Part:BBa_K3002215
L2 spectinomycin resistance + L1_connector_Pos2 + ccA_MHETase_SP20HA
This composite part contains a spectinomycin resistance (BBa_K3002102) and the MHETase with the cCA (BBa_K3002114) fused with an SP20HA-tag for easy detection via HA-antibody and enhanced secretion.
The SP20 HA tag in combination with the MHETase and secretion signal cCA leads to glycosylation of the MHETase and enhances the secretion of the enzyme significantly. It was crucial to secrete the MUT-PETase. The glycosylation worked very well but might influence the activity of the MHETase.
The Chlamy Yummy Project Collection
We are proud to present our MoClo part collection for C. reinhardtii - the Chlamy Yummy project collection.
These 67 parts are all parts used during our project and were specifically designed and codon optimized for Chlamydomonas. Among them are basic parts (L0) of a novel mutant of the PETase (BBa_K3002014), the wildtype PETase and MHETase as well as a variety of functional composite parts (L1+2). Containing different tags as well as selection markers, this collection serves as a perfect base for plastic degradation projects with Chlamydomonas. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. Because this is a MoClo collection, the parts are highly standardized for worldwide application. The combination with other part collections works fast and easy. While in MoClo, nomenclature is a bit different from the iGEM BioBricks, it is quickly explained:
Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
Level 1 parts are combinations of basic parts and usually form functional transcription units.
Level 2 parts are combinations of Level 1 parts, in case you want to transfer multiple transcription units at once. For example, you can pair your gene of interest with a selection marker.
The great thing about the Kaiser Collection and MoClo is that the ligation works in a one pot, one step reaction, as the Type IIs restriction enzymes cut out their own recognition sites. This way, multiple constructs can be combined linearly in a fixed order to create complex structures. This is ensured by the standardized overlaps that assign the parts one of 10 positions in the final constructs. After trying MoClo once, you won’t go back to traditional ligation. It is incredibly easy and reliable. Visit our parts site to get an overview over all parts.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3738
Illegal PstI site found at 4062
Illegal PstI site found at 4405
Illegal PstI site found at 5215 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2660
Illegal PstI site found at 3738
Illegal PstI site found at 4062
Illegal PstI site found at 4405
Illegal PstI site found at 5215
Illegal NotI site found at 4073 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4983
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3738
Illegal PstI site found at 4062
Illegal PstI site found at 4405
Illegal PstI site found at 5215 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3738
Illegal PstI site found at 4062
Illegal PstI site found at 4405
Illegal PstI site found at 5215
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 3671
Illegal NgoMIV site found at 4132
Illegal NgoMIV site found at 4162
Illegal NgoMIV site found at 4477 - 1000COMPATIBLE WITH RFC[1000]
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