!
"
#
$%
$&
$&
$
$'
$(
$
&$
&
&'
&
%
&
'
"
)*
' +* ! ,
+* ! ,
-. !
/
0
1
/
*)
' +*
1
1
1
1
+
1
#
'$
'
''
"
'
'#
33
(&
,
2
/
)
(
(#
(
"
4
6 )4
6
5
7
4
86 4 9
#
#&
!
8
9
,
).
)
"
:
1
;
"
<
"
:
"
""
6 =
"
1
" 9
>
8
/
!
)
$# '
8$#( % 9
1
:
= -
/
+
=
7
!
,
/
)
/
"
/
8
:
? //
"
A $#
@
1
!
9
+
" <
+
/
<
"
/
?
/
"
; /
"
+
1
:
/
"
/
.- .
, ) ,
, B !
C
!
C
/
1
$9
C
!
)
"
!
,
/
<
A+
<
A
!
) 1
% A' *&A'
A
8)
1
<
9
1
' % *$% %
<
+
/ <
:
% %%%
<
&*$% D $% $&
:
+
(%E A +
&9
/
$A *( D $% $&
1
C
8
9
!
/
F
+
F
A
+
1
A
/
//
;
1
"
G
?A
+
/
/
/
;
,
/
;
1
;
H
1
1
/
% A$*% A' µ1
%
?
83 D $% I
<
$&
D
<
D $% $
9H 1
$ ="
C
1
+
/
9
8
)
9
1
1
&% % *&' %
+
!) A
) A
)) A
6)
)
-
&
$A'
3
-9
!8
+
) 1
""
J
"
<
;
) 1
/
A
" "
1
/
1
/
)
&%
)
/
1
*&% K
"/
;
/
/
9
"/
:
$A'
1
/
/
/
/
A
1
"
, )!
'9
A
"
!
1
<
+
/ ""
/
)
G
1
)
L
) *
( &K 1
!
/
+
""
( A&
+
39
A
L
A
?
L
/
/
=
/
+
'%%
"
+
'% -
$% %
(9
-
/
+
;
!
!
1
-
/
8
/
1
$%
""
!
= "9
!
+
/
<
!
/
2
:
F
/ A"
F
""
A
1
$µ
A$ A$
/
A $%
/
!
L ,- !
4C
B .
!- , ).
1
!
,
!
/
/
F
7
F
:
F /
/
!
:
/
"
F
,
! .
-, !
), - .
"
F
8)
F
1
)!2
F
F
;
9
:
) 1
/
<A
$ ="
;
) 1
/
<
/
) 1 ,
/
8
+
@
/ <
F
F A
9
!
, B - ! . 6!
) 1
)
7
7
M
0
) 1
8
4
1
)
9
1
-.
),-
. )) !)) , .
C
4 , , )!+
('* %K
' +* +
3 % *3 ' K
+*' +
& I$% % K
(%* %K
+*' +
% +I$% % K
( &*( K
+*' +
& % +I#' K
('* %K
"
)!2
"
#+I#( A' K
&$+I#( A' K
.-,
. 6 !- - ! , C
! ,!B .
,
$9
A
!
8!
9
,
!
#' K
1
<
# *
:
6
:
6
;
6
1
#3 K
+!
//
'
:
/
,
?
/
/
+
*' K
1
F
F
;
:
)
+,
8K 9N
D 86 O 9 O & D 8!O)9
:
:
! /
8( &*( K 9
/
+
/
I
H
<
&% % %
/
1
&9
"
F
<F
9
$
&&
"9
H
/
""
/
""
?
"
"
A
/
6 /
/ ""
/ ""
9
'%K
"
3'K
1
/
9
8
"
1
' % *3 % E
<
/
6666669
9
4
9
7
/
/
A
/
/
F8 $
1
A
""
$A &
&A $
&9
F
*
, .
C
)!-
"
!B .
;
,
!
1
:
1
!
A
A/
/
/
1
"
A
/
/ A
!
/
!
+
:
1
/
. - , . )) - B B ! , C
! ,!B .
,
+
/
I
/
"
A
+ /
9
C
1
"
/
8
!
1
"9
.
?
;
"
9 :
"
/
/
"
G
+
:
/
"
: 1
;
9
C
"
+
;"
!
? :
/
1
/
/
1
!
/
!
9
"
1
<
F
F
""
!
/
.
2
C
,
!
/
<
/
< )
/
"
1
/
/
A
""
!
/
""
;
/
/
;
,
/
D
/
"
H
/
/
"
/
8,
+
!
*
F
A ,
&' & $A
7 -
FA
4
9
C
:
) 1
"
/
/ <
/
*
.
!6 6 .
4 !- - , )
<
1
"
A
?
/
/
/
) 1
$9
+
A
+
+
A
1
/
/ <
+
G
/
""
/
&9
) 1
/ <
1
+
!
?
+
8
9 G
"
F"
*
F
*
<
"
$9 C
+
8
/ <
+→' +
/
"
F"
*
F
)
9
8
!9
/
+
)
!
/
=
&9
"
*
+
!
/ <
8
"
9
<
K
7 A
9
!
C
/
)
8F)
/ <
9
*
/
F9
!
"
*
!
+
1
< '+
9
8
+
P
G
/
<
"
HinDIII
5' - NNNAAGCTT
CCATGGNNN - 5'
Amplificazione
con PCR
5' - NNNAAGCTT
3' - NNNTTCGAA
Kpn I
GGTACCNNN - 3'
CCATGGNNN - 5'
6 ,6 ,
! 6 , ,)
! .
,
1
!
;
: 1
<
8
9
"
H
1
1
:
1
,+
/
"
/
"
1
/
""
&K
"
,
!"# $ "% &
' % $
(
)
+
$,"
&
$
'
'
' ' &
$
$
"
(' *
- $ %. # & /*
(
$
"
"#
! )*
/
,
C.
)
C
*
:
1
/
!
! ,+
+
F
G
/
!
!
F
"
/
+
!
,+
"
A
A
)*
/
/
:
!
!
8
9 : "
G
+
!
+
!
!
/
+
!
$9
/
6
+
G
!
!*
!
<
1
A
?
!
08!90
8 )9
!
;
1
:
+
+
H
1
&9
6
.
F
F
1
/
/
"
!
/
G
:
"
/
/
/
?
/
/
:
"
:
"
/
!
F
+
!
*
!
4
F
P
!
!6
9
F
F
D
/
1
/
+
!
"
!
/
!
1
+
! "
:
"
1
8
!
1
;
8 )9 A
:
1
A
9
L ,- !
, , CB .
,
C
! )*
! ' +* ! , 8
C
!
"
!,
;
/
+
9
1
)*
:
'+
!
/
"
A/
F
!8
F
+
>
*
9
?
>
+
1
/
G
4
*
/
!
!
+
P
?
?
' +* ! ,
9
"9
*
9
+
:
!
1
/
1
+
1
G
/
;
1
:
A/
A
8
9
"
'+
!
L ,- !
, , CB .
,
,
! ' +* ! ,
! +* ! ,
!
;
+
/
"
!A
<
!
!A
!
+
+
!
1
! "
+* ! , /
/ 8&K
9
+* ! ,
L ,- !
, , CB .
,
,
! +* ! ,
-. !
8- D
.
*
;
!
!9
1
1
1
: 1
F
F
"
0
H
A
?
1
/
A
!
?
1
!
,
!
?
"
1
;
8
A
" "
9
P
A
1
A
+
/
A +
<
1
"
<
/
-. !
/
8
!
1
/
9
H,
<
;
"
1
:
" "
<
/
/
" "
<
1
;
"
C
/
8
"
A
" "
/
< 9
"
/ <
1
G
/
9
/
8
<
"
1
F
/
F
<
8
"
9
1
A
A
"
-. !
0
/
"
+
<
F- . ! F
:
1
G
" "
/
<
$9 C
1
"
? 1 /
;
"
H
/
)
0 A!
A
A6 A
AL
A
0 A6
)0
"
M
/
A
A!
3
&9
$( " H
< "
9
1
F
F
/
+
9
A
"
"
)
/
8
D
8
9
9 :
!A
P
'9 )
8
<
+9
-
)
AL
A 0 A6
"
:
"
)0
"
A
0 A!
:
"
A6 A
4 !- - , )
"
C
6L
"
/
:
!
$*& = "
+
/
$9
/
!
I
!
/
L
:
"
/
"
/ 1
&
//
"/
"
8
/
<
9
!
/
<
/
1
?
'9 -
!
I
/
H
K - OO9
! ,+
+
,+
1
39
+
!
"
1
) 1
/
/ <
"
"
+
1
+
@
A
+
) 19
8
+
+
/ <
K
+*' +
A
"
"
,D
1
)
8
K ) 1
0
A
0
9
A
!F
G
M,
,
F
:
1
,+
7 @
!
1
F
1
FA
!
A
/
A
A
,+
9
!
"9
9
1
/
!
" "
1
/
<
:
"
!
$% #
<
"
8
#
D $% 9
1
"
" "
1
/
:
H
<
<
!
*
/
8
F
*
=" 1
F9
L ,- !
, , CB .
,
C
!
M,
,*
44, , ) !
!Q *
/
/
!
!
/
A
/
8
1
"
8
9 C
"
1
9
8
$&
9:
0!
P
!
C
A:
$%
"
+
"
/
1
/
/
/
I
/
!
"
1
A
"
!
"
?
A
!
-
1
A
A
A
A
/
A
/
A
"
A
M ! )!6 6
*
,
*
:
/
1
$9
0
/
?
0!O 8% A' *$A% µ 9
&9
"
1
/
/
?
!
/
"
9
A:
1
/
9
1
+
"
/
+
!
/
"
""
. 2 ,-
,
*
!
"
A
+
:
/"
"
'+
$%
$% %
1
$% % "
'%% "
!
/
!
""
?
P
P
>
<
>
/
!
?
1
/
*
/
>
H
?
"
/
"
+:
"
<
""
/
4
/
) 1
C
!
/
-
1
/
/
"
*
/
/
!
G C ! ) )!) M !
?
!
!
/ 9
: 1
8G
/
1
"
;
/
:
A
A
/
P
>
:
$% *&% E
?/
"
<
:A
1
/
A
1
8
/
:
<
"
8
*
9
/
1
9
"
C
"
<
:1
/
?
G
+
:
/
/
"
<
F
+
*
F
1
/
/
P
/
/
1
/
F
"
<
/
F /
/
/
/
G
F
:
@
F
/
+
1
"
/
/
;
9A β*
8
6!
A
!
/
;
: 1
+
<
L
, ,-
.
)*
,- G C ! ) )!) M !
,! ) - ,
G
/
/
/
0
+
?
"
8
/
/
9
+
"
:
+
<
/
8
9
G
?
1
/
/
8
9 C
/
!
1
1
/
/
< 1
+
1
1
1
C
+
1
/
/
1
:
""
"
/
"
<
/
+
?
1
L ,- !
4C
B .
!- , ).
- ((%%
,
),- ! !2
, ,-
$9
,!B .
-
G C ! ) )!) M !
8
9
+
@
/ 8
)
1
86 !
<
L
1
/
&9
9
!
9
-
'9
!
*
/
*
39
(9
9
/
@
@
9
!
!
. .
,
C )!)
"
/
,
Il più basso
possibile
all'interno della
regione di crescita
esponenziale
/
/
R N
/
Q N/
/
1
80 N
/
/
8
D O "9
1
9
/
N
*"
Valore relativo
ad uno standard
endogeno
/
/
/
1
quantizzazione
relativa…
/
, ,-
.
, , )!B .
,
,
C )!)
!
-
!B . , ! ,
C ,!
"
A
! ) !- ), ' +
/
,+
1
/
A
"
1
+
H
1
?
A
/
/
1
/
+
/ < '+
) 1
!
P
+
!
1 A
)
A
""
+
8
F4
F9
<
/
/
+
PA
<
A1
+
/ <
/
<
) 1
< "
+
1
<
8
"
G
9
< "
1
<
/
/
:
"
/
/
8 K 4 !M
+
+
9H
<
"
8
9
G
+
1
+
+
A
+
"
+
4
"
"
<
/
A
"
A
/
1
+
1
<
:
"
H
1
;
'+
"
/
/
9
:
1
) 1
8
A
//
/
1
P+
/
;
!
- ,).
L,
,6
!
,G C , B !- , ).
C ,
,G C , B !- , ).
,6
!
C
1
,
:
;
/
+
/
<
<
A
/
A
/ A
/
A
+
+
1
"
<
A
A
/
/
/ I
+
/
1
P
1
"
!
/
"
/
/
1
- D
*6 "
A1
;
1
1
1
/
/
,G C , B !- , ). L 8- D *6 "
9
.
G
"
1
!
! /
/
< 8
+
/
1
8
9
/
/
1
H
:
A
<
< ' +9A
<
:
"
" A
P
/
8
/
+
"
"
/
"
/
/
1
:
F F9
-
1
8- D
*6 "
9
,G C , B !- , ). , B - !)
8
9
.
1
/
A
!
1
?
/
1
/
/
- D
*6 "
/
1
?
"
/
1
+
8) 1
!
,
A
A =
A )(
7
!
I
<
1
!
9 A
-
1
8
9
,G C , B !- , ).
"
/
. !C ). - !) B B !).
/
/
1
1
:
"
/
"
<
I!
I,
/
"
<
/
1
/
!
/
8
9
/
/
1
"
1
P
/
,
1
/
F
1
F
,
1
"
82 ! )9
!
- ,).
L, ,
. ), ,
! . CB . ,
.-2 ! )
.
CB .
,
. ),
,
.-2
9
8
,+
! )
"
/
/
"
/
/
?
/
9
H
1
+
9
/
1
/
;
+
,
+
1
?
/
'9
/
I
+
+
G
"
"
/
,
,
0
/
A
/
A2
/
+
1
2
I
"
1
A
A
),,
,
,
.
,
,
:
>
/
A
/
1
;
?
:
"
/ <
*
:
8
:
,
/
K
A
9
1
""
!
H
8
$% =
9
/
F
F
/
/
8
K
9
?
:
/
/
1
>
"
"
8
9
?
) /
/
1
!
1
<
/
"
<
8
:
/
+
/ <
9
/
,
/
/
<
<
1
1
1
/
F
Promotore RBS
(Inducibile)
FH
Epitopo
riconosciuto
da Anticorpo
commerciale
Sito di
taglio per
proteasi
ATG Seq. Fusa
(Poly-His)
Origine di
Replicazione
ori
Proteina
TGA Multiple
Cloning
Sequence
Gene per la
Resistenza
all'antibiotico
4
/
/
"
/
G
1
F
;
+
<
F
/
?
1
+
H
;
"
/
!
1
/
"
/
"
H
4
?
?
<A
!
1
4 !6 9
8
K
0
A
)
/
/
:
<
8
9
81
/
9
;
1
:
"
"
"
/
"
<H
:
"
,
<
"
<
A
"
" "
<
!
1
"
F
F
F
F
/
:
1
1
/
""
/
;
"
/
1
/
1
H
<
/
8
1
F
9
/
F
<
"
"
1
8
/
/
/
K 9
$% I&% A% % % D
/
"
//
8C
I
A
6
8
9A
9
?
"
/
/
/
I
+
+
,
+
1
"
/
8 2 9
G
/
:
/
"
/
F
1
F
/
!
/
1
;
""
+
+
"
"
+
/
/
1
+ /
/
1
/
:
/
<
*
"
H
;
""
1
: 1
2! C . M
,+
C
/
2
+
/
/
!
"
1
!
:
0
M
8!
M9 G
"
+
:
?
"
/
F
"
F
8
9
.
//
4 ,B .
+
//
,
1
+
"
/
G
+
/
+
A
!
3
A "
//
+
/
/
/
/
D
"
+
+
+
"
1
/
"
/
/
+
/
! 8 % *&% % = "9 /
"
,
/
/
A1
;
!
/ 8
+
9
/
/
1
/
"
+
+
+
' I3
+
1
1
:
?
% *' % E
?
G
"
/
2
:
/
"
+
//
1
/
2
/
"
/
/
/
9
2
/
*
/
8
<
"
/
/
? //
8
A
A
/
9
2
/
"
! +
//
<
<
P
+
/
1
1
<
/
"
/
/
/
1
/
/
/
2
/
"
M ,)).
.
) ! 4,
2! C . M C
- , ).
,
/
,
$9 .
&9 -
+
9
39
C
(9
C
/
1
9
2
/
8
"
/
M ! )!6 6
9
+
2! C . M
C
/ <
P
/
9
*
'9
/
/
'%E
39
/
/
(9
8
9
9
,+
+
#9
;
"
$% 9
"
:
//
$$9 ,
/
1
,
C ,
- !- - 4 , . . - ,
,
,
. ,
),- !
/
H 1
"
A
A
/
"
8
A
K
9
A
+
"
)
"
!A
!
"
"
"
+
/
+
!
/
,!,*
) !
4 ,B .
) !
, ) ,
) !
4 ,B .
)!2
,
)
"
8
9
/
1
!
"
:
1
"
/
/
""
1
!
/
"
/
1
/
)
/
$*
/
+
"
/
1
"
@
/
"
<
"
H
.
) .
, ,
,
.
) !
, )
/
/
/
/
/
"
/
8
1
/
.
) .
A !)A 6 4 A β*
8
/
/
, ,
,
.
/
@
""
8
"
!9
/
9
/ <
)!2
/
!
9
9 +
+
86 $ A
/
/
/
""
@
"
"
/
:
+
C
/
!
), !B .
. ),
!I
. ),
!
),
. . 6 , , . ),
), !B .
. . 6 , , . )C
.
. ), !I . ), !
1
/
"
,
,
/
$&9
1
/
+
$ 9 C
/
+
+
9
H
1
:
G
?
I
+
+
?
:
"
),-
.
.
2
.
2
G
/
0
/
+
//
/
//
1
*
/
8
/
*
1
F
F9
8!
2
A 2 9
< +
/
A1
/
"
1
/
/
A !
/
/
1
2
8"
9A
+!
"
:
:
!
+
8
A1
09
/
/
/
"
<
!
+
"
"
:
S
1
+!
2
A
A
/
/
F
: //
+
"
"
"
+
+
1
F
4C
B .
!- , ). , M ! )!6 6
/
"
/
/
"
!
"
G
"
/
1
/
/
9
A
,
I 2
"
+!
! 1
A
8
/
!
"
/
/
1
/
+
A
+
/
+
"
+!
2
! 1
+
/
+
2
1
<
<
<
+!
1
8
/
/
+
< 1
9
/
/
,
,
-, )
$9
,M , ) M
:
/
"
I
+
0
/
1
0I!
:
9
8
/
I 2
"
/
/
/
?
8
/
/
/
&9
/
/
9
<
:
1
<
"
/
/
+
/
/
0
"
F
/ F
+
!9 2
85
29 -
2
2
9
/
"
0 8
9
9
+
0"
/
"
F 7
0
F
7
!
! )
. !
,
,
7
*
0
/
?
/
+
H
1
1
//
"
/
+
*
"
8
K
9
+
"
H
"
?
A
A
<
+
"
:
"
/
1
*
"
"
0
0
+
8
"
+
1
*
+
:
8
"
9
"
/
9
/
,
C
"
?
*
!
1
8
A
I
1
F
F
!9
8
9
*
1
2
!
:
8
1
!9
P +
81
G
9 /
1
<
! "
+
+
+
1
+
<
*
1
1
<
!
+
*
1
+
:
8
+
< +
*
?
1
9
+
/
/
"
1
+
"
/
/
@
:
/
/
*
/
/
A
/
P
/ /
+
/
+
,
1
/
*
/
A
+
"
A
"
"
/
:
2
/
+
:
1
:
/
/
+
F
/
FA
+
/
/
1
/
"
+
+
"
/
0
A
+
+
G
/
/
C
1
/
<
/
/
/ :
8
/
%
%9
1
/
8
" "
;
;
+
9
A
/
1
"
1
+
/
/
"
"
"
0 /
I
6. .
2
.
M
1
! G
/
1
P
"
0
!
/
/
!
/
1
+
/
/
/
1
1
!
/
/
G
1
F.
C
,*!
*!*L ! 4 *L Q 2
F
+
!
"
8
9 !
1
1
1
+
81
9
/
"
0
:
"
),
!
, F4 ! 5 , ),
4C . , .
6
* *
F .
6 )
. ),
:
"
G
/
1
+
"
/
/
7
1
8
1
/
<
+
+
I6 )9
"
! 1
"
:
"
/
/
/
"
C
7
"
8
1
6 )9
!
,
*
/ :
*6 )9
? 1
8
/
/
/
+
+
"
/
/
"
! 6 ) 4C
.
. ),
C
>
.5
+
/
/
"
" 9/
8
6 )
"
1
<
/
I
+
/
/
/
"
8"
/
9
""
1
"
!
1
8
9
G
/
A
7
"
, ) 4 !B . ,
), !B .
. ), , ) !- ), . - - C . ,
G
:
4 !
)!B .
,
;
+
/
;
"
//
) /
+
; ""
1
;
+
1
H
?
+
""
/
/
+
/
/
+
/
+
+
! 8
69
/ <
/
"
/
/
8
/
/
"
"
<
+
//
9H
/ A
/
/
/
!
?
F
+
1
F
/
/
F
F
"
?
1
8 1
9
1
,
<
F
?
1
F
"
1
"
!
! F6 ,,
4 C. ,
,+
, )
>
T
! 1
"
0
'%#
/
! 1
/
"
8 :/
/
9
F 86 4 9
+
OO "
&3 A
=
8&
8 (%
! 1
9
?
/
8
4 ,)9 1
L
,+
. ),
9
"
U*"
$$ U*
*
"
:
*3 ' I)0 *3 3 I6 0*3 (
*
9 L
%
/
/
"
#%
"
8
"
:
"
64
C
;
*
64 :
7 0
C
:
1
<
/
! 1
'%#
1
G
/
<
"
/
+
64
/
64
+
8
+
9
/
?
F
D
F
,+
1
/
:
"
/
<
/
F
"
64
?
F
<
/
"
/
/
M
64
M
/
64
1
/
/
"
,
/
/
/
64
"
/
64
!
8 *)
A *)
A
9
M
/
64
8
K
/
/
A
"
H
?
AL M 9
64
A
"
*
64
/
64
/
1
A
/
/
/
"
8
9
/
//
/
/
1
I
64
/ /
+
/
"
H1
8'
8/
"
9
9
/
64
*
!
1
I