LIC TAL Effector Assembly Kit
(Kit # 1000000023 )

Depositing Lab:   Veit Hornung

This kit provides a LIC (ligation-independent cloning)-ready TALE repeat unit library of 2-mer fragments that can be assembled into an expression ready TALEN construct in 2 hierarchical assembly steps.

This kit consists of one 96-well plate, and will be shipped as bacterial glycerol stocks on dry ice. Samples should be frozen at -80 °C immediately upon arrival.

Original Publication

A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V. Nat Biotechnol 2012 Dec 16. doi: 10.1038/nbt.2460. PubMed PMID 23242165.

Description

Transcription activator-like effector (TALE) proteins derived from Xanthomonas species have emerged as powerful scaffolds for engineering DNA binding proteins of user defined specificity. Their DNA recognition is governed by an array of modular repeat units in which two hypervariable amino acids, known as the repeat variable di-residue (RVD), determine binding to a specific nucleobase. The repeat units detect their target bases in a largely context-independent manner, which allows the generation of TALE proteins of de novo specificity based on the recently deciphered RVD - nucleobase code. Synthetic TALE proteins can be functionalized with a nuclease domain, allowing facile editing of eukaryotic genomic loci.

The Hornung lab has developed a simple high-fidelity ligation-independent cloning (LIC) technique that enables the semi‐automated generation of TALE genes at high throughput merely requiring the combinatorial mixing of fluids. To this effect, they generated a LIC-ready TALE repeat unit library of 2-mer fragments that can be assembled into an expression ready TALEN construct in 2 hierarchical assembly steps. In the first assembly reaction three 2-mer fragments are assembled into a 6-mer fragment, of which three fragments are assembled into an 18-mer in the second reaction. The 18-mer fragment will already be contained in an expression ready backbone with the last half repeat unit and a C-terminal nuclease domain, giving rise to an 18.5 RVD TALEN construct.

Here is a short movie describing the LIC assembly technology:

The whole set of 2-mer fragments and the corresponding backbones are available in a 96-well plate. We recommend generating a pre-digested and pre-chewed fragment library for all subsequent assembly reactions based on the layout of the library plate. The pipetting directions obtained from the assembly calculator tool are based on these coordinates.

Kit Documentation

LIC TAL Kit 1.8 MB

Protocols

LIC TALEN Protocols 132.1 KB

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:
"The plasmid kit used for generation of TALENs was a gift from Veit Hornung (Addgene kit # 1000000023)"

For your Reference section:

A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V. Nat Biotechnol 2012 Dec 16. doi: 10.1038/nbt.2460. PubMed PMID 23242165.

LIC TAL Effector Assembly Kit - #1000000023

Inventory

Well	Plasmid	Resistance
A / 1	TAL-1AA2	Ampicillin
A / 2	TAL-1AC2	Ampicillin
A / 3	TAL-1AG2	Ampicillin
A / 4	TAL-1AT2	Ampicillin
A / 5	TAL-1CA2	Ampicillin
A / 6	TAL-1CC2	Ampicillin
A / 7	TAL-1CG2	Ampicillin
A / 8	TAL-1CT2	Ampicillin
A / 9	TAL-1GA2	Ampicillin
A / 10	TAL-1GC2	Ampicillin
A / 11	TAL-1GG2	Ampicillin
A / 12	TAL-1GT2	Ampicillin
B / 1	TAL-1TA2	Ampicillin
B / 2	TAL-1TC2	Ampicillin
B / 3	TAL-1TG2	Ampicillin
B / 4	TAL-1TT2	Ampicillin
B / 5	TAL-3AA4	Ampicillin
B / 6	TAL-3AC4	Ampicillin
B / 7	TAL-3AG4	Ampicillin
B / 8	TAL-3AT4	Ampicillin
B / 9	TAL-3CA4	Ampicillin
B / 10	TAL-3CC4	Ampicillin
B / 11	TAL-3CG4	Ampicillin
B / 12	TAL-3CT4	Ampicillin
C / 1	TAL-3GA4	Ampicillin
C / 2	TAL-3GC4	Ampicillin
C / 3	TAL-3GG4	Ampicillin
C / 4	TAL-3GT4	Ampicillin
C / 5	TAL-3TA4	Ampicillin
C / 6	TAL-3TC4	Ampicillin
C / 7	TAL-3TG4	Ampicillin
C / 8	TAL-3TT4	Ampicillin
C / 9	TALEN-ID12-A	Ampicillin
C / 10	TALEN-ID12-C	Ampicillin
C / 11	TALEN-ID12-G	Ampicillin
C / 12	TALEN-ID12-T	Ampicillin
D / 1	TAL-2AA3	Ampicillin
D / 2	TAL-2AC3	Ampicillin
D / 3	TAL-2AG3	Ampicillin
D / 4	TAL-2AT3	Ampicillin
D / 5	TAL-2CA3	Ampicillin
D / 6	TAL-2CC3	Ampicillin
D / 7	TAL-2CG3	Ampicillin
D / 8	TAL-2CT3	Ampicillin
D / 9	TAL-2GA3	Ampicillin
D / 10	TAL-2GC3	Ampicillin
D / 11	TAL-2GG3	Ampicillin
D / 12	TAL-2GT3	Ampicillin
E / 1	TAL-2TA3	Ampicillin
E / 2	TAL-2TC3	Ampicillin
E / 3	TAL-2TG3	Ampicillin
E / 4	TAL-2TT3	Ampicillin
E / 5	TAL-4AA1	Ampicillin
E / 6	TAL-4AC1	Ampicillin
E / 7	TAL-4AG1	Ampicillin
E / 8	TAL-4AT1	Ampicillin
E / 9	TAL-4CA1	Ampicillin
E / 10	TAL-4CC1	Ampicillin
E / 11	TAL-4CG1	Ampicillin
E / 12	TAL-4CT1	Ampicillin
F / 1	TAL-4GA1	Ampicillin
F / 2	TAL-4GC1	Ampicillin
F / 3	TAL-4GG1	Ampicillin
F / 4	TAL-4GT1	Ampicillin
F / 5	TAL-4TA1	Ampicillin
F / 6	TAL-4TC1	Ampicillin
F / 7	TAL-4TG1	Ampicillin
F / 8	TAL-4TT1	Ampicillin
F / 9	TAL-4A1	Ampicillin
F / 10	TAL-4C1	Ampicillin
F / 11	TAL-4G1	Ampicillin
F / 12	TAL-4T1	Ampicillin
G / 1	TAL-BBL1-ID14	Kanamycin
G / 2	TAL-BBL1-ID43	Kanamycin
G / 3	TAL-BBL1-ID32	Kanamycin
G / 4	TAL-BBL1-ID21	Kanamycin

Data calculated @ 2024-11-15