Part:BBa_K4012006

pTDH3

it is a promotor that starts the sequence that is resposible for the production of Ginkgo biloba chalcone synthase

Sequence and Features

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]

Obtaining the pTDH3 fragment and BsaI digested verification

Figure 1: [1] Results of yeast toolkit plasmids enzyme-digested verification

We construct pGAL1 with vector type8-pSB1K3-GFP and proceed digested verification using BsaI. According to Fig.1, we obtain two clear bands after BsaI digested, the length of the vector(1622bp) and the inserted fragments pTDH3(689bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

pTEF1 in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence AaDFR

The construction schematic of CrCPR sequence demonstrated as Fig.2. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, typr9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.7 The band length 3300bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pTDH in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

pGAL1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.