Part:BBa_K4012005

pRET2

it is a promoter that starts the sequence that is resposible for the production of tyrosine ammonia lyase

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 195
  
• 21
  INCOMPATIBLE WITH RFC[21]

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

  Illegal AgeI site found at 50
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

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

Obtaining the pRET2 fragment and BsaI digested verification

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

We construct pRET2 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 pRET2(709bp) matched with previous expectations by compared with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

pRET2 in Level1 plasmid assembly

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

The construction schematic of RtTAL sequence demonstrated as Fig.2. The initiation of the RtTAL sequence is done by promoter pRET2, with termination done by tSSA1. 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.3B. The band length 3432bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

pRET2 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

pRET2 is also involved in assembly of synthesis pathway of naringenin, shown by Fig.3.