test multiple variable aa components in a protein sequence cloned on an expression vector

create designs for assebling your variants

single protein, single expression vector

we recommend at least 5-7 aa long, if it's shorter you can frame it with invaribale aa to create a stretch

two CSV or excel

1st a single row with name and dna sequence of the invariant expression vector, indictaor linear/circular seq, and other descriptors are optional. If it's circular, make sure that the linear seq you provided ends where the variable aa will be inserted

imagine this is already a seq or part in the inventory (in that case make sure is the exact same info as in your library so the system recognizes is the same)

as many rows as variable aa sequences: name, aa sequence, name assigned, and other optional descriptors (same considerations for the registry)

For the variable region, it is recommended to have a fragment size of at least 5-7 amino acids. If your sequence is shorter, a good practice would be to frame it with a section of the invariant part.

🚨NOTE: Ensure that the backbone sequence is uploaded as a nucleotide sequence and the variable regions are uploaded as amino acid sequences.

Once our input files are set up, let's start building the designs. First, we'll go to the Designs library on the Molecular Biology Toolkit, click on "Import from external DB", and select the "Constant and Variable part files".

A pop-up window will appear, where we can upload our CSV files and select a name for our design. > subir los dos csvs y select name for the design > next > seleccionar > upload

Click on search and then...

if there are any duplicated sequences...

review the design done vemos el vector como constant region and the variable region (veremos que less hizo reverse translation a DNA sequences

**as default it uses a default high abundance human codon map but you can define others by editing the program code

by default it assigns all parts a direct synthesis forced assembly strategy y creates a direct synthesis firewall for each bin

aqui añadir una nota de que si no los teníamos en el inventario los va a crear, pero no AA sequences or parts will be created to avoid overloading the library, si necesitas verlos puedes usar the ove para hacerlo.

automatically selected GGA with BsaI, puedes modificarlo si lo necesitas y antes de submit puedes update the naming scheme

submit for assembly - check the assembly report

y ahora puedes it al lab!!

NODERED

this is only for admins

settings > integrations > replace the codon...