Difference between revisions of "Part:BBa K4399010"
| Line 14: | Line 14: | ||
===Results=== | ===Results=== | ||
The DNA elements (golden gate compatible, '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''')， '''SbDEL''' ('''BBa_K4399004'''), '''T<sub>nos</sub>''' ('''BBa_K4399015''')) were synthesized by Gensript based on their sequences and sub-cloned into pUC57 (level-0 vectors). | The DNA elements (golden gate compatible, '''P<sub>LexA35S</sub>''' ('''BBa_K3900024''')， '''SbDEL''' ('''BBa_K4399004'''), '''T<sub>nos</sub>''' ('''BBa_K4399015''')) 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: '''pEC47751: P<sub>LexA35S</sub>-SbDEL-T<sub>nos</sub>'''( '''BBa_K4399010'''), according to the protocol: | + | The three level-0 vectors were then used to construct Level-1 vector: '''pEC47751: P<sub>LexA35S</sub>-SbDEL-T<sub>nos</sub>''' ( '''BBa_K4399010'''), according to the protocol: |
{| class="wikitable" style="margin:auto" | {| class="wikitable" style="margin:auto" | ||
Latest revision as of 02:37, 10 October 2022
PLexA35S-SbDEL-Tnos
Induciable expression box of SbDEL(BBa_K4399004).
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1266
Illegal BglII site found at 678
Illegal BamHI site found at 921
Illegal BamHI site found at 990 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1266
Illegal PstI site found at 17 - 1000COMPATIBLE WITH RFC[1000]
Results
The DNA elements (golden gate compatible, PLexA35S (BBa_K3900024)， SbDEL (BBa_K4399004), Tnos (BBa_K4399015)) 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: pEC47751: PLexA35S-SbDEL-Tnos ( BBa_K4399010), 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 ul of the liagations were used to transform E. coli. Positive clines were then confirmed by PCR and sequencing (Fig 1):
