Part:BBa_K4399013
The negative control for anthocyanin biosynthesis
It is used as a negative control for inducible anthocyanin biosynthesis study, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box and an inducible SbDEL expression box. However, as there is not a XVE box, the activator for PLexA35S, so neither SbMYB75 nor SbDEL can be expressed.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal NheI site found at 1244
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal BglII site found at 4113
Illegal BglII site found at 4925
Illegal BamHI site found at 3547
Illegal BamHI site found at 3849
Illegal BamHI site found at 5168
Illegal BamHI site found at 5237 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 1000COMPATIBLE WITH RFC[1000]
Results
Construction of level-0 vectors
The DNA elements (golden gate compatible, promoters: PAtUBI5, PLexA35S, P2×35S; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: Tmas, Thsp18.2, Tnos, T35S) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57.
Construction of level-1 vectors
The level-0 vectors were then used to construct Level-1 vectors: pEC47732: PAtUBI5-GFP-Tmas, pEC47742: PLexA35S-SbMYB75- Thsp18.2, pEC47751: PLexA35S-SbDEL-Tnos, pEC47761: P2×35S-XVE-T35S, according to the protocol:
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 μl of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing.
Construction of level-2 vectors
Level-2 vectors were constructed based on level-1 vectors:
IA | NC | |
---|---|---|
volume / μL | volume / μL | |
level-2 empty vector (200 ng/μL) | 1.0 | 1.0 |
L1-P1 | 1.5 | 1.5 |
L1-P2 | 1.5 | 1.5 |
L1-P3 | 1.5 | 1.5 |
L1-P4 | 1.5 | - |
ELE-X | 1.5 | 1.5 |
NEB T4 buffer | 1.5 | 1.5 |
BSA (10×) | 1.5 | 1.5 |
T4 ligase (NEB) | 0.5 | 0.5 |
BsaI | 0.5 | 0.5 |
ddH2O | 7.5 | 9 |
the whole volume | 20.0 | 10.0 |
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 confirmed by sequencing and PCR.
None |