PMC:4307275 / 27530-28609 JSONTXT

{"project":"2_test","denotations":[{"id":"25561231-3554144-50799918","span":{"begin":228,"end":230},"obj":"3554144"},{"id":"25561231-3556162-50799919","span":{"begin":231,"end":233},"obj":"3556162"},{"id":"25561231-2041747-50799920","span":{"begin":234,"end":236},"obj":"2041747"},{"id":"25561231-11076863-50799921","span":{"begin":314,"end":316},"obj":"11076863"},{"id":"25561231-3045756-50799922","span":{"begin":510,"end":512},"obj":"3045756"},{"id":"25561231-18211686-50799923","span":{"begin":794,"end":796},"obj":"18211686"},{"id":"T72055","span":{"begin":228,"end":230},"obj":"3554144"},{"id":"T94156","span":{"begin":231,"end":233},"obj":"3556162"},{"id":"T58759","span":{"begin":234,"end":236},"obj":"2041747"},{"id":"T137","span":{"begin":314,"end":316},"obj":"11076863"},{"id":"T36548","span":{"begin":510,"end":512},"obj":"3045756"},{"id":"T78836","span":{"begin":794,"end":796},"obj":"18211686"}],"text":"4.1. Vector Constructs\nThe DNA fragments of both rRTp and sGFP were amplified from a pSB-RTG plasmid [14]; the sGFP is identical to commercially available eGFP (Clontech, USA). The Kz sequence (AACAATGGC) identified for plants [16,17,18] and the recombination sites (attB1 and attB2) for a gateway cloning system [30] were introduced as a non-homologous overhang in the designed PCR primers, and the chimeric fragments of sGFP, K-sGFP and KR-sGFP with attB1-B2 sites were prepared by an overlap extension-PCR [31]. The PCR primers used in this study are listed in the Table S1. All PCR reactions were performed using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA).\nThe three chimeric gene fragments (sGFP, K-sGFP, and KR-sGFP) were cloned into the pB2GW7 vector [32] containing a 35S promoter for constitutive expression using a gateway cloning system. Gateway cloning procedures were performed using Gateway® BP Clonase® II Enzyme Mix and Gateway® LR Clonase® II Enzyme Mix following the manufacturer’s instructions (Invitrogen, Waltham, MA, USA)."}