Methods

The complete open reading frame of CG17415 was amplified using the primers:

5¢GCGCTAGCCTGCCGCCACCATGAGCGACCAGATTGGCAACACC and 5¢GCGAAGCTTTCATACCTTCTCTCCTGGCAG. The first primer incorporated a Kozak sequence (underlined) and a NheI. A 1332 bp fragment was TA cloned into pGEMT-Easy vector (Promega) and double digested (NheI/NotI) and subcloned into the pcDNA5/FRT vector (Invitrogen).

We sequenced this fragment and found that it agrees with Accession number- NM_165979:

MSDQIGNPNATFSGSGSGSGTNVASIAESVAESGPDFDALRAACETRLNASGQLAGSGGPGAEAGTHCAGTFDGWLCWPDTAVGTSAYELCPDFITGFDPARYAHKECGLDGEWFKHPLTNKTWSNYTTCVNLEDLNWRHTVNLISEVGYGTSLLAILLSLAILGYFKSLKCARITLHMNLFASFAANNSLWLVWYLLVMPNSELLHQSPMRCVALHITLHYFLLSNYSWMLCEGFYLHTVLVAAFISEKRLVKWLIAFGWGSPAIVIFVYSMARGLGGTPEDNRHCWMNQTNYQNILMVPVCISMFLNLLFLCNIVRVVLLKLNAPASIQGSCGPSRTVLQAFRATLLLVPLLGLQYILTPFRPAPKHPWENTYEIISAFTASFQGLCVAILFCFCNGEVIAQMKRKWRMMCFSNRPRTNSYTATQVSFVRCGPPLPGEEKV

 

ATGAGCGACCAGATTGGCAACCCCAATGCAACATTCAGTGGCAGTGGCAGTGGCAGTGGCACCAACGTCGCCTCCATCGCCGAAAGTGTGGCGGAGAGCGGACCGGACTTCGATGCACTTCGGGCCGCCTGCGAAACGCGGCTAAATGCCAGCGGTCAACTGGCGGGCTCCGGAGGACCAGGCGCAGAAGCAGGAACCCACTGTGCCGGCACCTTTGATGGCTGGCTTTGTTGGCCGGATACGGCTGTCGGCACTTCCGCCTACGAACTCTGCCCGGACTTCATCACGGGATTCGATCCAGCAAGATATGCCCACAAGGAATGCGGTCTCGATGGCGAGTGGTTCAAGCATCCGCTGACCAATAAAACATGGTCCAACTACACAACCTGCGTAAATCTCGAAGACCTCAACTGGAGGCACACAGTGAATCTGATCTCCGAGGTTGGCTACGGCACCTCACTGCTGGCCATTCTGCTGTCGTTGGCCATATTGGGTTATTTCAAATCCCTGAAGTGCGCCCGCATCACGCTGCACATGAATCTGTTCGCCTCGTTCGCTGCCAATAACTCGTTGTGGCTGGTCTGGTACCTGCTGGTCATGCCGAATTCGGAGCTACTGCATCAGAGTCCGATGCGCTGCGTTGCCCTGCACATAACGCTACACTACTTCCTCCTGTCCAATTACTCCTGGATGCTCTGCGAGGGATTCTATCTGCACACCGTCCTCGTCGCCGCATTCATTTCCGAGAAGAGGCTGGTCAAATGGCTCATCGCATTCGGCTGGGGCTCCCCGGCCATCGTCATATTCGTCTATAGCATGGCTCGCGGTCTGGGCGGCACGCCCGAGGACAATCGTCACTGCTGGATGAACCAAACCAACTACCAAAACATTCTTATGGTGCCTGTGTGTATCTCCATGTTCCTGAACCTCCTGTTCCTGTGCAACATTGTGCGAGTGGTCCTTTTGAAACTGAATGCCCCGGCCAGTATTCAGGGCAGCTGCGGTCCATCGCGAACGGTTTTGCAAGCATTTCGGGCAACGCTGCTTTTGGTTCCTCTGCTCGGCCTCCAGTACATCCTTACGCCCTTTCGTCCTGCACCCAAACATCCCTGGGAGAATACATACGAAATCATCTCGGCGTTTACGGCCTCATTTCAAGGTCTGTGCGTTGCCATTTTGTTCTGCTTTTGCAACGGCGAGGTGATTGCCCAAATGAAGCGCAAGTGGCGGATGATGTGCTTCAGCAACCGACCGCGGACCAATTCCTATACAGCCACTCAGGTCTCGTTCGTGCGCTGCGGACCGCCACTGCCAGGAGAGGAGAAGGTATGA

We designed primers for CG4875 to flank the predicted ORF and that incorporated restriction sites for directional cloning. An amplicon derived from PCR using a Drosophila adult head cDNA library using a Superscript kit (Invitrogen, San Diego, USA) as template was cloned into the pGEM-T Easy vector (Promega, Madison). A 712 bp BamHI/NotI fragment was subcloned into the pcDNA5/FRT vector (Invitrogen). The first primer includes a BamHI site and the Kozak sequence (underlined) upstream of the start codon:

5¢CGGGATCCAGATCTGCCGCCATGCAGATTCTACAGAAGATCAA

5¢CTACCCGAGGTTTTCTCCATCTGG

 

We sequenced this fragment and found that it agrees with Accession number - NM_132947:

MQILQKIKSTKKKFGMRNLATVTYEALQYLEESPCKTQTRENIMNYVKDL

SSYRLKSQEILQMINDPPTSALHTQLLIDDNKAPLTDEENEKIIQLSYKH

FHGGETKGAAKETPAEKPAESTAKAGKQSGVKRNAQAKSNPAEQIAPADK

TTPGDGVTPANVATPSSEATTTTPAEEASPVETSSSENNATPANEATTAP

ATDAPTSMETKSNPTPDGENLG

ATGCAGATTCTACAGAAGATCAAAAGCACCAAAAAGAAGTTCGGCATGCGCAACTTGGCCACGGTTACCTACGAGGCACTGCAGTATCTGGAGGAGTCGCCCTGCAAGACACAAACTCGTGAGAACATTATGAATTACGTTAAAGACCTGTCTTCTTATCGTCTAAAGTCCCAAGAGATCCTGCAAATGATCAACGATCCGCCTACAAGTGCACTACACACGCAACTGCTCATCGACGACAATAAAGCTCCACTAACCGATGAGGAGAACGAGAAAATCATTCAGCTGAGCTACAAACACTTCCATGGTGGAGGAACCAAAGGCGCTGCTAAGGAAACACCGGCGGAAAAGTCTGCGGAGTCCACTGCGAAGGCCGGCAAGCAATCTGGCGTAAAGCGTAATGCCCAGGCAAAATCCAATCCAGCGGAACAAATAGCTCCAGCGGACAAAACCACGCCAGGTGATGGAGTTACTCCTGCGAATGTCGCTACTCCTTCAAGTGAAGCCACTACTACTACTCCAGCAGAAGAAGCAAGCCCAGTAGAAACATCGTCTTCAGAAAATAATGCTACTCCAGCAAATGAAGCCACTACAGCGCCTGCAACGGATGCACCCACTTCAATGGAAACCAAGTCCAATCCGACACCAGATGGAGAAAACCTCGGGTAG