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7.13 Experimental Microbial Genetics
Fall 2008
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pT9
pT9 insert is a truncated form (1st 1142 nt) of xxxY from P. aeruginosa cloned into pPL17 using EcoR1(5’) and BamH1 (3’)
Primers used to amplify insert from pTF2 (full xxxY clone):
5’ EcoRI xxxY 825: (5’ TO 3’) GGA ATT CCC GCC ACC ATG TCA ATG TCC (In bold is the EcoRI site, in italics is the RBS sequence, rest is coding sequence) 3’ BamHI xxxY 1979: (5’ TO 3’) CGG GAT CCA TGG GCG TCT TAA AGT TG (Rev Comp of 3’ BamHI xxxY 1979: CAACTTTAAGACGCCCATGGATCCCG
BamHI site is in bold, xxxY sequence is in italics and the additional T between xxxY sequence and BamHI site for maintaining correct reading frame for GFP fusion protein)
Insert sequence cloned:
atgtccatgtccgacaaccttttttctgacgatgaggtactttcaatttcctcaagcccagaacagcgatcttctccgttctacctcaatatatcgc ccatgtcccacggatcagacaattctcagattaatacagtcatcattaattcgaagaaattgccctcaaatcaagcagacataagtttaaaaaa ctcttctggggctgctataaaaattgttaattccctttcacacaagaagaaagagaacacaaacgttaataatgcccaaaaagaccccctctca ctcaccaatactactgcaagcacttgtggcgccaaaagcagcatctcagaggggaaattgtcttctcctccgtccacctcacacacatatgag gggaaattactcacaaaacttactcacacacacacagactttagaggcgccaaaacgagcgatgcaatgggaagtttcccctctctctcgca cagcgacaatagcatagagaaaaatctgagttcttccaccaaaattggaccaaacgcttcttcccctccttctcatgcacacactcacactagc aaatccactgatataagcttagaaagccgctcaaaacatcccgcgcttgccaatacggacgcacgctctataaaagccaatgctaatgacaa tggggaaattttctcctcacttatacaaattgacgaacgcaagcaagaggaaaggccttgcacaactatcaacgctttttggtctatttttaaacc caagccggacgttactaaactaagtctaaagaggaaacccaccaatcccactaaaaacactgggaaaaaatgcatctcccctcataaaaag agcgcttatttatgcccttccgctcaggatgatttaaatttaaatttaaaccccaaatctagcgccaagcccactgtggtgaatttaccagctgcc cgcatcctaagccggcctgcagccaagcgggatttatttaaatcatcatcctcccgaagcccagacgagcagcctatgagtttttcggaagt ggtcgctggcacgggttcaatttttgcggcaccctgtgtcccggcacctttaacgaaaactccaggcaagcggacaaacgacgatctggac tgctccaactttaagacgccca
HpaI
IIId H ni
pa
RIoc E
I I Sac Cla I
ohX
IIId Hin
Ieh
N
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pT9
Armadillo Prom
oter
EGFP
Kanamycinre
sistance
pUCori
T9
XbaI H I
Eco RV
AatII XhoI
II Sac BspEI
NotI
EI Bsp
XbaI
SalI
I
SacII
KpnI
1000
2000
3000 4000
5000
6000
7000
