccgtagaagt tgttggtcaa gtggccttcg cccctccaga aactcttccg cctgcaggac caccaacatt cagctcagcg agaggaactg cccagagcag gacgcctgag tttacctgtt ctgtgacttc caccggggtg ccaagttttg Ufa 'NkClLL~iv\ ccctgcaccg ggthCtgag ccgagcttcc ggaccccggc nggatgagg caggcgcgga gtgchLch acgcctcatc gaatatcacg caccaaagtt Ctggcagggc ctcttcccag cagcctcacc tgcggcctca agtctactcc aggggacaga gcttgtgcca ccagcctgtc tccagagagc gaagcattca ctcactcggc ttcgcaccta tccaggtctc gtgggaacca tgtattcttc L A 3. Design primers (forward and reverse) Forward Primer chtcchgc tgtgacagcc anggCtgtg aatttctatg ctggccctgc CCgtgggagC actctgcttc gctgctccac aatttcctcc tgaccaggtg caccctcccc ccatggacac aactctgaga gagagcagct accctgcaaa ccatcaggga acgggcatgg tgaagacagg aacctcattg Lgtcchccc gagtcctgga ctgaacactg cctggaagag tgtcggaagc ccctgcagct gggCtCtggg tccgaacaat ggggaaagct tgtccacctg cgccactcct tccagtgcca tctaaggatg ttaaactcag atttgatgcc caggatgacc gcactccctt atgggggCtg acaagaactg Identify 15-20 bases at the start of the mature protein: gccccaccacgcctcatc Since we are producing a C—terminal fusion to the maltose binding protein (malE section in vector), we can assume that the ribosomal binding site or Shine-Delgarno sequence @ b already there. Add restriction enzyme site as well as an overhang to allow the restriction enzyme to be able to attach and cut the sequence: Overhang RE Site Gene of Interest TGCAGGTGAA GGA T T T Cgccccacc acgcctc Reverse primer Identify last 15-20 bases through (including) the stop codon: aggac aggggacaga tga Find the reverse complement of these bases: TCATCTGTCCCCTGTCCT Add restriction enzyme site and overhang to the 5’ end: Overhang EcoRl Reverse complement TGCATGCGAA TTC TCATCTGTCCCCTGTCCT 2. Structure of EPO using PDB: lBUY 2 Disulfide bonds shown in cyan (light blue) w :3 50 Z bUHm/Q (Ltfl wué—ktcx ( (j \ {Doc 14! . Boo, '4 7N1? £64 [ m 70 UL t" ...
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