cruzi ubiquitin intergenic region (TcUIR – 278 bp) and the cassette containing the T. cruzi Dm28c pol
I promoter (617 bp) followed by a TcUIR and a hexahistidine tag were synthesized in vitro (GenScript, Piscataway, USA) (Figure 6). The third DNA segment, represented by the RfA cassette (Invitrogen) (1711 bp), was PCR-amplified from pCR-Blunt and was inserted into pBluescript(r) II KS+. Restriction sites were placed in specific positions of the sequence, to insert the various cassettes or remove some segments of DNA, such that new segments could be inserted for the construction of new vectors. Figure EPZ-6438 in vitro 6 Schematic drawing showing the vector construction steps. The elements shown are the neomycin (NEO) and hygromycin (HYGRO) resistance genes, the T. cruzi intergenic region from ubiquitin locus (TcUIR), the attachment sites for Gateway(r) recombination (attB1, attB2, attR1 and attR2), the chloramphenicol resistance gene (CmR), the gene for negative selection during cloning (ccdB), the fusion tags (6xhis, GFP, YFP, CFP, TAP and c-myc) and the ribosomal promoter (PR). In A, the steps for vectors construction are represented. In B, the
vector reading frame with start and stop codons are shown. The plasmid containing the three cassettes was named pTc6HN. We constructed some derivative vectors from pTc6HN, by replacing the polyhistidine tag with a TAP tag, the sequence of the c-myc epitope or with genes coding check details for fluorescent proteins (EGFP, CFP and YFP). All tags were amplified from plasmid vectors with the exception of c-myc, which was synthesized as two single-strand oligonucleotides (Additional file 5 – Table S2). For c-myc strands hybridization, 1.3 μg of each strand was used. The single strands
were incubated in 10 mM NaCl Clomifene buffer at 95°C for 10 min. The temperature was then slowly lowered to allow hybridization. After N-terminal tag insertion, the original vectors were identified as pTcTAPN, pTcGFPN, pTcCFPN, pTcYFPN, selleck compound pTcMYCN and pTcGFPH (neomycin resistance was replaced with hygromycin resistance in pTcGFPN). All of the constructs were sequenced by the commercial Macrogen facility (Macrogen, Seoul, Korea). The analysis of ab1 files was performed on SeqMan software (DNASTAR, Inc., Madison, USA). The sequences are available in GenBank under accession numbers HM162840 (pTcYFPN), HM162841 (pTcMYCN), HM162842 (pTcTAPN), HM162843 (pTcGFPN), HM162844 (pTcGFPH), HM162845 (pTcCFPN) and HM162846 (pTc6HN). Oligonucleotides used for the construction and sequencing of vectors are listed in Additional file 5 – Table S2 and Additional file 6 – Table S3, respectively. Validation of vectors Five T. cruzi genes were used in the validation process: TcRab7 (Tc00.1047053508461.270), PAR 2 (Tc00.1047053511215.119), a putative centrin (Tc00.1047053506559.380), Tcpr29A (Tc00.1047053506167.40), and TcrL27 (Tc00.1047053506817.30).