Table 2
Oligonucleotides for the amplification of the dual promoter region, the plasmid backbone, and the two target cDNAs and their cloning
Region to amplify
Primer name
Primer sequence
Native constructs (pFastBac Dual, pKL. or pAC8_MF)
Dual promoter module
Prom-F (PH)
GGTGGATCCGCGCCCGATGG
Prom-R (p10)
GGTGGCTCGAGATCCCGGGTG
Plasmid backbone
Backbone-R (PH)
TCTAGAGCCTGCAGTCTCG
Backbone-F (p10)
GCTAGCAGCTGATGCATAG
Gene under pH promoter
pH-Gene-START-F
CATCGGGCGCGGATCCACC/ATG.
. .first 15–21 nt of the gene (BamHI+Kozak)
pH-Gene-STOP-R
CGAGACTGCAGGCTCTAGA/STOP.
. .RC(last 15–21 nt of the gene) (XbaI)
Gene under p10 promoter
p10-Gene-START-F
CACCCGGGATCTCGAGCCACC/ATG.
. .first 15–21 nt of the gene (XhoI+Kozak)
p10-Gene-STOP-R
CTATGCATCAGCTGCTAGC/STOP.
. .RC(last 15–21 nt of the gene) (NheI)
C-terminal histidine-tagged construct (pKL-PH—3C-10His-Cter)
Plasmid backbone
Backbone-3C-R (PH)
TCTAGACTGGAAGTTCTGTTTC
Gene under pH
pH-Gene-10His-3C R
GAAACAGAACTTCCAGTCTAGA.
. .RC(last 15–21 nt of the gene) (XbaI)
The NheI, XhoI, BamHI, and XbaI restriction sites are underlined. Note that oligonucleotides Prom-F and Prom-R contain a kozak consensus sequence (CCACC in bold).
pKL-PH-3C-10His-Cter is a pKL derivative designed to co-express a cleavable C-terminally histidine-tagged protein under the control of the PH promoter with an untagged
protein under the control of p10. For this vector, the gene under PH promoter is amplified using the pH-Gene-START-F/pH-Gene-10His-3C-R primer pair and the plasmid
backbone with the Backbone-3C-R (PH)/Backbone-F (p10) pair. The gene under p10 promoter is amplified with the p10-Gene-START-F/p10-Gene-STOP-R pair. RC stands for
reverse and complement (see Figs. 4 and 5)
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Paola Rossolillo et al.
Oligonucleotides for the amplification of the dual promoter region, the plasmid backbone, and the two target cDNAs and their cloning
Region to amplify
Primer name
Primer sequence
Native constructs (pFastBac Dual, pKL. or pAC8_MF)
Dual promoter module
Prom-F (PH)
GGTGGATCCGCGCCCGATGG
Prom-R (p10)
GGTGGCTCGAGATCCCGGGTG
Plasmid backbone
Backbone-R (PH)
TCTAGAGCCTGCAGTCTCG
Backbone-F (p10)
GCTAGCAGCTGATGCATAG
Gene under pH promoter
pH-Gene-START-F
CATCGGGCGCGGATCCACC/ATG.
. .first 15–21 nt of the gene (BamHI+Kozak)
pH-Gene-STOP-R
CGAGACTGCAGGCTCTAGA/STOP.
. .RC(last 15–21 nt of the gene) (XbaI)
Gene under p10 promoter
p10-Gene-START-F
CACCCGGGATCTCGAGCCACC/ATG.
. .first 15–21 nt of the gene (XhoI+Kozak)
p10-Gene-STOP-R
CTATGCATCAGCTGCTAGC/STOP.
. .RC(last 15–21 nt of the gene) (NheI)
C-terminal histidine-tagged construct (pKL-PH—3C-10His-Cter)
Plasmid backbone
Backbone-3C-R (PH)
TCTAGACTGGAAGTTCTGTTTC
Gene under pH
pH-Gene-10His-3C R
GAAACAGAACTTCCAGTCTAGA.
. .RC(last 15–21 nt of the gene) (XbaI)
The NheI, XhoI, BamHI, and XbaI restriction sites are underlined. Note that oligonucleotides Prom-F and Prom-R contain a kozak consensus sequence (CCACC in bold).
pKL-PH-3C-10His-Cter is a pKL derivative designed to co-express a cleavable C-terminally histidine-tagged protein under the control of the PH promoter with an untagged
protein under the control of p10. For this vector, the gene under PH promoter is amplified using the pH-Gene-START-F/pH-Gene-10His-3C-R primer pair and the plasmid
backbone with the Backbone-3C-R (PH)/Backbone-F (p10) pair. The gene under p10 promoter is amplified with the p10-Gene-START-F/p10-Gene-STOP-R pair. RC stands for
reverse and complement (see Figs. 4 and 5)
22
Paola Rossolillo et al.