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\subsection*{Cloning and subcloning of the fly T-type Ca\textsuperscript{2+} channel (DmCa\textsubscript{v}3)} DmCa\textsubscript{v}3}  Full length cDNA clone of We generated a full-length  DmCa\textsubscript{v}3 (CG15899) was generated cDNA  by piecemeal  PCR amplification and subcloning. amplification.  Total RNAwere  extracted from adult heads using Trizol reagents (Invitrogen) and was  reverse transcribedinto cDNA  using RevertAid First Strand cDNA Synthesis Kit (Fermentas). Six adjacent DNA fragments(clone 1 – clone 6)  that cover full-length the entire  DmCa\textsubscript{v}3 CDS cDNA  were obtained by PCR amplification using cDNA as a template. amplification.  Primer sets were designed based on the  FlyBasegene  annotation. \textcolor{red}{Which version of FlyBase? FlyBase changes over time.}  Hind III and Xba I sites  were inserted at the  5' end  of clone 1 and 3' end  of clone 6, respectively. Primer sets used : sets:  clone 1 (5'-CGAGATAAGCTTAAAATGCTGCCACAGCCA-3' and (5'-CGAGATAAGCTTAAAATGCTGCCACAGCCA-3',  5'-GCATCAGACTACATCGCTGTC-3'), clone 2 (5'-CTGGACACGCTGCCCATGCTG-3' and (5'-CTGGACACGCTGCCCATGCTG-3',  5'-TTCCAGCTCCTCCACTTGCAC-3'), clone 3 (5'-CAACGGTGGCTCCAACAGTCG-3' and (5'-CAACGGTGGCTCCAACAGTCG-3',  5'-CCACTGGCGGAAGCTCATGCC-3'), clone 4 (5'-GCCACGCCTCTCCAAGATCCG-3' and (5'-GCCACGCCTCTCCAAGATCCG-3',  5'-GACGATAAGAGCGTTTGCACG-3'), clone 5 (5'-TCTGAAACTAGTCGTGCAAAC-3' and 5'-TGGAAGTACTGGACGGTCTGC-3') (5'-TCTGAAACTAGTCGTGCAAAC-3', 5'-TGGAAGTACTGGACGGTCTGC-3'),  and clone 6 (5'-AATCCCAGCCTGACCAGCTCG-3' and (5'-AATCCCAGCCTGACCAGCTCG-3',  5'-TCTAGATTAGTCCATGGAGGATTGGGGTGA-3'). Amplified PCR fragments were sequenced and assembled into pBlueScript II KS (+) vector by using sequential  restriction enzyme digestion sequentially.  Among various isoform-specific fragments of clone digests.  Clones  2 and clone 3, RC-specific fragments 3 contained isoform-specific exons.  Of the combinations that  were used. amplified by PCR, we chose to proceed to assembling the RB and RC isoforms.  Frequent A to G RNA editing sites were found identified  in clone clones  3 and 5. One RNA editing site in clone 3(5'-AGTTCAGAGC-3') 3 (5'-AGTTCAGAGC-3')  was reversed to A by site-directed mutagenesis based on genomic sequence.  Due to several RNA editing mutagenesis.  Since there were too many edited  sites in clone 5, we reverted to using  genomic DNA was used a  as template for clone 5 instead of cDNA. Assembled full length \textcolor{red}{Why did you do this? If it came from  cDNA and you found the same edits several times, then what makes you think it wasn't supposed to be there? Maybe this  was why your UAS rescue didn't work?}  The final assembled full-length cDNAs were  cut out of the vector by digestion of with  HindIII and /  XbaI andthen  subcloned into pcDNA3-HE3 where 5' untranslated region downstream  of the 5'-UTR from the  \emph{Xenopus laevis} $\beta$-globin was included for better gene to improve  expression in \emph{Xenopus} oocytes.