Part:BBa_K4399009

PLexA35S-SbMYB75-Thsp18.2

Induciable expression box of SbMYB75 (BBa_K4399003).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 17
  Illegal PstI site found at 1437
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 17
  Illegal PstI site found at 1437
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1371
  Illegal BamHI site found at 805
  Illegal BamHI site found at 1107
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 17
  Illegal PstI site found at 1437
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 17
  Illegal PstI site found at 1437
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

The DNA elements (golden gate compatible, P_LexA35S (BBa_K3900024)， SbMYB75 (BBa_K4399003), T_hsp18.2 (BBa_K4399006)) were synthesized by Gensript based on their sequences and sub-cloned into pUC57 (level-0 vectors). The three level-0 vectors were then used to construct Level-1 vector: pEC47742: P_LexA35S-SbMYB75- T_hsp18.2( BBa_K4399009), according to the protocol:

PCR reaction system of level-1 vector BBa_K4399009 construction

	volume / μL
level-1 empty vector (200 ng/μL)	1.0
promoter	1.5
CDS	1.5
terminator	1.5
NEB T4 buffer	1.5
BSA (10×)	1.5
T4 ligase	0.5
BsaI	0.5
ddH2O	10.0
the whole volume	20.0

The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min， 32 cycles；16℃ 15 min；50℃ 5 min，80℃ 5 min. 10 ul of the liagations were used to transform E. coli. Positive clines were then confirmed by PCR and sequencing (Fig 1):

Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399009. Line 1, negative control; Line 2, positive control; Line 3-6, PCR results of 4 single colonies of BBa_K4399009 using P_LexA35S (BBa_K3900024) forward primer and SbMYB75 (BBa_K4399003) reverse primer; Line 7, marker.