The pET-44 vectors are designed for cloning and high-level expression of peptide sequences fused with the 495 aa Nus?Tag? protein. Compared to the pET-43.1 series, the pET-44 vectors encode an additional N-terminal His?Tag. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand. Therefore, single stranded sequencing should be performed using the COLIDOWN primer (Cat. No. 70845-3). Vector encoded sequence can be completely removed when cloning into the PshA I or Sma I sites (as shown below) by cleaving the Nus?Tag fusion protein with enterokinase or thrombin, respectively.