Part:BBa_K3781304

Scar B4/B5 [GGTTCG], MocloMania

This basic part encodes a scar that acts as a connector between individual B4 and B5 basic parts within the Modular Cloning system as established by Weber et al.

It is important to recognize that this part does not actually correspond to an existing DNA fragment, but is rather a theoretical sequence that is required for the accurate representation of MoClo L1 constructs within the systematic framework of the iGEM Parts Registry. In reality, the connection scars between individual L0 basic parts in a L1 construct result from the nucleotide overhangs that are incorporated into the ends of every L0 part sequence.

For a L1 assembly, all desired basic parts are digested using BsaI. This generates four nucleotide overhangs that are adapted towards the cloning position the respective part is supposed to occupy. At the connection point between individual cloning positions, such as B4 and B5, the overhangs are complementary. This guarantees that all basic parts within a reaction mix will assemble in a pre-determined order.

Because the reading frame has to be cohesive across the entire length of the fusion protein, some of the overhangs require additional base pair extension in order to ensure correct transcription of the assembled genetic unit.

Cloning positions B4 and B5 are connected by the overhang TTCG, meaning that TCG has to be a functional codon within the reading frame, see Figure 1. This scar allows for the insertion of an additional glycine fused to a serine [GGTTCG], eabling the construction of correctly fused L1 constructs within the iGEM Registry framework. In reality, the [GGT] extension is pre-attached to the 3' end of every B4 part, whereas the [TCG] extension is pre-attached to the 5' end of every B5 part.

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  Figure 1 | MoClo Overhangs
  blue boxes | MocloMania cloning positions
  white boxes | respective four nucleotide overhangs
  underlined letters | bases that have to be read as a codon for reading frame maintenance
  italic letters | additional bases added to the overhangs for reading frame maintenance

The MocloMania collection

This L1 construct was assembled using basic parts from the MocloMania collection, the very first collection of genetic parts specifically designed and optimized for Modular Cloning assembly and recombinant protein expression in the protozoan parasite Leishmania tarentolae.

Are you trying to express complexly glycosylated proteins? Large antibody side chains? Human proteins that require accurate post-translational modification? Then Leishmania might be just the right organism for you! Leishmania tarentolae’s glycosylation patterns resemble those of human cells more closely than any other microbial expression host, while still delivering all the benefits of microbial production systems like easy transfection and cultivation. So instead of relying on mammalian cell lines, try considering Leishmania as your new expression host of choice!

Our MocloMania collection will allow you to easily modify your protein of choice and make it suitable for downstream detection and purification procedures - all thanks to the help of Modular Cloning. This cloning system was first established by Weber et al. in 2011 and relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequence, hereby generating four nucleotide overhangs. Every basic part in our collection is equipped with a specified set of overhangs that assign it to its designated position within the reading frame. These so-called cloning positions are labelled B2-B5 from upstream to downstream. By filling all positions with the basic parts of your choice, you can easily generate variable genetic constructs that code for the fusion protein of your desire.

We furthermore provide a specifically domesticated Leishmania expression vector, named weird_plex, which will package your fusion construct into a functional transcriptional unit that is optimized for high expression in Leishmania.

The best part? Because of the type IIS restriction properties and the specifity of the generated overhangs, restriction and ligation of your construct can all happen simultaneously in a simple one-step, one-pot reaction. This will safe you a lot of time and frustration in your cloning endeavours!

Do we have your attention? In the table below you can find some basic information on how our cloning system, along with most other MoClo systems, is set up. Please feel free to check out our wiki to find more information on Leishmania and Modular Cloning as well as to understand how the part that you are looking at integrates into our part collection. See you there!

MOCLO | Important nomenclature and parameters

Level	What does this level contain?	antibiotic resistance	Enzyme used for ligation
L0	The foundation to every MoClo construct which are basic genetic units, such as coding sequences, promoters, terminators	spectinomycin	BbsI
L1	Several L0 parts assembled into a functional transcriptional unit, e.g. consisting of promoter, coding region and terminator	ampicillin	BsaI
L2	Multiple transcriptional units added into one multi-gene construct, e.g. a protein of interest fused to a selection marker	kanamycin	BbsI

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]