Part:BBa_K4012001

pTEF1

A promoter sequence in genome of the Saccharomyces cerevisiae genome, which is used to activate the CHS sequence from Ginkgo biloba in our reconbined plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 714
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3
  Illegal BsaI.rc site found at 724
  

Obtaining the pTEF1 fragment and BsaI digested verification

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

We construct pTEF1 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 pTEF1(709bp) 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 GbCHS

The construction schematic of GbCHS sequence demonstrated as Fig.2. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 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.2 The band length 2500bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pTEF1 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

pTEF1 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.