a.Target site duplication
b. Tol1 left arm
nt. 1-157 of GenBank D42062
c. Multicloning sites
GATCC GAATTC GATATC GGTACC CTGCAG TCTAG
BamHI EcoRI EcoRV KpnI PstI XbaI
d. Tol1 right arm
nt. 1750-1855 of GenBank D42062
e. Tartget site duplication
nt. 391-460 were changed to:
CCAGTGAAT GTCGAC CATGC AAGCTT GGCGTAAT
g. CMV promoter + EGFP + Poly(A) signal
nt. 1-1632 of GenBank U55763
Inserted at the EcoRV site of the muticloning sites.
h. Entire coding sequence for transposase
nt. 31-2817 of GenBank AB264112
i. pCS2 (+), carrying CMV promoter, SP6 promoter, MCS 1, 3' UTR, and MCS2.
j. nt. 31-995 of GenBank AB264112
k. nt. 996-1001 (ATGAAA for methionine and lysine) were changed to two stop codons (TAGTAA).
l. nt. 1002-2817 of GenBank AB26411
<How to recover plasmid DNA>
1. Cut out one of the circles of the paper and immerse it in water or TE in a microfuge tube. Other circles are for backup.
2. Mix by tapping.
3. Centrifuge for 1 minute at >10 krpm.
4. Transform competent bacterial cells (commonly used strains, such as JM109, DH5a and XL1-Blue) with a small amount of supernatant.
5. Spread the bacteria on an LB/agar plate containingl ampicillin, and incubate the plate at 37 oC for >12 hours.
6. Pick up a single colony.
7. Amplify bacteria in liquid media.
8. Extract plsmid DNA by the standard method.