deletions | additions       diff --git a/Methods_knockin_alleles.tex b/Methods_knockin_alleles.tex  index 49282e1..eeb5e9b 100644  --- a/Methods_knockin_alleles.tex  +++ b/Methods_knockin_alleles.tex 
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\subsection*{Generation of knock-in alleles}  5' and 3' homologous arms surrounding the \emph{DmCa\textsubscript{v}3} locus were PCR amplified using \emph{w\textsuperscript{1118}} genomic DNA with the following primers: 5'-CGAGATGAATTCTAGCCTCATCAACTGAGC-3', 5'-CGAGATGCGGCCGCGAGCAAGCACTAATAGCA-3', 5'-GAGATACTAGTCATGCTACAATGTCAGCA-3', 5'-CGAGATCTCGAGGGCCACGTATAGGGATGC-3'.  The homologous arms were then inserted into the \emph{pGX-attP} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1293}{DGRC #1293}). \#1293}).  P\{Donor\} flies were generated by P-element based transgenesis of \emph{pGX-attP} containing the homologous arms into the \emph{w\textsuperscript{1118}} genetic background (Genetic Services, Inc., US) and crossed to Flp I-Sce I flies for homologous recombination.  Candidate for proper targeting (i.e., flies with red or mosaic eyes) were selected and verified by PCR.  The \emph{white} marker was removed from a verified strain via Cre-mediated recombination.  The resulting \emph{white}\textsuperscript{-} line was used as a founder (\emph{DmCa\textsubscript{v}3\textsuperscript{Founder,w-}}) for site-specific DNA integration.  \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}}, \emph{DmCa\textsubscript{v}3\textsuperscript{Rescue}} and \emph{GFP::DmCa\textsubscript{v}3} lines were generated by $\phi$C31 integrase-mediated site-specific integration.   The Gal4 insert (i.e., splice acceptor-Gal4 CDS-poly A) was amplified from the \emph{pBS-KS-attB1-2-GT-SA-GAL4-Hsp70pA} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1325}{DGRC #1325}) \#1325})  with the following primers: 5'-CGTACTCCACGAATTTCTAGAAGTCGATCCAACAT-3' and 5'-ACCGGCGCGCCTCGACTCTAGAACTAGTGGATCTA-3'. The resulting amplified DNA fragment was sequenced and inserted into the \emph{pGE-attB\textsuperscript{GMR}} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1295}{DGRC #1295}) \#1295})  using the EZ-FusionTM cloning kit (Enzynomics, South Korea). The Rescue insert was PCR amplified from \emph{w\textsuperscript{1118}} genomic DNA with the following primers: 5'-GCAGAATTCAATCGATTCCATAGATCCGC-3' and 5'-GCACTCGAGAATTTTGCAACAGGCAGCTA-3'.  The resulting fragment was inserted into the EcoR I/Xho I site of the \emph{pGE-attB\textsuperscript{GMR}} vector.   The GFP insert along with a (Gly-Gly-Ser)x4 linker was amplified from the \emph{pBS-KS-attB1-2-PT-SA-SD-1-EGFP-FIAsH-StrepII-TEV-3xFlag} vector (\href{https://dgrc.bio.indiana.edu/product/View?product=1306}{DGRC #1306}) \#1306})  with the following primers: 5'-GCACCCCAGAAAATGGTGTCCAAGGGCGAGGAGCT-3' and 5'-CGCTGGCTGTGGCAGGGAACCTCCGCTTCCACCGC-3'. The resuling fragment was inserted downstream of the ATG start site in the Rescue construct by inverse PCR (5'-CTGCCACAGCCAGCGGCAGCG-3', 5'-CATTTTCTGGGGTGCCAACTA-3') using the 5X In-Fusion HD Enzyme Premix (Clontech).   \emph{pGE-attB\textsuperscript{GMR}} vectors containing the Gal4, Rescue, and GFP-tagging constructs were injected into \emph{DmCa\textsubscript{v}3\textsuperscript{Founder,w-}} embryos (Rainbow Transgenic Flies, Inc., US) for $\phi$C31-mediated site-specific integration into the \emph{attP} landing site in the DmCa\textsubscript{v}3 locus.  The white-markers of the \emph{DmCa\textsubscript{v}3\textsuperscript{Gal4}} and \emph{DmCa\textsubscript{v}3\textsuperscript{Rescue}} lines were removed for further behavioral analysis.