Table 1.

Sequences of Triad1 oligonucleotides


Name

Sequence (5′-3′)
AO  GTCGCGAATTCGTCGACGCG (T)15 
AP1  GTCGCGAATTCGTCGACGCG  
AP2  ACTCACTATAGGGCTCGAGCGGC  
T1B  TGCCTGGTTCCAGCTGTCAGA  
T1D  TGCTCTGAGGCTGAGGTGTG  
T1K  GCAGACAGCTATGACAGAGG  
T1O  AAAGGTGAGCCACAGACATG  
T1P  TGCCAGTGTCTCTCAGGAGT  
T1V  TTTGATCCCGAGGAGTACCA  
T1W  GTTCACTTGCTTGACCTACA  
T1X  TGTAGGTCAAGCAAGTGAAC  
T1Y  TGGTACTCCTCGGGATCAAA  
T1Z  TGATCTGCAACTTGGCTGGA  
T1AB  GATGGGATGTGGGAACATGT  
T1exp1  GATCGGATCCATGTCAGTGGACATGAATAGCCAG  
T1exp2  TCGCGAATTCTTAGGTGTCATGGAAATCTTTCAGC  
T1G1  GGATCCTTAGGATGGGATGTGGGAACATGT  
T1G2 GGATCCTACAAGTCCAATTCTGCTCA  
T1M4  TCGCGGATCCTTACAGGTCCTCCTCGCTGATCAGCTTCTGCTCGGTGTCATGGAAATCTTTCAGC  
T1M5  GATCGAATTCATGCTAAGATGTCAGTGG  
T1qF  TGGTCCTTTCTTTTGTGCCAGTA  
T1qR  GCAGCAAGACAGTGTCAGGTTT  
T1qP  CCAAATGTGACCCTGCTCAGAGCTATACCAC  
H158AF  TCTCTGGCCTGTCAGGCCCAGTTTTGCCGCAGC  
H158AR  GCTGCGGCAAAACTGGGCCTGACAGGCCAGAGA  
C161AF  TGTCAGCACCAGTTTGCCCGCAGCTGCTGGGAG  
C161AR
 
CTCCCAGCAGCTGCGGGCAAACTGGTGCTGACA
 

Name

Sequence (5′-3′)
AO  GTCGCGAATTCGTCGACGCG (T)15 
AP1  GTCGCGAATTCGTCGACGCG  
AP2  ACTCACTATAGGGCTCGAGCGGC  
T1B  TGCCTGGTTCCAGCTGTCAGA  
T1D  TGCTCTGAGGCTGAGGTGTG  
T1K  GCAGACAGCTATGACAGAGG  
T1O  AAAGGTGAGCCACAGACATG  
T1P  TGCCAGTGTCTCTCAGGAGT  
T1V  TTTGATCCCGAGGAGTACCA  
T1W  GTTCACTTGCTTGACCTACA  
T1X  TGTAGGTCAAGCAAGTGAAC  
T1Y  TGGTACTCCTCGGGATCAAA  
T1Z  TGATCTGCAACTTGGCTGGA  
T1AB  GATGGGATGTGGGAACATGT  
T1exp1  GATCGGATCCATGTCAGTGGACATGAATAGCCAG  
T1exp2  TCGCGAATTCTTAGGTGTCATGGAAATCTTTCAGC  
T1G1  GGATCCTTAGGATGGGATGTGGGAACATGT  
T1G2 GGATCCTACAAGTCCAATTCTGCTCA  
T1M4  TCGCGGATCCTTACAGGTCCTCCTCGCTGATCAGCTTCTGCTCGGTGTCATGGAAATCTTTCAGC  
T1M5  GATCGAATTCATGCTAAGATGTCAGTGG  
T1qF  TGGTCCTTTCTTTTGTGCCAGTA  
T1qR  GCAGCAAGACAGTGTCAGGTTT  
T1qP  CCAAATGTGACCCTGCTCAGAGCTATACCAC  
H158AF  TCTCTGGCCTGTCAGGCCCAGTTTTGCCGCAGC  
H158AR  GCTGCGGCAAAACTGGGCCTGACAGGCCAGAGA  
C161AF  TGTCAGCACCAGTTTGCCCGCAGCTGCTGGGAG  
C161AR
 
CTCCCAGCAGCTGCGGGCAAACTGGTGCTGACA
 

For amplification of overlapping Triad1 RT-PCR fragments 2, 3, 4 (Figure 1) primer combinations T1V-T1AB, T1Z-T1P, and T1K-T1O were used, respectively. For 3′-RACE primers AO was used for cDNA synthesis followed by PCR using primers AP1 and T1D, followed by heminested PCR using primers AP1 and T1B. For 5′-RACE primers AP2 and T1X were used in PCR followed by heminested PCR using primers AP2 and T1Y. T1M4 and T1M5 were used to for the construction of a C-terminally myc tagged Triad1 PCR construct. The 30-bp myc-tag encoding sequence is underlined. Primers H158AF, H158AR, C161AF, and C161AR were used to construct Triad1 H158A and Triad1 C161, respectively. For generation of GST-Triad1 and GFP-Triad1 primer pairs T1exp1 and T1exp2 were used. For cloning full-length Triad1 in pCDNA3.1 and retroviral vectors primers T1M4 and T1exp2 were used. For cloning Triad1-119 and Triad1109-493 in pGAD vector primer combinations T1exp1-T1G1 and T1G2-T1exp2 were used, respectively. For Triad1 qPCR primers T1qF and T1qR and probe (TET-label) T1qP were used. BamHI and EcoRI sites introduced by PCR for cloning purposes are indicated (bold italics).

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