Part:BBa_K4344085

Shortened Transgen Sequence UL19

Composite part made from BBa_K4344028 (Kozak sequenz), BBa_K4344020 (HSV-UL19) and BBa_K4344065 (SV40 Poly Terminator). Used for recombinant protein production.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 312
  Illegal NgoMIV site found at 459
  
• 1000
  COMPATIBLE WITH RFC[1000]

Kozak sequenz

A missing Kozak-sequence was inserted to enhance the translation of HSV UL19 AA816:1148-6xHis via PCR. The HSV UL19 fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd., HindIII-HSV UL19-rev. and roughly 1 ng of pEX-A258-HSV-UL19-6xHis, the total reaction volume being 20 µL, for this purpose. The success of the PCR was evaluated on a 1.2 % TAE-Agarose gel stained with ethidium bromide. Successful PCR products were pooled and purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.

HSV-UL19

The sequence of UL19 encoding for HSV-1 major capsid protein was extracted from the complete https://www.ncbi.nlm.nih.gov/nuccore/KF498959.1 human herpesvirus 1 genome. The sequence was human codon optimised using https://www.benchling.com/ Benchling. The sequence was screened for published antibody binding sites and functional structure elements using the https://www.uniprot.org/ UniProt database. We identified the amino acids 862 to 880 in UL19 as a published antibody binding site (Han et al., 2019). Since it is a published antibody site, we anticipate that the corresponding DNA sequence is highly conserved in the HSV genome. Therefore, we chose a sequence for cloning and expression including this structure element. For UL19 bases 2446 to 3444 were selected resulting in a fragment of 999 bp in size. The fragment was further modified to mask unwanted restriction sites and ease primer design while keeping the amino acid sequence unchanged. UL19 was modified at the 16-18TCC>AGT and 21G>A. A His-tag (5’-CATCACCATCACCATCAC-3’) and a TAA stop codon were added at the 3’ sequence end.