## 2 Materials and Methods
### Isolation and DNA Sequence Analysis of CLCuKoV-Bu LIR Region  
The cloned CLCuKoV-Burewala genome (DNA-A) in this study was previously cloned and sequenced [29]. The CLCuKoV-Bu LIR region was PCR-amplified using the primer P1 5'-GCGGTACCTGACTTTGGTCAATTAGAGACAAC-3' to obtain the CLCuKoV-Bu fragment comprising nucleotide coordinates 292 to 268, with the addition of KpnI restriction enzyme at the 5' end, and primer P2 5'-GCGGTACCTAATTCCTAGCCCTTATTACCAG-3' to obtain the CLCuKoV-Bu fragment comprising nucleotide coordinates from 2595 to 2618, with the addition of KpnI restriction enzyme at the 5' end. The amplicons harboring the CLCuKoV-BuC1 and CLCuKoV-BuV1 promoters were separated by agarose gel electrophoresis, visualized, eluted and was cloned separately into the Invitrogen TA plasmid vector using PCR 2.10 TA available in the TA cloning kit. To verify the successful TA-cloning of the bidirectional promoter (CLCuKoV-Bu-C1 and CLCuKoV-Bu-V1), the clones were digested using KpnI and visualized by gel electrophoresis, as well as by PCR amplification (Table 2).  
\begin{table}
\begin{tabular}{l c c} \hline \hline
**Biological Materials** & **Relevant Features** & **Reference or Source** \\ \hline \multicolumn{3}{c}{**Bacterial Strains**} \\ \hline LBA4404 and & _Agrobacteriumrium tumefaciens_ strain used for plant & [27] \\ GV1301 & transformation & \\ \hline DH5\(\alpha\) & _Escherichia coli_ strain used for cloning & [28] \\ \hline TOP10 & _Escherichia coli_-based cells used for cloning & Invitrogen \\ \hline CEMB-Cry1 Ac & _Bacillus thuringiensis \(\delta\)-endotoxin gene_ Cry1 Ac & \begin{tabular}{c} CEMB Patent: \\ (K140649-aroA) \\ \end{tabular} \\ \hline \multicolumn{3}{c}{**Viral Species**} \\ \hline CLCuKoV-Bu & \begin{tabular}{c} Circular, single-stranded, DNA-A genome of the \\ cotton leaf curl Kokhran virus-Burewala \\ \end{tabular} & [29] \\ \hline LIR & \begin{tabular}{c} Large intergenic region of the CLCuKoV-genome \\ possesses bidirectional promoter \\ \end{tabular} & This study \\ \hline CLCuKoV-BuC1 & C1 gene promoter of CLCuKoV-Bu genome & This study \\ \hline CLCuKoV-BuV1 & V1 gene promoter of CLCuKoV-Bu genome & This study \\ \hline \multicolumn{3}{c}{**Plasmids**} \\ \hline PIA2 & \begin{tabular}{c} Source plasmid vector containing CaMV35S \\ promoter and Cry1 Ac-NOS expression cassette \\ \end{tabular} & [30,31] \\ \hline pCAMBIA1301 & \begin{tabular}{c} binary vector used for cloning of gene expression \\ cassettes (promoter +Cry1 Ac-NOS) \\ \end{tabular} & This study \\ \hline PK2-Ac & \
\begin{tabular}{c} Positive control plant expression vector \\ containing CaMV35S promoter and \\ Cry1 Ac-NOS cassette \\ \end{tabular} & [32] \\ \hline CLCuKoV51-Ac & 5′- CLCuKoV51-Cry1 Ac-NOS-3′ plasmid & This study \\ \hline CLCuKoVV1-Ac & 5′- CLCuKoVV1-Cry1 Ac-NOS-3′ plasmid & This study \\ \hline pBluescript II KS/SK & Cloning vector harboring CLCuKoV-Bu genome & [29] \\ (+) & & \\ \hline \multicolumn{3}{c}{**Plant Materials**} \\ \hline _Nicotiana tabacum_ & Tobacco model plant used for agrorinfiltration & This study \\ \hline _Gossypium hirsutum_ &
\begin{tabular}{c} Cotton cultivar MNH-786 used for \\ transformation \\ \end{tabular} & This study \\ \hline \hline \end{tabular}
\end{table}
Table 1: Bacterial strains, viral species, plasmid vectors and plant species.