Table 1

Primers used for vector construction.

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

TTTTTT

    GCGGCCGC
TTAGC

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

AACCTA

    CCTAGG
ACT
    TTA

    ATTAA
TGTTATTTAATTCG

TCAGTG

GCTT

    TTAATTAA
GTTCAA

CTATTTCACTATCGATTGT

TATT

GTCT

    TTAATTAA
CCAAGC

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

TTGTCA

    TTAATTAA
GCTA

GCAAAGGAGAAGAAC

TTTACT

    CCTAGG
TTATTTG

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

ATATGA

    GGGCCC
AACTGA

AGGCGGGAAACGACAATC

GACCACTTTATGGAGGTT

    CGTACG
TCTAGGGGATCC

GGTGCAG

AACCTCCATAAAGTGGTC

    ACTAGT
ATCGTTCAAACA

TTTGGC

ATTATG

    CCTGCAGG
AGCT

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

TAATAA

    CGTACG
ATGGAAC

GAGCTATACAAG

TTTTTT

    ACTAGT
TTACTCG

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.


Liu and Kearney BMC Biotechnology 2010 10:88   doi:10.1186/1472-6750-10-88

Open Data