Table 2. Assessment of various sequences used as the junction between the GOI and the selectable marker.

Rowa	Plasmidb	Type of Junctionc	Description or Sequence of Junction	No. of Transformants/µg DNAd	No. of Venus-Positive Clonese
1	pMO448	N/A	Control (no 5′ ORF as GOI)	∼2500	N/A
2	pMO518	Promoter-containing	Control (conventional two-promoter construct; see Figure 1A)	∼400f	∼1/16f
3	pMO455	IRES	EMCV (porcine encephalomyocarditis virus) IRES (586 bp)	39	0/16
4	pMO467	IRES	crTMV (crucifer-infecting tobamovirus) IRES-CP (148 bp)	13	1/16
5	pMO463	IRES	PVY (potato virus Y) long (91 bp)	35	0/16
6	pMO464	IRES	PVY short (66 bp)	21	0/16
7	pMO466	IRES	PFBV (Pelargonium flower break virus) (78 bp)	21	1/16
8	pMO473	IRES	PLRV (potato leafroll virus)g	64	10/16
9	pMO474	IRES	PLRVmut11h	73	8/16
10	pMO449	Non-IRES	TAGCATi	327	15/16
11	pMO471	Non-IRES	TAGCCATi	359	14/16
12	pMO470	Non-IRES	TGATAGCCATi	452	13/16

^aUsing CrVENUS-3FLAG as a sample GOI and APHVIII (conferring resistance to paromomycin) as the selectable marker.

^bFor additional details, see Table 1 and/or Materials and Methods.

^cWith or without an IRES.

^dExcept 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.

^eIn each case, 16 transformants were examined by fluorescence microscopy for expression of the Venus protein.

^fpMO518 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.

^gTAA 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.

^hTAA 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).

ⁱNote (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.