Fig.S1 Cloning strategy of the lentiviral vectors expressing DTA and fluorescent protein. The steps of the DNA manipulations are indicated with the target fragments and the applied technology or enzymes.

Fig.S2 The coordinate expression cassette of fluorescent protein and DTA is driven by the CMV promoter in lentiviral vector A and B, whereas the expression cassette is driven by the Surp1430 in lentiviral vector C and D.

Fig.S3. Procedure for the generation of IDLV(RRK262263264AAH) expressing DTA and fluorescent protein coordinately. IDLV(RRK262263264AAH), the coordinate expression of DTA and fluorescent protein driven by CMV or Surp1430, was generated after the co-transfection of lentiviral vectors, pMDLg/pRRE(RRK262263264AAH), RSV-Rev and PMD.G into HEK293T[mEF-2(G717R)], which was obtained after the overexpression of mutated EF-2 at amino acid residue 717.

Fig.S4. DTA-expressingIDLVs cannot be produced in HEK293T. As shown in the following figures, we concluded that the expression of the fluorescent protein was totally inhibited by DTA in the wild-type HEK293T cells and no fluorescence was detected through fluorescent microscopy.

Supplementary Table 1. Primers used for deciding the level of Survivin and DTA with quantitative PCR

Genes / Primer sequence(5’-3’)
β-actin / Forward:5’-CATGTACGTTGCTATCCAGGC-3’
Reverse:5’-CTCCTTAATGTCACGCACGAT-3’
Survivin / Forward:5’-AGAACTGGCCCTTCTTGGAGG-3’
Reverse:5’-CTTTTTATGTTCCTCTATGGGGTC-3’
DTA / Forward:5’-GGCGTGGTCAAAGTGACGTA-3’
Reverse:5’-CTTGCTCCATCAACGGTTCA-3’

Supplementary Table 2. Primers used for the construction of Luciferase reporters

Luciferase
reporters / Primer sequence(5’-3’)
pGL3-Surp269-
Luciferase / Forward:5’-GGGGTACCCGCGTTCTTTGAAAGCAGTC-3’
Reverse:5’-CTAGCTAGCTGCCGCCGCCGCCACCTCTG-3’
pGL3-Surp1430-Luciferase / Forward:5’-GGGGTACCAAATTGACATCGGGCCGGGC-3’
Reverse:5’-CTAGCTAGCAAATCTGGCGGTTAATGGCG-3’

Supplementary Table 3. The primers and 2A oligos used for the construction of DTA-expressing lentiviral vectors, and the cloning strategy follows the steps in Fig.S1. The resultant PCR products comprising two specific sites of the restricted enzyme, which are underlined, suffered enzyme digestion and ligation to generate lentiviral vectors.

Target genes or fragments / Primer sequence(5’-3’)
ApaI-SurP1430-NheI / Forward:5’-gcgggcccAAATTGACATCGGGCCGGGC-3’
Reverse:5’-ctagctagcAAATCTGGCGGTTAATGGCG-3’
AgeI-GFP-NotI / Forward:5’-GCACCGGTGCCACCATGGTGAGCAAGGGCGAGG-3’
Reverse:5’-ATAGTTTAGCGGCCGCGGGCCCTCTAGACTTGTACAGCTCGTCCATGC-3’
AgeI-RFP-NotI / Forward:5’-GCACCGGTGCCACCATGGCCTCCTCCGAGGAC-3’
Reverse:5’-ATAGTTTAGCGGCCGCGGGCCCTCTAGACAGGAACAGGTGGTGGCG-3’
ApaI-DTA-NotI / Forward:5’-GCGGGCCCATGGACCCTGATGATGTTG-3’
Reverse:5’-ATAGTTTAGCGGCCGCTTAGAGCTTTAAATCTCTGTA-3’
2Aoligo / Forward:5’-CTAGAGCCCCTGTGAAGCAGACCCTGAATTTCGATCTGCTGAAGCTGGCCGGCGACGTGGAGTCTAATCCTGGGCC-3’
Reverse:5’-CAGGATTAGACTCCACGTCGCCGGCCAGCTTCAGCAGATCGAATTCAGGGTCTGCTTCACAGGGGCT-3’
XbaI-2A-ApaI / 5’-ctagagcccctgtgaagcagaccctgaatttcgatctgctgaagctggccggcgacgtggagtctaatcctgggcc-3’

Supplementary Table 4. The primers used for EF-2 and integrase clone, directed mutations and the construction of corresponding plasmid. Nucleotides in red and bold highlight the nucleotides designed for mutations of the corresponding amino acids residual.

Target genes or fragments / Primer sequence(5’-3’)
EcoRI-EF-2-BamHI / Forward:5’-CGGAATTCGCCACCATGGTGAACTTCACGGTAGAC-3’
Reverse:5’-CGGGATCCCTACAATTTGTCCAGGAAGTTG-3’
EF-2(G717R) / Forward:5’-GACGCCATCCACCGCCGAGGGGGCCAGATCA-3’
Reverse:5’-TGATCTGGCCCCCTCGGCGGTGGATGGCGTC-3’
AgeI-Integrase-BspT1 / Forward:5’-accggtacatggagtgtattatg-3’
Reverse:5’-TCCGGAATTCCATGTGTTAATCC-3’
Integrase
(RRK262263264AAH) / Forward:5’-gtgacataaaagtagtgccagcaagaaaagcaaagatcatcaggg-3’
Reverse:5’-CCCTGATGATCTTTGCTTTTCTTGCTGGCACTACTTTTATGTCAC-3’
Forward:5’-cataaaagtagtgccagcagcaaaagcaaagatcatcag-3’
Reverse:5’-CTGATGATCTTTGCTTTTGCTGCTGGCACTACTTTTATG-3’
Forward: 5’- gtagtgccagcagcacacgcaaagatcatcag-3’
Reverse: 5’- CTGATGATCTTTGCGTGTGCTGCTGGCACTAC-3’