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. |