We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. Both antibiotic resistance genes are efficiently...

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy ...

Double-stranded RNA (dsRNA) can induce post-transcriptional gene silencing in a wide variety of organisms. Commonly, inverted repeats are used to produce dsRNA to silence genes of interest. However, cloning inverted repeats still remains a rate-limiting step for widely applying this technique. Here we describe a pGEM-T-based vector, pGEM-WIZ, designed to produce inverted repeats for any Drosoph...

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multi...

background and objectives: during past decades hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer. materials and methods: the corresponding nucleotide sequences for the expression of heterologous genes in hansenula poylmorpha were extracted and as...

Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. ...

Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the C...

Here we report the use of exonuclease to expose complementary DNA between an insert and vector such that annealing becomes independent of restriction site compatibility. We demonstrate that unusual and, in some cases, previously impossible cloning strategies can be readily and efficiently achieved as long as the flanking sequences of the linear vectors are highly related. Furthermore, we show t...

Background and aims: Laron syndrome is a disease that treated by Insulin-like Growth Factor 1 (IGF-1). This protein is a single chain and has three disulfide bonds. People with Laron syndrome have low rates of cancer and diabetes, although they appear to be at increased risk of casual death due to their stature. IGF-1 is synthesized by many tissues and is secreted from liver as an endocrine hor...

Bluetongue (BT) is a viral disease causing morbidity and mortality in sheep, cattle and wild ruminants, including deer, sambar and bluebull. In this study we present the internal gene of outer coat protein, VP2 of bluetongue virus serotype 1 of Indian isolate was amplified from cDNA synthesized through RT-PCR using total RNA with specific forward and reverse primers. The PCR product was cloned ...