A method of preparing a shuttle vector comprising a selection marker gene and a low copy bacterial replication of origin, and an adenovirus genome comprising a 5' ITR, a 3' ITR, and an L5 gene said method comprising the steps: a) preparing an adenovirus shuttle vector by performing a one-step three-way ligation to equal proportions of the following three fragments: i) a vector fragment comprising a selection marker gene and a low copy bacterial replication of origin, wherein the 5' end of the vector fragment starts with a first restriction enzyme site and terminates at the 3' end of the vector fragment in a second restriction enzyme site, ii) a 5'-arm comprising the 5' end of the adenovirus genome including the 5' ITR, wherein the 5' end of the 5' arm starts with a second restriction enzyme site and terminates at the 3' end of the 5' arm with a third restriction enzyme site, iii) a 3'-arm comprising the 3' end of the adenovirus genome including the 3' ITR and the L5 gene, wherein the 5' end of the 3' arm starts with a third restriction enzyme site and terminates at the 3' end of the 3' arm with a first restriction enzyme site, thereby joining: the 3' end of the 3' arm (fragment iii) to the 5' end of the vector fragment (fragment i) at the first restriction enzyme site, the 3' end of the vector fragment (fragment i) to the 5' end of the 5' arm (fragment ii) at the second restriction enzyme site, and the 3' end of the 5' arm (fragment ii) to the 5' end of the 3' arm (fragment iii) at the third restriction enzyme site, to form a circularised shuttle vector arranged as a first restriction enzyme site followed by a vector fragment followed by a second restriction enzyme site followed by a 5' arm, followed by a third restriction enzyme site followed by a 3' arm, b) introducing at least one unique restriction site and/or transgene into the shuttle vector in a location: i) between the L5 gene and an E3 site, ii) between the L5 gene and an E4 site, or iii) between the L5 gene and an E3 site, and also between the L5 gene and an E4 site. a) preparing an adenovirus shuttle vector by performing a one-step three-way ligation to equal proportions of the following three fragments: i) a vector fragment comprising a selection marker gene and a low copy bacterial replication of origin, wherein the 5' end of the vector fragment starts with a first restriction enzyme site and terminates at the 3' end of the vector fragment in a second restriction enzyme site, ii) a 5'-arm comprising the 5' end of the adenovirus genome including the 5' ITR, wherein the 5' end of the 5' arm starts with a second restriction enzyme site and terminates at the 3' end of the 5' arm with a third restriction enzyme site, iii) a 3'-arm comprising the 3' end of the adenovirus genome including the 3' ITR and the L5 gene, wherein the 5' end of the 3' arm starts with a third restriction enzyme site and terminates at the 3' end of the 3' arm with a first restriction enzyme site, thereby joining: the 3' end of the 3' arm (fragment iii) to the 5' end of the vector fragment (fragment i) at the first restriction enzyme site, the 3' end of the vector fragment (fragment i) to the 5' end of the 5' arm (fragment ii) at the second restriction enzyme site, and the 3' end of the 5' arm (fragment ii) to the 5' end of the 3' arm (fragment iii) at the third restriction enzyme site, to form a circularised shuttle vector arranged as a first restriction enzyme site followed by a vector fragment followed by a second restriction enzyme site followed by a 5' arm, followed by a third restriction enzyme site followed by a 3' arm, i) a vector fragment comprising a selection marker gene and a low copy bacterial replication of origin, wherein the 5' end of the vector fragment starts with a first restriction enzyme site and terminates at the 3' end of the vector fragment in a second restriction enzyme site, ii) a 5'-arm comprising the 5' end of the adenovirus genome including the 5' ITR, wherein the 5' end of the 5' arm starts with a second restriction enzyme site and terminates at the 3' end of the 5' arm with a third restriction enzyme site, iii) a 3'-arm comprising the 3' end of the adenovirus genome including the 3' ITR and the L5 gene, wherein the 5' end of the 3' arm starts with a third restriction enzyme site and terminates at the 3' end of the 3' arm with a first restriction enzyme site, the 3' end of the 3' arm (fragment iii) to the 5' end of the vector fragment (fragment i) at the first restriction enzyme site, the 3' end of the vector fragment (fragment i) to the 5' end of the 5' arm (fragment ii) at the second restriction enzyme site, and the 3' end of the 5' arm (fragment ii) to the 5' end of the 3' arm (fragment iii) at the third restriction enzyme site, to form a circularised shuttle vector arranged as a first restriction enzyme site followed by a vector fragment followed by a second restriction enzyme site followed by a 5' arm, followed by a third restriction enzyme site followed by a 3' arm, b) introducing at least one unique restriction site and/or transgene into the shuttle vector in a location: i) between the L5 gene and an E3 site, ii) between the L5 gene and an E4 site, or iii) between the L5 gene and an E3 site, and also between the L5 gene and an E4 site. i) between the L5 gene and an E3 site, ii) between the L5 gene and an E4 site, or iii) between the L5 gene and an E3 site, and also between the L5 gene and an E4 site.