Part:BBa_K1431301:Design

TRE-3G promoter+SV40 PolyA, an ideal controller of mammalian gene expression with Tet-On 3G protein

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

For BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.

Fig.1 The Machanism of Our Standard

If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

Fig.2 PCR Product Sequence

And the reason why we designed that because we want to make a seamless gather between promoter and protein or any number nucleotides. That depend on which will make the highest efficiency.

Protocol

1. Design the primers show below:
   TRE-3G promoter forward: T TCTAGA G TTTAAACTTTACTCCCTATC
   TRE-3G promoter reverse: ACGCA GGATCC AT GAAGAC GATTTACGAGGGTAGGAAGTG
   SV40 PolyA forward: ACGCG AGATCT AT GAAGAC CCAACTTGTTTATTGCAGCTTA
   SV40 PolyA reverse: AAAACTGCAG CGGCCGC T ACTAGT A TCCATGCCGAGAGTGATGAA
2. Dissolve the primers into 50pmol/μl
3. PCR TRE-3G promoter and SV40 PolyA by the protocol below:
   
   Thermocycling Conditions for a Routine PCR:
   
4. By Gel Purification Kit, purify TRE-3G promoter and SV40 PolyA
5. Using Nanodrop to get each sample’s concentration
6. Digest TRE-3G by XbaI and BamHI, SV40 PolyA by BglII and PstI overnight. The digest protocol shows below:
   
7. Purify TRE-3G promoter and SV40 PolyA again
8. Ligase vector, promoter and PolyA at 16℃ for 45min and inactive at 60℃ for 10min. Ligation protocol shows:
   
9. Transfect the plasmid into DH5α supercompetent cells

Sequencing Results

We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.

Sequence of sequencing primer we used:
VF2: tgccacctgacgtctaagaa
VR: attaccgcctttgagtgagc

The result shows the same sequence with our ideal design.

Source

The plasmid was from our instructor, Huangwei's lab. And we design primers to copy down by PCR.

References

[http://www.clontech.com/US/Products/Inducible_Systems/Tetracycline-Inducible_Expression/Tet-On_3G Clontech]