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A spin-off built around those patents, Roslin Biomed, was soon sold to the US-based Geron, which brought cloning together with stem cell research.
This offers a rapid pipeline for more streamlined cloning together with a flexible and trackable reporter system.
The identification of these RNAs by cloning, together with the existence of corresponding precursor sequences, suggests that these sRNAs are, in fact, miRNAs from Physcomitrella.
Map-based cloning, together with transgenic complementation and RNA-interference tests, revealed that TBP1 is a member of the somatic embryogenesis receptor kinases (SERKs) family involved in BR signaling.
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An XhoI-3xFlag-SBP-Venus-15-alanine-glycine-linker-HindIII fragment was amplified from synthetic DNA and cloned together with both homology arms into the NotI sites of pAAV-MCS (Stratagene).
Myc-CcaIncA1 220 was cloned together with GFP into an IRES vector to simultaneously express a transfection marker (40% average transfection efficiency).
These constructs were cloned together with LdTopIΔS deletion in the biscistronic pESC -URA vector, expressed and purified from EKY3 transformed yeast according to the above-mentioned method.
A neomycin resistance cassette (Neo) driven by the PGK promoter (in 3' to 5' direction) flanked by loxP sequences was cloned together with a short arm into SpeI/XbaI-digested pBluescript vector (pBS) (short arm-loxP-Neo-loxP).
The pAntryp1-luc fragment was cloned, together with an Actin5C-EGFP marker cassette, within the arms of the minos transposable element, to generate transformation vector pMinLuc [21](Figure 1a).
This 420-bp 5'-fragment was cut with BamHI/SacI at the ends and cloned, together with a 4.2-kb SacI/SpeI Xa21 3'-fragment, into the pBluescript II SK- vector to create a promoterless full-length Myc-Xa21 gene.
The ORF of POM152 also was amplified and cloned together with the GAL1 /10 promoter into pRS305 to create pRS305- GAL1 - POM152 (pSJ1655).
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