The first step in Gateway cloning is the preparation of a Gateway Entry clone. There are a few different ways to make entry clone. Gateway attB1 and attB2 sequences are added to the 5' and 3' end of a gene fragment, respectively, using gene-specific PCR primers and PCR amplification.

Gateway cloning is a highly efficient alternative to restriction cloning and does not require the use of restriction enzymes. Instead, an insert is moved into a vector through a two-step …

The attB1 site is indicated in bold and the ATG initiation codon for the protein of interest is underlined. Inclusion of the Shine-Dalgarno and Kozak consensus sequence allows protein expression in both E. coli and mammalian cells.

Recombination of attP and attB sites creates attL and attR sites. Gateway vectors contain modified versions of the att sites so that scientists can easily clone in their desired DNA sequences. Gateway technology relies on the two reactions described below:

您可以利用这些载体克隆含有attB序列 (InvitrogenTM pDONRTM 载体) 或特异限制性位点 (InvitrogenTM pENTRTM 载体) 的引物扩增的PCR 产物。 使用PCR 生成入门克隆,两个短的人工合成的attB序列 (attB1 和attB2) 必须插入您的目的基因并添加至用于扩增目的基因的特定引物中。

nucleotide changes within the 25-bp recognition region (Fig. 1C) has been used to engineer different subtypes of att sites that recombine only with each other (i.e., attB1 sites with attP1 sites, but not with attP2 or attP3 sites).

What are the sequences of the attB1 and attB2 sites that I need to add to my PCR product for Gateway® cloning?

Learn how to simulate Gateway® cloning in Geneious Prime, including how to automatically design attB-linked oligonucleotide primers, simulate BP and LR reactions. Geneious Prime provides all of the tools required for primer design and in silico simulation of Gateway® cloning.

Jan 11, 2021 · After second-strand synthesis and end polishing reactions a phosphorylated attB1 adapter is ligated to the 5′ end of the cDNA. The presence of biotin prevents the attB1 adapter from ligating to the 3′ end of the cDNA.

Set whether the insert is recombined in the forward (attB1 → Insert → attB2) or reverse (attB2 → Insert → attB1) orientation. Set the desired target melting temperature (T m) for the new PCR primers.