AttII

STEP 1.

STEP 2.
pGEM-T backbone vector 2219bp
AatII/NotI digest
isolate 2958bp 2958bp
5177bp fragment

NotI

NotI AatII
AatII

SfiI

Trasgenic Fragment 1 vector 503bp

AatII/NotI digest TGF1 + backbone
2220bp Ligate
isolate 2960bp
5683bp fragment 2960bp 5904bp

AatII
NotI
SfiI STEP 3.

Digest with SfiI

CIP

Trasgenic Fragment 2 vector 2252bp Ligate

SfiI digest
isolate 2252bp 2745bp AatII
4997bp fragment

SfiI

SfiI

EcoRI EcoRI
TGF1 +TG2 + backbone

8143bp

41bp
Trasgenic Fragment 3 vector EcoRI/NotI digest
1985bp
isolate 1985bp MluI
fragment 2689bp
4715bp

NotI
SfiI
NotI
STEP 4.

Digest with Isolate

EcoRI
EcoRI MluI and NotI 8106bp
fragment

37bp
38bp
Trasgenic Fragment 4 vector EcoRI/MluI digest 8106bp
2237bp
isolate 2237bp
4953bp fragment 2680bp
STEP 5.

MluI
Ligate
AatII
Final Product I

EcoRI SfiI SfiI

54bp
Trasgenic Fragment 5 vector EcoRI/SfiI digest
2685bp
isolate 2846bp
5585bp fragment 2846bp

MluI
pDestXtrans R1-R2

12308bp
EcoRI
SfiI

STEP 7.

Final Product II Ligate

EcoRI NotI
EcoRI

STEP 6.
Digest with
EcoRI and SfiI Isolate
6225bp
fragment

MluI 2252bp
pDestXtrans R4-R3
3857bp

9071bp 6225bp

SfiI

AatII