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Table 1 Primers used for vector construction.

From: An efficient Foxtail mosaic virus vector system with reduced environmental risk

Plasmid Primer Oligonucleotide sequence (5'-3') Purpose
pFoMV/
JL22
FoMV 5' term
UP
(pFoMV nt.
1-21)
FoMV756
NotI
DOWN
(pFoMV nt.
737-757)
P-GAAAACTCTTCCGAA
ACCGAA
TTTTTTGCGGCCGCTTAGC
CAGTTTAGGTCCTTA
The 5' end of FoMV was amplified by PCR with primers FoMV5'termUP and FoMV756NotDown and cut with PmlI. The 3' end of FoMV was digested with PmlI and XbaI. Both 5' and 3' end fragments of FoMV were cloned into the JL22 backbone cut with StuI and XbaI.
pFECT0
pFECT22
pFECT40
FoMV Up
(pFoMV nt
3044-3063)
FoMV+0sgp
Down
(pFoMV nts.
4114-4131)
FoMV+22sgp
Down
(pFoMV nts.
4124-4153)
FoMV+40sgp
Down
(pFoMV nts.
4150-4169)
GTGGGCATGTGCAGATGA
GG
AACCTACCTAGGACTTTA
ATTAATGTTATTTAATTCG
TCAGTG
GCTTTTAATTAAGTTCAA
CTATTTCACTATCGATTGT
TATT
GTCTTTAATTAACCAAGC
TTTGTTAGTCGTTC
To create ΔTGB/ΔCP mutants, PacI and AvrII cloning sites were introduced by PCR amplified with two primers (FoMVUp and FoMV+0sgp Down). PCR with mutated start codon of TGB was cut with BamHI and AvrII and cloned into pFoMV vector backbone to create pFECT0. Other two downstream primers (with PacI site) were used to save 22nts and 40nts 5' end of TGB DNA sequence. PCR fragments were cloned in pFECT0 vector backbone cut with BamHI and PacI to generate pFECT22 and pFECT40.
pFECT0/
GFP
pFECT22/
GFP
pFECT40/
GFP
PacGFPUp
GFPAvrDown
TTGTCATTAATTAAGCTA
GCAAAGGAGAAGAAC
TTTACTCCTAGGTTATTTG
TAGAGCTCATCCA
To clone the GFP ORF into the pFECT vector. Primer PacGFPUp adds a PacI site (underline) at the 5' end, and primer GFPAvrDown adds an AvrII site (underline) to the 3' end.
pFECT40/
GFP/
PnosTnos
ApaI Pnos
UP
PnosBsiWI-
overlapDN
TnosSpeI-
overlapUP
SbfI Tnos DN
ATATGAGGGCCCAACTGA
AGGCGGGAAACGACAATC
GACCACTTTATGGAGGTT
CGTACGTCTAGGGGATCC
GGTGCAG
AACCTCCATAAAGTGGTC
ACTAGTATCGTTCAAACA
TTTGGC
ATTATGCCTGCAGGAGCT
GGCATGCAAGCTGTCGAGG
To add PnosTnos in pFECT40, and create BsiWI and SpeI in between Pnos and Tnos. Inner primers PnosBsi-overlapDN and TnosSpe-overlapUP have overlap sequence and BsiWI and SpeI sites. Two inner primers pair with outer primers ApaPnosUP (ApaI at 5' end) and SbfTnosDN (SbfI at 3' end) to generate two PCR products. The two products were fused using outer primers and cloned into pFECT/GFP.
pFECT40/
GFP/p19
BsiWI/p19
UP
p19SpeI
DOWN
TAATAACGTACGATGGAAC
GAGCTATACAAG
TTTTTTACTAGTTTACTCG
CTTTCTTTTTCGAAGG
To clone the p19 ORF into pFECT40/GFP/PnosTnos vector. Primer Bsip19UP adds a BsiWI site (underline) at the 5' end and primer p19SpeDown adds a SpeI (underline) site at the 3' end of the ORF. The amplified DNA fragment was cloned into pFECT40/GFP/PnosTnos vector backbone cut with BsiWI and SpeI.