Table 2 Assessment of various sequences used as the junction between the GOI and the selectable marker
RowaPlasmidbType of JunctioncDescription or Sequence of JunctionNo. of Transformants/µg DNAdNo. of Venus-Positive Clonese
1pMO448N/AControl (no 5′ ORF as GOI)∼2500N/A
2pMO518Promoter-containingControl (conventional two-promoter construct; see Figure 1A)∼400f∼1/16f
3pMO455IRESEMCV (porcine encephalomyocarditis virus) IRES (586 bp)390/16
4pMO467IREScrTMV (crucifer-infecting tobamovirus) IRES-CP (148 bp)131/16
5pMO463IRESPVY (potato virus Y) long (91 bp)350/16
6pMO464IRESPVY short (66 bp)210/16
7pMO466IRESPFBV (Pelargonium flower break virus) (78 bp)211/16
8pMO473IRESPLRV (potato leafroll virus)g6410/16
9pMO474IRESPLRVmut11h738/16
10pMO449Non-IRESTAGCATi32715/16
11pMO471Non-IRESTAGCCATi35914/16
12pMO470Non-IRESTGATAGCCATi45213/16
  • a Using CrVENUS-3FLAG as a sample GOI and APHVIII (conferring resistance to paromomycin) as the selectable marker.

  • b For additional details, see Table 1 and/or Materials and Methods.

  • c With or without an IRES.

  • d Except for pMO518 (note f), the numbers of total and Venus-positive transformants shown here were taken from a single experiment that included all constructs; it used strain CMJ030 and the Bio-Rad electroporator with cells in in TAP medium plus 40 mM sucrose, followed by selection on TAP agar containing 20 µg/ml paromomycin. The numbers shown are consistent with those from multiple other experiments using strain CMJ030 or CC-124 with various subsets of the constructs.

  • e In each case, 16 transformants were examined by fluorescence microscopy for expression of the Venus protein.

  • f pMO518 was not included in the particular experiment that yielded the other data shown in this table. Thus, the numbers indicated are estimates based on several other experiments in which transformant numbers and numbers of GOI-positive transformants could be compared directly among pMO518, pMO448, and/or pMO449.

  • g TAA C ATG ATT ATG ACT CCG ATG AGG ATT ACG GTC TGG AGA GAG AGG CTG CAA CAA ATG ATG. Additional potential start codons, in frame with that of APHVIII itself, are in bold face; the CrVENUS-3FLAG stop codon and the APHVIII start codon are indicated by italics.

  • h TAA C ATG ATT ATG ACT CCG ATG AGG ATT ACG GTC TCC TCT CTC TCC CTG CAA CAA ATG ATG. Additional start codons, in frame with that of APHVIII itself, are in bold face; the CrVENUS-3FLAG stop codon and the APHVIII start codon are indicated by italics. The sequence altered by mut11 is underlined (Jaag et al. 2003).

  • i Note (1) that the linkers contain one (pMO449 and pMO471) or two (pMO470) additional stop codons in frame with that of Cr-VENUS-3FLAG itself, and (2) that the APHVIII start codon is in frame with the stop codons in pMO449 but out of frame in pMO471 and pMO470.