Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Table 1 Primers used for the amplification of either ACLSV FL-cDNA or the different PCR fragments used for ACLSV FL-cDNA cloning by homologous recombination in Saccharomyces cerevisiae

Primer name	Sequence 5'-3'^a	Amplified fragment	Size (kbp)
FLAC5 FLAC3	TAATACGACTCACTATAG TGATACTGATACAGTGTACACTCACG T ₍₃₀₎ GTAGTAAAATATTTAAAAGTCTACAG	T7-FL-cDNA^b	7.5
30ANotvec T7ACR	A₍₃₀₎GCGGCCGCTCTAGCTAGAAGCTTTTGTTCCCTTTAGTG GTATCAGTATCA CTATAGTGAGTCGTATTA AGATCGGACCCTGGCGTAATAGC	Yeast-pBS70T^b	5.2
ACLSVF FLAC3	TGATACTGATACAGTGTACACTCACGTCGTGAG T ₍₃₀₎ GTAGTAAAATATTTAAAAGTCTACAG	FL-cDNA^c	7.5
30ACNOSF AC35SR	TAAATATTTTACTACA(30) CGGGTACCGAGC CGACGTGAGTGTACACTGTATCAGTATCA CCTCTCCAAATGAAATGAACTTCCTTATA	Yeast-pBS70T^c	5.2
ACPCR1F FLAC3	AGAGG TGATACTGATACAGTGTACACTCACG T ₍₃₀₎ GTAGTAAAATATTTAAAAGTCTACAG	FL-cDNA (PCR1)^d	7.5
ACPCR2F PCR2	ACA ₍₃₀₎ GAGCTCGAATTCGCTGAAATCACC TCGAGTCGTATCGGGCTACCTAGCA	Fragment of pBIN61 (PCR2)^d	10.7
PCR3F PCR3R	TGCTAGGTAGCCCGATACGACTCGA GGGGGTGGAGCTTCCCATTGCG ATCAACTCGAGTCGGTCGAAAAAAG	Fragment of Yeast-pBS70T (PCR3)^d	2
PCR4F ACPCR4R	CTTTTTTCGACCGACTCGAGTTGAT GGCGGTCCTGGGGGCTA TGTACACTGTATCAGTATCA CCTCT CCAAATGAAATGAACTT	Fragment of Yeast-pBS70T (PCR4)^d	2
LEV-R AC-F2	CGGCTCGTATGTTGTGTGGA TTTCTACTACGCCTGAAGTGG	Junction between 35S promoter and ACLSV FL-cDNA	0.5

a: The regions of homology introduced in the various primers to allow homologous recombination between fragments are underlined while ACLSV sequences are indicated in bold.
b: The amplified fragments have been used for cloning the ACLSV T7-FL-cDNA in Yeast-pBS70T vector
c: The amplified fragments have been used for cloning ACLSV 70S-FL-cDNA in Yeast-pBS70T shuttle vector
d: The amplified fragments have been used for cloning ACLSV 35S-FL-cDNA in ternary Yeast-E. coli-A. tumefaciens shuttle vector

ISSN: 1743-422X