Part:BBa_J100419
rClone mScarlet
This is the same sequence as the original rClone Red plasmid, but this plasmid has the mScarlet fluorescent protein inserted in place of the RFP. rClone Red: https://parts.igem.org/Part:BBa_J119384 mScarlet: https://parts.igem.org/Part:BBa_J100398
rClone mScarlet (see Eckdahl et al. 2017) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter and GFP with the new RBS or riboswitch. Transcription will be initiated in the direction of the mScarlet coding sequence. The level of expression of mScarlet will depend on the efficiency of the newly cloned RBS or riboswitch.
When designing oligonucleotides for use with rClone Red, make sure they result in 5' overhang sticky ends that are CGAC (left) and GCGG (right). Also make sure the oligonucleotides do not contain binding sites for BsaI. Finally, make sure the RBS element ends immediately before the GCGG right sticky end. This will ensure a spacing of 6 bases between the RBS and the ATG start codon of RFP. Below is an example.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 923
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 923
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 923
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 923
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 923
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 859
Illegal BsaI.rc site found at 52
| None |