Does the pNEBR-X1 expression plasmid have an ATG?
pNEBR-X1 is designed to express your gene of interest as an unfused protein. The plasmid does not contain an initiation codon nor ‘Kozak’ consensus sequences for optimal translation initiation. However, there is an ATG as part of the ApaI site in the polylinker and this ATG can be used as an initiation codon.