PROTEIN ACTIVATION USING PHOSPHO-BRET IMAGING SENSOR

AND METHODS THEREOF

FIELD OF THE INVENTION The present invention relates to molecular biology techniques, and more particularly to methods and systems for detection of protein activation using phospho-BRET sensor.

BACKGROUND Monitoring cellular signaling events in live cells in a timely manner is a critical strategy for developing therapeutically potential chemical or biological molecules for all major diseases. One of the major cellular signaling events is that of protein phosphorylation in which a protein existing in the cell pool undergoes phosphorylation at a specific characteristic amino acid position or positions, as a result of which the protein is said to get activated. The activated proteins trigger signaling cascades downstream in the cell signaling pathway, and thus play crucial role in the regulation of nearly every aspect of cellular life. One of the mechanisms employed by the activated proteins to regulate a cell’s life is by interacting with self, i.e. by forming a homodimer or by interacting with other regulatory proteins i.e. by forming a heterodimer. The precise measurement of phosphorylation of the proteins is pivotal, and desired, in delineating protein activation.

Usually, protein kinases transfer phosphate groups from ATP to serine, threonine, or tyrosine amino acids on protein peptide substrates, which directly affects the activity and function of the target. As suggested earlier, this crucial post-translational modification regulates a broad range of cellular activities including the cell cycle, differentiation, metabolism, and neuronal communication. Therefore, abnormal phosphorylation events are implicated in many disease states.

Several methods to detect and measure phosphorylation of a desired protein have been reported and are in use. Such methods include kinase activity assays, phospho-specific antibody assays, western blotting, ELISA, flow cytometry, mass spectrometry and other such methods. Of late, in vivo methods are preferred, where intracellular imaging is deployed to determine, detect and measure signaling pathways and events. Current such approaches rely on the two systems - a. Fluorescence Resonance Energy Transfer (FRET) and b. Bioluminescence Resonance Energy Transfer (BRET). FRET presents itself with several limitations, as the major being dependence on external illuminations. The limitations of FRET are resolved by Bioluminescence Resonance Energy Transfer (BRET).

Standard BRET systems include a luciferase, which in the presence of its bioluminogenic substrate, acts as a resonance energy donor, and a fluorescent protein, which is the resonance energy acceptor. To successfully translate a BRET assay from cell culture to living subjects, it is critical that the BRET system is characterized for efficient energy transfer, excellent spectral resolution, a BRET donor with high bioluminescence quantum yield, and a red light-emitting BRET acceptor. The bioluminescent donor, usually a luciferase, does not excite the fluorophore using light, but transfers resonance energy through dipole-dipole coupling. To transfer resonance energy, the donor must be within a range of 1-lOnm of the acceptor and in the proper orientation making the technique useful for measuring proteins in close proximity. This makes BRET a critical methodology to detect and measure dynamic events such as phosphorylation of a protein. STAT3 is a key oncogenic molecule whose activation and signaling is necessary for cancer cells to survive, grow and migrate to distant organs. STAT3 activation is triggered in response to external stimuli which via a series of events gets activated at its key post- translational modification sites (Y705, S727 amino acids) and as a result STAT3 forms homo-dimer. Dimerization of STAT3 is a rate limiting step for its oncogenic activity and works as a control switch. Therefore, identifying potential inhibitors that can effectively block STAT3 signaling by blocking its phospho-PTM specific activation is crucial.

Hyper-activation of AKT is a prominent signature in many human cancers which leads to phosphorylation of plethora of proteins that promote tumour cell survival and apoptosis resistance. Complete activation of AKT is a multistep process which includes i) interaction with phosphatidylinositol 3,4,5-trisphosphate (PIP3) via pleckstrin- homology (PH) domain, followed by ii) translocation to membrane AND then iii) phosphorylation of AKT at T308 by PDK and S473 by other kinases. Multiple mechanisms regulating several physiological process such as growth factor signaling through various receptor tyrosine kinases, G-protein coupled receptor signaling, integrin signaling through focal adhesion kinase (FAK), cytokine signaling all impinge on activation of Phosphatidylinositol 3-Kinases (PI3Ks) resulting in membrane translocation of AKT and hence its activation.

Therefore, it is critical to develop dynamic assays for time-dependent monitoring of membrane translocation as a result of AKT phosphorylation, as well as for measuring/monitoring STAT3 phosphorylation to assess effect of new therapeutic molecules on AKT and STAT3 signaling.

SUMMARY OF THE INVENITON

Accordingly, a fusion construct comprising a first nucleotide sequence encoding a Nanoluciferase polypeptide and a second nucleotide sequence encoding a fluorophore polypeptide selected from group consisting of TurboFP635, TagRFP and mOrange, wherein the nanoluciferase polypeptide and the fluorophore polypeptide are operably linked to form a bioluminescence resonance energy transfer (BRET) sensor-based donor-acceptor pair.

The fusion construct is inserted into a pCMV empty vector comprising a flexible GGSGGSxGGSGGS linker using Xhol and Barn HI restriction sites and a suitable mammalian selection gene. The nanoluciferase polypeptide is inserted at the C- terminus, and the fluorophore polypeptide is inserted at the N-terminus of a plasmid. The vector includes the fusion construct which is transfected or transduced via viral vector mediated gene transfer procedure into a cell line.

In another aspect, a method to quantify and visualize STAT3 phosphorylation including the step of expressing the bioluminescence resonance energy transfer (BRET) sensor-based donor- acceptor pair of claim 1 in a cell line by transfecting through a PCMV vector wherein STAT3 is fused with C-terminus of donor (Nanoluciferase) or acceptor (Fluorophore) reporter proteins. The fluorophore is TurboFP635. In yet another aspect, a method to quantify and visualize AKT activation phosphorylation comprising expressing a Nanoluc-Fluorophore BRET pair in a cell line by transfecting through a PCMV vector, wherein AKT is fused with C-terminus of donor (Nanoluciferase) or acceptor (Fluorophore) reporter proteins. In another aspect, the PH domain of AKT is fused to the N-terminal of nanoluciferase. In yet another aspect, a cell line expressing BRET sensor pair with suitable BRET donor and acceptor selected from list of available luciferases and fluorescence proteins as donor-acceptor pair. Further, an assay for screening activator, inhibitory, mutants or cell lines using STAT3 Phospho-BRET sensor. Furthermore, an assay for screening activator, inhibitory, mutants or cell lines using AKT Phospho-BRET sensor. DESCRIPTION OF THE DRAWING

Fig. 1 illustrates schematic of working principle of STAT3 Phospho-BRET sensor, according to an embodiment herein;

Fig. 2 illustrates schematic working principle of AKT Phospho-BRET sensor, according to an embodiment herein;

Fig. 3 illustrates AKT activation monitoring using the method described in Fig. 1, according to an embodiment herein;

Fig. 4 illustrates development of STAT3 phosphorylation and dimerization sensor using Niue and TurboFP BRET pair, according to an embodiment herein; Fig. 5A-5D illustrates ligand-induced BRET change in STAT3-Phospho BRET sensor, according to an embodiment herein;

Fig. 6 illustrates abrogation of STAT3 activation in presence of STAT3 pathway blockers or inhibitors, according to an embodiment herein; and

Fig. 7 illustrates schematic mechanism of drug action based on research findings of specific inhibitor of STAT3 signaling pathway using the Phospho-BRET sensor, according to an embodiment herein;

Fig. 8 illustrates a map of the expression vector pCMV-Nluc-(K) a. a)-STAT3, according to an embodiment herein;

Fig. 9 illustrates a map of the expression vector pCMV-STAT3-(14 a. a)-Nluc, according to an embodiment herein;

Fig. 10 illustrates a map of the expression vector pCMV-TurboFP-(10 a. a)-STAT3, according to an embodiment herein; Fig. 11 illustrates a map of the expression vector pCMV-TurboFP-(12 a. a)-STAT3, according to an embodiment herein;

Fig. 12 illustrates Phospho-STAT3 BRET sensor as HTS compatible live cell screening tool for STAT3 activator/ inhibitor molecules.

DESCRIPTION OF THE INVENTION

Various embodiment of the present invention provides a system and a method for aerial visualization. The following description provides specific details of certain embodiments of the invention illustrated in the drawings to provide a thorough understanding of those embodiments. It should be recognized, however, that the present invention can be reflected in additional embodiments and the invention may be practiced without some of the details in the following description. The various embodiments including the example embodiments will now be described more fully with reference to the accompanying drawings, in which the various embodiments of the invention are shown. The invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, the sizes of components may be exaggerated for clarity.

The subject matter of example embodiments, as disclosed herein, is described with specificity to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. Rather, the inventors have contemplated that the claimed subject matter might also be embodied in other ways, to include different features or combinations of features similar to the ones described in this document, in conjunction with other technologies. Generally, the various embodiments including the example embodiments relate to a system and method for aerial visualization.

The present methods are based on the biophysical principle of resonance energy transfer (RET). The present embodiment provides comprehensive systems and methods for intracellular sensing to affirm protein homo-or-heterodimer formation in living cell. The general principle of the present BRET assay system is generating a BRET effector signal as an indicator of the occurrence of a target protein phosphorylation in living cell. The methods herein disclose a Phospho-BRET sensor to determine and measure phosphorylation by generating a BRET effector signal as an indicator of the phosphorylation of a protein. The phospho-BRET [p-BRET] sensor and method as provided herein is exemplified via two examples. In a first embodiment, STAT3 phosphorylation event is detected, in which the indicative signal is resultant of activation followed by homodimer formation, which is a key rate limiting step for STAT3 signalling activation. In a second embodiment, AKT phosphorylation event is detected, in which the resultant signal amplification is due to AKT translocation to cell membrane resulting in activation of AKT itself. The measurement may be accomplished by standard instruments like a microplate reader or light sensing in vivo imaging equipment. Fig. 1 illustrates schematic of working principle of STAT3 p-BRET sensor, according to an embodiment herein. The STAT3 p-BRET sensor assay employs Nanoluc- TurboFP635 BRET reporter pair to develop a versatile STAT3 phosphorylation sensor that can capture the kinetics of the STAT3 activation and homo-dimerization. STAT3 fused with C-terminus of donor (Nanolucif erase) or acceptor (TurboFP635) reporter proteins shows distinct acceptor specific signal amplification due to STAT3 homo dimerization in response to its ligands such as IL6 and EGF as depicted in Fig. 1. In an embodiment, several cell lines are established and distributed in different sized plates (typically a 96 well or 384 well plate) suitable for imaging/ photon counting using in vivo optical imager or luminescence plate reader respectively. As a result of phosphorylation of STAT3 protein at its specific sites (serine727 and tyrosine705 amino acids), increased BRET ratio (i.e. ratio of acceptor/donor) in a panel of different cancer cell lines (MCF7, PC3, A549, HT1080) are observed. This method allows quantitative measurements from just a few thousands live cells and is compatible with various detection instruments including photomultiplier tube (PMT) based detection tool like microplate reader and intensely cooled CCD equipped in vivo optical imaging equipment. Thus, the methodology developed can be readily adapted for high- throughput screening (HTS) of chemical compounds using live cell format. HTS compatibility by using 384 well plate is demonstrated.

In an embodiment, fusion constructs of full length Nanolucif erase with different flurophores is prepared by cloning PCR amplified nanoluc (516bp) into a pCMV empty vector containing flexible GGSGGSxGGSGGS linker using Xhol and Bam HI restriction sites. Followed by nanoluc insertion at the C-terminus, flurophores (TurboFP635, TagRFP and mOrange) are PCR amplified without stop codon flanked by EcoRI and Bglll restriction sites and inserted at the N-terminus of the plasmid with a separation of 12 amino acids between donor and acceptor molecule. For dipole orientation related studies, PCR amplified fragment of XhoI-mOrange- BamHI is cloned at the C-terminus of pCMV-GGS vector while nanoluc is inserted at the N- terminus using EcoRI and Bglll restriction sites separated by a linker of 12amino acid. mOrange-Nluc (12aa) fusion construct is also prepared as above. Optimization of linker length is achieved by ligating nanoluc and mOrange in pCMV vector containing GGS amino acid repeat linker of length varying from 12aa to 24aa. Expression vectors pSTAT3-Nluc and pSTAT3-TurboFP635 coding for STAT3 fusion protein are generated by inserting a Nhel-Sall fragment of the full length human Stat3 cDNA without the stop codon into the pCMV-GGS-Nluc and pCMV-GGS-TurboFP635 vectors, resulting in a linker separation of 10-12 amino acids between STAT3 and nanoluc/TurboFP635. Wild type STAT3 is converted to mutant STAT3 (Y705F, S727A etc.) by site directed mutagenesis as per prior art to test verify if the existence of activation site mutants are capable to form STAT3 homodimer.

HT1080 and PC3 cells are cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. A549 and MCF7 cells are maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. All cells are maintained at 37° C in a 5% C02 humidified atmosphere as in prior art. A day before transfection 1c10 ^L 5 cells may be seeded in a 12 well-flat bottom plate. Transfection is carried out at an optimal confluency of 80% using suitable transfection reagent as per the manufacturer’s instructions. For BRET studies expression vectors coding for donor and acceptor plasmid are transfected in 1: 1 ratio.

Using the same Nanoluc-TurboFP635 BRET reporter protein pair as employed in the first embodiment, another assay system where AKT translocation to membrane leads to its own phosphorylation by PDK and other kinases in response to stimulation of growth factor receptor tyrosine kinases is determined and measured. The PH domain of AKT is fused to the N-terminal of nanoluciferase (Niue) which when transfected in a cell expressing membrane-localized TurboFP635 fluorescent protein leads to generation of specific BRET signal upon stimulation with Insulin, IGF- 1 and EGF in MCF7 and A2780 cell lines as shown in Fig. 2. In an embodiment, two inhibitors, Wortamanin, an irreversible PIK3CA inhibitor and Picropodophyllin, showed approximately 3.46 fold (Wortmanin) and 3.35 fold (PPP) reduction in BRET output. In yet another embodiment, introduction of a single point mutation in the PH domain (K14A) abrogated PIP3-AKT-PH domain interaction and reduced the BRET signal output to basal level. The A2780 cells are engineered to stably express the AKT BRET sensor pair that enables us to correctly detect the effect of various inhibitors as well as to monitor inherent modulation in AKT activation in diseased condition. Modulation in active IGF1R/PI3KCA/AKT is known to drive acquirement of resistance in ovarian cancer cells. In indigenously developed platinum-taxol resistant cellular models of ovarian cancer cells (OvCa), enhanced IGF1R expression at the onset of resistance development is reported, and which subsequently decreased at final/late stage of resistance, which is captured by the AKT sensor as developed herein, across stages of resistance, as shown in Figure 3. The embodiment described herein are therefore amenable for capturing true biological modulation in AKT activation as well as high throughput screening of various novel inhibitors in live cells in a simplified manner.

The presently developed STAT3 p-BRET sensor is able to successfully detect the activation or inhibition of STAT3 pathway in the presence of its specific ligands or known and unknown inhibitor molecules. This may also be used for characterizing the PTM specific STAT3 dimerization and activation status across various cancer cell types. It may be easily extended in the multiplexing format to identify other potential STAT3 interacting partners (for example various GPCR molecules) in live cells. The AKT phosphorylation BRET sensor detects the dynamics of AKT activation in multiple cell lines when challenged with various activators and inhibitors. Along with its ability to provide a high throughput drug/cell/phosphor-site specific mutant screening platform, this technology may also provide hints on the mechanistic and biochemical interaction features of a specific inhibitors molecule aimed to inhibit either the interaction between AKT and PIP3 or its translocation to the membrane from cytoplasm.

Both STAT3 and AKT phosphorylation sensor as provided herein use pairing of Nanoluciferase, and Turbo635 fluorescent protein in the current form, but can work with any known or novel BRET pair as well. The currently used BRET sensor pair generate specific BRET signal while reducing noise due to improved spectral resolution of 140 nm.

Fig. 8 illustrates a map of the expression vector pCMV-Nluc-(10 a. a)-STAT3, according to an embodiment herein. The vector is represented by SEQ ID NO 1. Fig. 9 illustrates a map of the expression vector pCMV-STAT3-(14 a. a)-Nluc, according to an embodiment herein. The vector is represented by SEQ ID NO 2.

Fig. 10 illustrates a map of the expression vector pCMV-TurboFP-(10 a. a)-STAT3, according to an embodiment herein. The vector is represented by SEQ ID NO 3.

Fig. 11 illustrates a map of the expression vector pCMV-TurboFP-(12 a. a)-STAT3, according to an embodiment herein. The vector is represented by SEQ ID NO 4.

EXAMPLES:

EXAMPLE 1: DEVELOPMENT AND VALIDATION OF STAT3 PHOSPHORYLATION BRET SENSOR IN TRANSIENT VS STABLE CELLS

For developing the STAT3 BRET sensor, STAT3 was fused to either Nanoluc or TurboFP and was combined in all possible orientations i.e. Nluc-STAT3 & TurboFP- STAT3, Nluc-STAT3 & STAT3-TurboFP635, STAT3-Nluc & STAT3-TurboFP635 and STAT3-Nluc & TurboFP-STAT3 as depicted in Figure 4. Of all the above four combinations tested upon EGF trigger in a concentration dependent manner in HT1080 cells, Nluc-STAT3+TurboFP-STAT3 pair showed the maximum energy transfer and thus gain in BRET signal. Similar observation was replicated in PC3 cell line which is null for endogenous STAT3. In the stable cell line established for acceptor (TurboFP- STAT3), the STAT3 phospho- BRET sensor showed positive gain of BRET signal upon EGF treatment in a concentration dependent manner. Also, in acceptor stable cells, the maximum BRET signal was achieved even with lOng EGF because of stable over-expression of the TurboFP-STAT3 protein as shown in Fig. 5. EXAMPLE 2: MONITORING STAT3 ACTIVATION IN MULTIPLE CANCER CELL TYPES AND WITH DIFFERENT LIGANDS

STAT3 activation and overexpression can be activated via various ligands. To study STAT3 activation in different cancer types, the developed STAT3 phospho-BRET sensor was transfected in panel of cancer cell lines i.e. HT1080 (fibrosarcoma), A549 (human lung cancer), PC3 (prostate cancer) and MCF7 cells (breast cancer) followed by serum starvation for 18hrs (Figure-5B). Upon treatment with two different ligands, IL6 and EGF, at variable concentration the stimulated cells showed a significant gain in BRET signal as compared to nonstimulated counterpart indicating activation of STAT3 pathway. Furthermore, the strength of BRET signal correlated with the expression of EGFR receptor in different cancer cell lines.

EXAMPLE 3: ABROGATION OF STAT3 ACTIVATION IN PRESENCE OF STAT3 PATHWAY BLOCKERS OR INHIBITORS To further expand the applicability of STAT3 phospho-BRET sensor, cells stably expressing the Nluc-STAT3 and transfected with TurboFP-STAT3 counterpart were serum starved for 16hrs followed by treatment with two different STAT3 inhibitors, like Stattic (that binds to the SH2 domain of STAT3) and Niclosamide (blocks STAT3 Y705 phosphorylation) at variable concentrations for 12hrs. The BRET output signal was monitored either in EGF +/- conditions following substrate addition. The innate ability of the STAT3 phospho-BRET sensor to capture the inhibition on STAT3 signaling following disruption of STAT3 homo-dimerization was recorded as a drastic drop in BRET signal in presence of inhibitors and in a concentration dependent manner as shown in Fig. 6 and Fig. 7. Further to show that EGF mediated STAT3 phosphorylation and activation is highly specific, the cells expressing the STAT3 phospho-BRET sensor were incubated with EGFR blocking antibody for 5hrs prior to trigger with EGF. With the loss of EGF binding to EGFR receptor the loss in BRET signal gain was clearly reflected as a true event of disruption in phosphorylation mediated STAT3 homodimerization. Further minimal numbers of cells being able to support BRET signal measurement maintaining the sensitivity was also determined - a step towards getting HTS readiness.

EXAMPLE 4: ENGINEERING A2780 CELLS STABLY EXPRESSING AKT PHOSPHORYLATION BRET SENSOR PAIR

To validate the BRET sensor expressing stable clones of A2780 cells and understand the effect of IGF1R ligand IGF-1 on AKT activation, cells were cultured in charcoal striped serum for 3-4 passage and then stimulated with various concentrations of ligands (Fig 3A). The BRET assay was further validated using western blotting (Fig 3B). To understand the effect of phosphoinositol-3-Kinase inhibitor on BRET signal output as a measure of AKT activation, the cells were pre-treated with wortmannin for 2 hours and the IGF- 1 induced BRET signal was monitored. Similarly to understand the effect of picropodophyllin (a natural IGF1R receptor) on AKT activation, cells were pre-treated with picropodophyllin for 24 hours followed by IGF-1 stimulation and BRET spectra were acquired. Application of both wortmannin and picropodophyllin reduced IGF-1 induced BRET signal and subsequently AKT activation (Fig 3A).

EXAMPLE 5: VALIDATION OF AKT BRET SENSOR BY MUTATING PIP3 INTERACTION DOMAIN OF PH-AKT

PH domain containing proteins interact with phosphoinositide phosphate moiety through conserved KxxR domain. Side directed mutagenesis was performed to replace this Lysine residue with Alanine. Insertion of K to A in the PH domain of AKT abolished its interaction with PIP3 and hence its activation as observed by reduced BRET signal output (Fig 3C).

EXAMPLE 6: COMPARISON OF AKT ACTIVATION ACROSS THE STAGES OF RESISTANCE IN OVARIAN CANCER CELLS

The BRET sensor developed was applied to compare ligand induced AKT activation across the stages of platinum-taxol resistance as described earlier. A2780 cells and its corresponding early resistant and late resistant cells were transfected with donor and acceptor plasmid and were seeded in 96 well plate. Ligand induced AKT activation was monitored across the stage of resistance by stimulating the cells with lOOOnM of IGF-1 (Fig 3D). Maximum BRET signal was observed in early resistant cells which possess highest IGF expression.

EXAMPLE 7: DEMONSTRATION OF HTS COMPATIBILITY OF THE METHOD DEVELOPED

Fig. 12 shows STAT3 p-BRET cell system developed were sufficiently sensitive for relaying the message of drug inhibitor action from very low number of cells which can be plated in 384 well plate. This ability essentially demonstrated that the p-BRET screening method is sensitive enough and can be adapted for HTS screen. Of the molecules tested, several are known drug inhibitors of STAT3 (e.g. Stattic, Niclosamide), or repurposed drug candidates (e.g. ERK inhibitor, Neratinib), and molecules like Curcumin are natural products. Fig. 12 shows a representative image of 96 and 384 well plate for screen of 12 different compounds against STAT3 BRET sensor at respective donor and acceptor filters. Fig. 12 further illustrates a chart representing corrected BRET ratio for each of the drug candidates tested as indicated in presence or absence of EGF conditions. While several implementations have been described and illustrated herein, a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein may be utilized, and each of such variations and/or modifications is deemed to be within the scope of the implementations described herein. More generally, all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific implementations described herein. It is, therefore, to be understood that the foregoing implementations are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, implementations may be practiced otherwise than as specifically described and claimed. Implementations of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the appended claims.

SEQ ID NO 1

Name location

CMV 1-582

GGS-1 Linker 592-632

Nluc 639-1151

GGS-1 Linker 1152-1181

STAT3 1182- 3491

GGS-1 Linker 3495-3501

Zeocin 4726-5100 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gCTAGCACCG GTAAGCTTGG TGGTAGTGGT GGTAGTGAAT TCGCCACCAT GGTCTTCACA CTCGAAGATT TCGTTGGGGA CTGGCGACAG ACAGCCGGCT ACAACCTGGA CCAAGTCCTT GAACAGGGAG GTGTGTCCAG TTTGTTTCAG AATCTCGGGG TGTCCGTAAC TCCGATCCAA AGGATTGTCC TGAGCGGTGA AAATGGGCTG AAGATCGACA TCCATGTCAT CATCCCGTAT GAAGGTCTGA GCGGCGACCA AATGGGCCAG ATCGAAAAAA TTTTTAAGGT GGTGTACCCT GTGGATGATC ATCACTTTAA GGTGATCCTG CACTATGGCA CACTGGTAAT CGACGGGGTT ACGCCGAACA TGATCGACTA TTTCGGACGG CCGTATGAAG GCATCGCCGT GTTCGACGGC AAAAAGATCA CTGTAACAGG GACCCTGTGG AACGGCAACA AAATTATCGA CGAGCGCCTG ATCAACCCCG ACGGCTCCCT GCTGTTCCGA GTAACCATCA ACGGAGTGAC CGGCTGGCGG CTGTGCGAAC GCATTCTGGC GAGATCTGGT GGTAGTGGTG GTAGTCTCGA GATGGCCCAA TGGAATCAGC TACAGCAGCT TGACACACGG TACCTGGAGC AGCTCCATCA GCTCTACAGT GACAGCTTCC CAATGGAGCT GCGGCAGTTT CTGGCCCCTT GGATTGAGAG TCAAGATTGG GCATATGCGG CCAGCAAAGA ATCACATGCC ACTTTGGTGT TTCATAATCT CCTGGGAGAG ATTGACCAGC AGTATAGCCG CTTCCTGCAA GAGTCGAATG TTCTCTATCA GCACAATCTA CGAAGAATCA AGCAGTTTCT TCAGAGCAGG TATCTTGAGA AGCCAATGGA GATTGCCCGG ATTGTGGCCC GGTGCCTGTG GGAAGAATCA CGCCTTCTAC AGACTGCAGC CACTGCGGCC CAGCAAGGGG GCCAGGCCAA CCACCCCACA GCAGCCGTGG TGACGGAGAA GCAGCAGATG CTGGAGCAGC ACCTTCAGGA TGTCCGGAAG AGAGTGCAGG ATCTAGAACA GAAAATGAAA GTGGTAGAGA ATCTCCAGGA TGACTTTGAT TTCAACTATA AAACCCTCAA GAGTCAAGGA GACATGCAAG ATCTGAATGG AAACAACCAG TCAGTGACCA GGCAGAAGAT GCAGCAGCTG GAACAGATGC TCACTGCGCT GGACCAGATG CGGAGAAGCA TCGTGAGTGA GCTGGCGGGG CTTTTGTCAG CGATGGAGTA CGTGCAGAAA ACTCTCACGG ACGAGGAGCT GGCTGACTGG AAGAGGCGGC AACAGATTGC CTGCATTGGA GGCCCGCCCA ACATCTGCCT AGATCGGCTA GAAAACTGGA TAACGTCATT AGCAGAATCT CAACTTCAGA CCCGTCAACA AATTAAGAAA CTGGAGGAGT TGCAGCAAAA AGTTTCCTAC AAAGGGGACC CCATTGTACA GCACCGGCCG ATGCTGGAGG AGAGAATCGT GGAGCTGTTT AGAAACTTAA TGAAAAGTGC CTTTGTGGTG GAGCGGCAGC CCTGCATGCC CATGCATCCT GACCGGCCCC TCGTCATCAA GACCGGCGTC CAGTTCACTA CTAAAGTCAG GTTGCTGGTC AAATTCCCTG AGTTGAATTA TCAGCTTAAA ATTAAAGTGT GCATTGACAA AGACTCTGGG GACGTTGCAG CTCTCAGAGG ATCCCGGAAA TTTAACATTC TGGGCACAAA CACAAAAGTG ATGAACATGG AAGAATCCAA CAACGGCAGC CTCTCTGCAG AATTCAAACA CTTGACCCTG AGGGAGCAGA GATGTGGGAA TGGGGGCCGA GCCAATTGTG ATGCTTCCCT GATTGTGACT GAGGAGCTGC ACCTGATCAC CTTTGAGACC GAGGTGTATC ACCAAGGCCT CAAGATTGAC CTAGAGACCC ACTCCTTGCC AGTTGTGGTG ATCTCCAACA TCTGTCAGAT GCCAAATGCC TGGGCGTCCA TCCTGTGGTA CAACATGCTG ACCAACAATC CCAAGAATGT AAACTTTTTT ACCAAGCCCC CAATTGGAAC CTGGGATCAA GTGGCCGAGG TCCTGAGCTG GCAGTTCTCC TCCACCACCA AGCGAGGACT GAGCATCGAG CAGCTGACTA CACTGGCAGA GAAACTCTTG GGACCTGGTG TGAATTATTC AGGGTGTCAG ATCACATGGG CTAAATTTTG CAAAGAAAAC ATGGCTGGCA AGGGCTTCTC CTTCTGGGTC TGGCTGGACA ATATCATTGA CCTTGTGAAA AAGTACATCC TGGCCCTTTG GAACGAAGGG TACATCATGG GCTTTATCAG TAAGGAGCGG GAGCGGGCCA TCTTGAGCAC TAAGCCTCCA GGCACCTTCC TGCTAAGATT CAGTGAAAGC AGCAAAGAAG GAGGCGTCAC TTTCACTTGG GTGGAGAAGG ACATCAGCGG TAAGACCCAG ATCCAGTCCG TGGAACCATA CACAAAGCAG CAGCTGAACA ACATGTCATT TGCTGAAATC ATCATGGGCT ATAAGATCAT GGATGCTACC AATATCCTGG TGTCTCCACT GGTCTATCTC TATCCTGACA TTCCCAAGGA GGAGGCATTC GGAAAGTATT GTCGGCCAGA GAGCCAGGAG CATCCTGAAG CTGACCCAGG TAGCGCTGCC CCATACCTGA AGACCAAGTT TATCTGTGTG ACACCAACGA CCTGCAGCAA TACCATTGAC CTGCCGATGT CCCCCCGCAC

TTTAGATTCA TTGATGCAGT TTGGAAATAA TGGTGAAGGT GCTGAACCCT CAGCAGGAGG GCAGTTTGAG TCCCTCACCT TTGACATGGA GTTGACCTCG GAGTGCGCTA CCTCCCCCAT GTGAGAGCTC Ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat taaggaggcg ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat

SEQ ID NO 2

Name location

CMV 1-582

GGS-1 Linker 592-596

STAT3 603-2912

GGS-1 Linker 2913-2948

Nluc 2955-3470

GGS-1 Linker 3471-3471

Zeocin 4696-5070

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gCTAGCGCCA CCATGGCCCA ATGGAATCAG CTACAGCAGC TTGACACACG GTACCTGGAG CAGCTCCATC AGCTCTACAG TGACAGCTTC CCAATGGAGC TGCGGCAGTT TCTGGCCCCT TGGATTGAGA GTCAAGATTG GGCATATGCG GCCAGCAAAG AATCACATGC CACTTTGGTG TTTCATAATC TCCTGGGAGA GATTGACCAG CAGTATAGCC GCTTCCTGCA AGAGTCGAAT GTTCTCTATC AGCACAATCT ACGAAGAATC AAGCAGTTTC TTCAGAGCAG GTATCTTGAG AAGCCAATGG AGATTGCCCG GATTGTGGCC CGGTGCCTGT GGGAAGAATC ACGCCTTCTA CAGACTGCAG CCACTGCGGC CCAGCAAGGG GGCCAGGCCA ACCACCCCAC AGCAGCCGTG GTGACGGAGA AGCAGCAGAT GCTGGAGCAG CACCTTCAGG ATGTCCGGAA GAGAGTGCAG GATCTAGAAC AGAAAATGAA AGTGGTAGAG AATCTCCAGG ATGACTTTGA TTTCAACTAT AAAACCCTCA AGAGTCAAGG AGACATGCAA GATCTGAATG GAAACAACCA GTCAGTGACC AGGCAGAAGA TGCAGCAGCT GGAACAGATG CTCACTGCGC TGGACCAGAT GCGGAGAAGC ATCGTGAGTG AGCTGGCGGG GCTTTTGTCA GCGATGGAGT ACGTGCAGAA AACTCTCACG GACGAGGAGC TGGCTGACTG GAAGAGGCGG CAACAGATTG CCTGCATTGG AGGCCCGCCC AACATCTGCC TAGATCGGCT AGAAAACTGG ATAACGTCAT TAGCAGAATC TCAACTTCAG ACCCGTCAAC AAATTAAGAA ACTGGAGGAG TTGCAGCAAA AAGTTTCCTA CAAAGGGGAC CCCATTGTAC AGCACCGGCC GATGCTGGAG GAGAGAATCG TGGAGCTGTT TAGAAACTTA ATGAAAAGTG CCTTTGTGGT GGAGCGGCAG CCCTGCATGC CCATGCATCC TGACCGGCCC CTCGTCATCA AGACCGGCGT CCAGTTCACT ACTAAAGTCA GGTTGCTGGT CAAATTCCCT GAGTTGAATT ATCAGCTTAA AATTAAAGTG TGCATTGACA AAGACTCTGG GGACGTTGCA GCTCTCAGAG GATCCCGGAA ATTTAACATT CTGGGCACAA ACACAAAAGT GATGAACATG GAAGAATCCA ACAACGGCAG CCTCTCTGCA GAATTCAAAC ACTTGACCCT GAGGGAGCAG AGATGTGGGA ATGGGGGCCG AGCCAATTGT GATGCTTCCC TGATTGTGAC TGAGGAGCTG CACCTGATCA CCTTTGAGAC CGAGGTGTAT CACCAAGGCC TCAAGATTGA CCTAGAGACC CACTCCTTGC CAGTTGTGGT GATCTCCAAC ATCTGTCAGA TGCCAAATGC CTGGGCGTCC ATCCTGTGGT ACAACATGCT GACCAACAAT CCCAAGAATG TAAACTTTTT TACCAAGCCC CCAATTGGAA CCTGGGATCA AGTGGCCGAG GTCCTGAGCT GGCAGTTCTC CTCCACCACC AAGCGAGGAC TGAGCATCGA GCAGCTGACT ACACTGGCAG AGAAACTCTT GGGACCTGGT GTGAATTATT CAGGGTGTCA GATCACATGG GCTAAATTTT GCAAAGAAAA CATGGCTGGC AAGGGCTTCT CCTTCTGGGT CTGGCTGGAC AATATCATTG ACCTTGTGAA AAAGTACATC CTGGCCCTTT GGAACGAAGG GTACATCATG GGCTTTATCA GTAAGGAGCG GGAGCGGGCC ATCTTGAGCA CTAAGCCTCC AGGCACCTTC CTGCTAAGAT TCAGTGAAAG CAGCAAAGAA GGAGGCGTCA CTTTCACTTG GGTGGAGAAG GACATCAGCG GTAAGACCCA GATCCAGTCC GTGGAACCAT ACACAAAGCA GCAGCTGAAC AACATGTCAT TTGCTGAAAT CATCATGGGC TATAAGATCA TGGATGCTAC CAATATCCTG GTGTCTCCAC TGGTCTATCT CTATCCTGAC ATTCCCAAGG AGGAGGCATT CGGAAAGTAT TGTCGGCCAG AGAGCCAGGA GCATCCTGAA GCTGACCCAG GTAGCGCTGC CCCATACCTG AAGACCAAGT TTATCTGTGT GACACCAACG ACCTGCAGCA ATACCATTGA CCTGCCGATG TCCCCCCGCA CTTTAGATTC ATTGATGCAG TTTGGAAATA ATGGTGAAGG TGCTGAACCC TCAGCAGGAG GGCAGTTTGA GTCCCTCACC TTTGACATGG AGTTGACCTC GGAGTGCGCT ACCTCCCCCA TGGTCGACAG ATCTGGTGGT AGTGGTGGTA GTCTCGAGGC CACCATGGTC TTCACACTCG AAGATTTCGT TGGGGACTGG CGACAGACAG CCGGCTACAA CCTGGACCAA GTCCTTGAAC AGGGAGGTGT GTCCAGTTTG TTTCAGAATC TCGGGGTGTC CGTAACTCCG ATCCAAAGGA TTGTCCTGAG CGGTGAAAAT GGGCTGAAGA TCGACATCCA TGTCATCATC CCGTATGAAG GTCTGAGCGG CGACCAAATG GGCCAGATCG AAAAAATTTT TAAGGTGGTG TACCCTGTGG ATGATCATCA CTTTAAGGTG ATCCTGCACT ATGGCACACT GGTAATCGAC GGGGTTACGC CGAACATGAT CGACTATTTC GGACGGCCGT ATGAAGGCAT CGCCGTGTTC GACGGCAAAA AGATCACTGT AACAGGGACC CTGTGGAACG GCAACAAAAT TATCGACGAG CGCCTGATCA ACCCCGACGG CTCCCTGCTG TTCCGAGTAA CCATCAACGG AGTGACCGGC TGGCGGCTGT GCGAACGCAT TCTGGCGTAA

Ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat taaggaggcg ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat SEQ ID NO 3

Name location

CMV 1-582

GGS-1 Linker 592-596

STAT3 603-2909

GGS-1 Linker 2910-2945

TurboFP 2946-3650

GGS-1 Linker 3654-3654

Zeocin 4879-5253

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gCTAGCGCCA CCatggccca atggaatcag

ctacagcagc ttgacacacg gtacctggag cagctccatc agctctacag cgacagcttc ccaatggagc tgcggcagtt tctggcccct tggattgaga gtcaagattg ggcatatgcg gccagcaaag aatcacatgc cactttggtg tttcataatc tcctgggaga gattgaccag cagtatagcc gcttcctgca agagtcgaat gttctctatc agcacaatct acgaagaatc aagcagtttc ttcagagcag gtatcttgag aagccaatgg agattgcccg gattgtggcc cggtgcctgt gggaagaatc acgccttcta cagactgcag ccactgcggc ccagcaaggg ggccaggcca accaccccac agcagccgtg gtgacggaga agcagcagat gctggagcag caccttcagg atgtccggaa gagagtgcag gatctagaac aggaaatgaa agtggtagag aatctccagg atgactttga tttcaactat aaaaccctca agagtcaagg agacatgcaa gatctgaatg gaaacaacca gtcagtgacc aggcagaaga tgcagcagct ggaacagatg ctcactgcgc tggaccagat gcggagaagc atcgtgagtg agctggcggg gcttttgtca gcgatggagt acgtgcagaa aactctcacg gacgaggagc tggctgactg gaagaggcgg caacagattg cctgcattgg aggcccgccc aacatctgcc tagatcggct agaaaactgg ataacgtcat tagcagaatc tcaacttcag acccgtcaac aaattaagaa actggaggag ttgcagcaaa aagtttccta caaagggtac cccattgtac agcaccggcc gatgctggag gagagaatcg tggagctgtt tagaaactta atgaaaagtg cctttgtggt ggagcggcag ccctgcatgc ccacgcatcc tgaccggccc ctcgtcatca agaccggcgt ccagttcact actaaagtca ggttgctggt caaattccct gagttgaatt atcagcttaa aatcaaagtg tgcattgaca aagactctgg ggacgttgca gctctcagag gatcccggaa atttaacatt ctgggcacaa acacaaaagt gatgaacatg gaagaatcca acaacggcag cctctctgca gaattcaaac acttgaccct gagggagcag agatgtggga atgggggccg agccaattgt gatgcttccc tgattgtgac tgaggagctg cacctgatca cctttgagac cgaggtgtat caccaaggcc tcaagattga cctagagacc cactccttgc cagttgtggt gatctccaac atctgtcaga tgccaaatgc ctgggcgtcc atcctgtggt acaacatgct gaccaacaat cccaagaatg taaacttttt taccaagccc ccaattggaa cctgggatca agtggccgag gtcctgagct ggcagttctc ctccaccacc aagcgagggc tgagcatcga gcagctgact acactggcag agaaactctt gggacctggt gtgaattatt cagggtgtca gatcacatgg gctaaatttt gcaaagaaaa catggctggc aagggcttct ccttctgggt ctggctggac aatatcattg accttgtgaa aaagtacatc ctggcccttt ggaacgaagg gtacatcatg ggctttatca gtaaggagcg ggagcgggcc atcttgagca ctaagcctcc aggcaccttc ctgctaagat tcagtgaaag cagcaaagaa ggaggcgtca ctttcacttg ggtggagaag gacatcagcg gtaagaccca gatccagtcc gtggaaccat acacaaagca gcagctgaac aacatgtcat ttgctgaaat catcatgggc tataagatca tggatgctac caatatcctg gtgtctccac tggtctatct ctatcctgac attcccaagg aggaggcatt cggaaagtat tgtcggccag agagccagga gcatcctgaa gctgacccag gcgctgcccc atacctgaag accaagttta tctgtgtgac accaacgacc tgcagcaata ccattgacct gccgatgtcc ccccgcactt tagattcatt gatgcagttt ggaaataatg gtgaaggtgc tgaaccctca gcaggagggc agtttgagtc cctcaccttt gacatggagt tgacctcgga gtgcgctacc tcccccatgG TCGACAGATC

TGGTGGTAGT GGTGGTAGTC TCGAGATGGT GGGTGAGGAT AGCGTGCTGA TCACCGAGAA CATGCACATG AAACTGTACA TGGAGGGCAC CGTGAACGAC CACCACTTCA AGTGCACATC CGAGGGCGAA GGCAAGCCCT ACGAGGGCAC CCAGACCATG AAGATCAAGG TGGTCGAGGG CGGCCCTCTC CCCTTCGCCT TCGACATCCT GGCTACCAGC TTCATGTACG GCAGCAAAAC CTTTATCAAC CACACCCAGG GCATCCCCGA CTTCTTTAAG CAGTCCTTCC CTGAGGGCTT CACATGGGAG AGGATCACCA CATACGAAGA CGGGGGCGTG CTGACCGCTA CCCAGGACAC CAGCCTCCAG AACGGCTGCC TCATCTACAA CGTCAAGATC AACGGGGTGA ACTTCCCATC CAACGGCCCT GTGATGCAGA AGAAAACACT CGGCTGGGAG GCCAGCACCG AGATGCTGTA CCCCGCTGAC AGCGGCCTGA GAGGCCATAG CCAGATGGCC CTGAAGCTCG TGGGCGGGGG CTACCTGCAC TGCTCCCTCA AGACCACATA CAGATCCAAG AAACCCGCTA AGAACCTCAA GATGCCCGGC TTCTACTTCG TGGACAGGAG ACTGGAAAGA ATCAAGGAGG

CCGACAAAGA GACCTACGTC GAGCAGCACG AGATGGCTGT GGCCAGGTAC TGCGACCTGC CTAGCAAACT GGGGCACAGC taaGgatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa

gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gattaaggag gcgccaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgattcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat

SEQ ID NO 4

Name location

CMV 1-582

GGS-1 Linker 592-596

STAT3 603-2909

GGS-1 Linker 2910-2945

TurboFP 2946-3650

GGS-1 Linker 3654-3654

Zeocin 4879-5253

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gCTAGCGCCA CCatggccca atggaatcag

ctacagcagc ttgacacacg gtacctggag cagctccatc agctctacag cgacagcttc ccaatggagc tgcggcagtt tctggcccct tggattgaga gtcaagattg ggcatatgcg gccagcaaag aatcacatgc cactttggtg tttcataatc tcctgggaga gattgaccag cagtatagcc gcttcctgca agagtcgaat gttctctatc agcacaatct acgaagaatc aagcagtttc ttcagagcag gtatcttgag aagccaatgg agattgcccg gattgtggcc cggtgcctgt gggaagaatc acgccttcta cagactgcag ccactgcggc ccagcaaggg ggccaggcca accaccccac agcagccgtg gtgacggaga agcagcagat gctggagcag caccttcagg atgtccggaa gagagtgcag gatctagaac aggaaatgaa agtggtagag aatctccagg atgactttga tttcaactat aaaaccctca agagtcaagg agacatgcaa gatctgaatg gaaacaacca gtcagtgacc aggcagaaga tgcagcagct ggaacagatg ctcactgcgc tggaccagat gcggagaagc atcgtgagtg agctggcggg gcttttgtca gcgatggagt acgtgcagaa aactctcacg gacgaggagc tggctgactg gaagaggcgg caacagattg cctgcattgg aggcccgccc aacatctgcc tagatcggct agaaaactgg ataacgtcat tagcagaatc tcaacttcag acccgtcaac aaattaagaa actggaggag ttgcagcaaa aagtttccta caaagggtac cccattgtac agcaccggcc gatgctggag gagagaatcg tggagctgtt tagaaactta atgaaaagtg cctttgtggt ggagcggcag ccctgcatgc ccacgcatcc tgaccggccc ctcgtcatca agaccggcgt ccagttcact actaaagtca ggttgctggt caaattccct gagttgaatt atcagcttaa aatcaaagtg tgcattgaca aagactctgg ggacgttgca gctctcagag gatcccggaa atttaacatt ctgggcacaa acacaaaagt gatgaacatg gaagaatcca acaacggcag cctctctgca gaattcaaac acttgaccct gagggagcag agatgtggga atgggggccg agccaattgt gatgcttccc tgattgtgac tgaggagctg cacctgatca cctttgagac cgaggtgtat caccaaggcc tcaagattga cctagagacc cactccttgc cagttgtggt gatctccaac atctgtcaga tgccaaatgc ctgggcgtcc atcctgtggt acaacatgct gaccaacaat cccaagaatg taaacttttt taccaagccc ccaattggaa cctgggatca agtggccgag gtcctgagct ggcagttctc ctccaccacc aagcgagggc tgagcatcga gcagctgact acactggcag agaaactctt gggacctggt gtgaattatt cagggtgtca gatcacatgg gctaaatttt gcaaagaaaa catggctggc aagggcttct ccttctgggt ctggctggac aatatcattg accttgtgaa aaagtacatc ctggcccttt ggaacgaagg gtacatcatg ggctttatca gtaaggagcg ggagcgggcc atcttgagca ctaagcctcc aggcaccttc ctgctaagat tcagtgaaag cagcaaagaa ggaggcgtca ctttcacttg ggtggagaag gacatcagcg gtaagaccca gatccagtcc gtggaaccat acacaaagca gcagctgaac aacatgtcat ttgctgaaat catcatgggc tataagatca tggatgctac caatatcctg gtgtctccac tggtctatct ctatcctgac attcccaagg aggaggcatt cggaaagtat tgtcggccag agagccagga gcatcctgaa gctgacccag gcgctgcccc atacctgaag accaagttta tctgtgtgac accaacgacc tgcagcaata ccattgacct gccgatgtcc ccccgcactt tagattcatt gatgcagttt ggaaataatg gtgaaggtgc tgaaccctca gcaggagggc agtttgagtc cctcaccttt gacatggagt tgacctcgga gtgcgctacc tcccccatgG TCGACAGATC

TGGTGGTAGT GGTGGTAGTC TCGAGATGGT GGGTGAGGAT AGCGTGCTGA TCACCGAGAA CATGCACATG AAACTGTACA TGGAGGGCAC CGTGAACGAC CACCACTTCA AGTGCACATC CGAGGGCGAA GGCAAGCCCT ACGAGGGCAC CCAGACCATG AAGATCAAGG TGGTCGAGGG CGGCCCTCTC CCCTTCGCCT TCGACATCCT GGCTACCAGC TTCATGTACG GCAGCAAAAC CTTTATCAAC CACACCCAGG GCATCCCCGA CTTCTTTAAG CAGTCCTTCC CTGAGGGCTT CACATGGGAG AGGATCACCA CATACGAAGA CGGGGGCGTG CTGACCGCTA CCCAGGACAC CAGCCTCCAG AACGGCTGCC TCATCTACAA CGTCAAGATC AACGGGGTGA ACTTCCCATC CAACGGCCCT GTGATGCAGA AGAAAACACT CGGCTGGGAG GCCAGCACCG AGATGCTGTA CCCCGCTGAC AGCGGCCTGA GAGGCCATAG CCAGATGGCC CTGAAGCTCG TGGGCGGGGG CTACCTGCAC TGCTCCCTCA AGACCACATA CAGATCCAAG AAACCCGCTA AGAACCTCAA GATGCCCGGC TTCTACTTCG TGGACAGGAG ACTGGAAAGA ATCAAGGAGG CCGACAAAGA GACCTACGTC GAGCAGCACG AGATGGCTGT GGCCAGGTAC TGCGACCTGC CTAGCAAACT GGGGCACAGC taaGgatcca ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gattaaggag gcgccaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgattcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac gggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat