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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
Thursday, March 20, 2008 2:29 PM
pT9
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Hpa
I
Bs
pE
I
7000
Xh
oI
ri
Co
pU
EI
Hind
Sa
cII
T9
Sac
II
Cla I
200
III
0
Ec o RI
HindIII
7051 bp
40
Bs
00
EGFP
Hpa
300
0
Kp
I Xb
aI
Not I
nI
tII I
A a S al
p
Nhe I
pT9
Xho I
Eco RV
5000
Kanamycin resistance
cII
Sa
00
10
er
ot
om
Pr
60
00
aI
Xb
Ar
m
ad
ill
o