Development of Sensitive FRET Sensors and Methods of Using the Same

Abstract

Intramolecular biosensors are disclosed, including PBP-based biosensors, comprising a ligand binding domain fused to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon binding ligand. At least one of the donor and fluorescent moieties may be internally fused to the biosensor such that both ends of the internally fused fluorophore are fixed. In addition, methods of improving the sensitivity of terminally fused biosensors are provided. The biosensors of the invention are useful for the detection and quantification of ligands in vivo and in culture.

Description

REFERENCE TO SEQUENCE LISTING

[0002] A computer readable text file, entitled "056100-5046-02-SequenceListing.txt," created on or about 18 Feb. 2016, with a file size of about 184 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

[0003] The invention relates generally to the field of molecular biology and metabolomics. More specifically, the invention relates to biosensors and methods for measuring and detecting ligand binding using intramolecular fluorescence resonance energy transfer (FRET).

BACKGROUND OF INVENTION

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0005] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0006] The field of metabolomics centers on the metabolic and biochemical events associated with a cellular or biological system. Metabolomics seeks to depict the steady-state physiological state of a cell or organism as well as dynamic responses of a cell or organism to genetic and environmental modulation. Metabolomic tools permit the detection of disease states, the monitoring of disease progression and patient response to therapy, the classification of patients based on biochemical profiles and the identification of targets for drug design.

[0007] An ideal metabolomic tool reveals the concentration of a particular molecular species of interest in a physiological environment. It allows one to visualize how its concentration varies across an organ, tissue or cell. It permits the detection of metabolite levels and the changes in metabolite levels in response to environmental stimuli, and allows these changes to be monitored in real time. Using various such tools should permit multiple analytes to be measured simultaneously, even analytes of different structural and functional classes.

[0008] No currently available technology addresses all these issues in a satisfactory manner. Non-aqueous fractionation is static, invasive, has no cellular resolution and is sensitive to artifacts, while spectroscopic methods such as NMRi (nuclear magnetic resonance imaging) and PET (positron emission tomography) provide dynamic data, but poor spatial resolution. The development of genetically encoded molecular sensors, which transduce an interaction of the target molecule with a recognition element into a macroscopic observable format, via allosteric regulation of one or more signaling elements, may facilitate some of the goals.

[0009] The most common reporter element employed in molecular sensors is a sterically separated donor-acceptor FRET pair of fluorescent proteins (GFP spectral variants or otherwise) (Fehr et al., 2002, Proc. Natl. Acad. Sci USA 99: 9846-51), although single fluorescent proteins (Doi and Yanagawa, 1999, FEBS Lett. 453: 305-7), enzymes (Guntas and Ostermeier, 2004, J. Mol. Biol. 336: 263-73) and bioluminescent molecules (Xu et al., 1999, Proc. Natl. Acad. Sci. USA 96: 151-56) have been used as well. FRET (fluorescence resonance energy transfer) refers to a quantum mechanical effect between a given pair of chromophores, consisting of a fluorescence donor and respective acceptor. Prerequisites for FRET are proximity of donor and acceptor, and overlap between the donor emission spectrum and the acceptor excitation spectrum. When the donor and acceptor are in close enough vicinity, the emission of the excited donor decreases while emission of the sensitized acceptor increases (see Fehr et al., 2004, Current Opinion in Plant Biology 7: 345-51, herein incorporated by reference in its entirety).

[0010] There are two general types of FRET used by biosensors: intermolecular and intramolecular (Truong and Ikura, 2001, Current Opinion in Structural Biology 11: 573-78, herein incorporated by reference). Intermolecular FRET occurs when the fluorescent donor and acceptor molecules are on different macromolecules. This form of FRET is difficult to quantitate because the stoichiometry of acceptors to donors can vary with transfection efficiencies and expression levels. Nevertheless, several examples of intermolecular FRET have been reported (for a review, see Truong and Ikura, 2001; and Wouters et al., 2001, TRENDS in Cell Biol. 11(5): 203-11).

[0011] Intramolecular FRET occurs when both the donor and acceptor molecules are fused to the same molecule. In this type of sensor, the binding domains must undergo conformational changes that are large enough to translate metabolite binding into a change in FRET. Ideally, sensor families should share similar three-dimensional structures but have different substrate specificities that cover a wide spectrum of substrates. Furthermore, ultra-high-affinity binding in the nanomolar range would facilitate the engineering of mutant "nanosensors" for different physiological detection ranges by site-directed mutagenesis.

[0012] Some molecular sensors additionally employ a conformational actuator (most commonly a peptide which binds to one conformational state of the recognition element), to magnify the allosteric effect upon and resulting output of the reporter element (i.e., Miyakawa et al., 1997, Nature 388: 882-87). The applicability of the method in the absence of a conformational actuator, and its generalizability to a variety of analytes, has recently been demonstrated using bacterial periplasmic binding proteins (PBPs) (Fehr et al., 2002; Fehr et al., 2003, J. Biol. Chem. 278: 19127-33; and Lager et al., 2003, FEBS Lett. 553: 85-9).

[0013] Members of the bacterial PBP superfamily recognize hundreds of substrates with high affinity (atto- to low micro-molar) and specificity (Tam and Saier, 1993, Microbiol. Rev. 57: 320-46). PBPs have been shown by a variety of experimental techniques to undergo a significant conformational change upon ligand binding. Fusion of individual sugar-binding PBPs with a pair of GFP variants has produced sensors for maltose, ribose and glucose (Fehr et al., 2002; Fehr et al., 2003; and Lager et al., 2003). Moreover, PBPs bind substrates with affinities in the nanomolar range (Fehr et al., 2004). Thus, PBPs satisfy many of the criteria important for an ideal biosensor. The sensors have been used to measure sugar uptake and homeostasis in living animal cells, and sub-cellular analyte levels have been determined using nuclear-targeted versions (Fehr et al., 2004, J. Fluoresc. 14: 603-9).

[0014] Intramolecular biosensors are typically designed by fusing donor and acceptor fluorescent molecules to the amino and carboxy terminal portions of the sensor domain, respectively, which undergo a venus flytrap-like closure of two lobes upon substrate binding (see, e.g., Fehr et al, 2002; Fehr et al., 2003; Lager et al., 2003; and Truong and Ikura, 2001). Bacterial PBPs comprise two globular domains and are convenient scaffolds for designing FRET sensors (Fehr et al., 2003). The binding site is located in the cleft between the domains, and upon binding, the two domains engulf the substrate and undergo a hinge-twist motion (Quiocho and Ledvina, 1996, Mol. Microbiol. 20: 17-25).

[0015] PBPs can be divided into two types based on different topological arrangements of the central .beta.-sheets and position of the termini (Fukami-Kobayashi et al., 1999, J. Mol. Biol. 286: 279-290). Maltose binding protein (MBP) is a type II binding protein, with termini being located at the distal ends of the lobes relative to the hinge region. A comparison of the crystal structures of bound and unbound states shows that the hinge-twist motion brings the termini closer together. As would be expected in the case of maltose sensor, the decrease in distance upon maltose binding leads to increased FRET between attached chromophores (Fehr et al., 2002).

[0016] In GGBP (D-GalactoseD-Glucose Binding Protein) (a type I PBP), termini are located at the proximal ends of the two lobes (Fehr et al., 2004). Thus, because of the different chromophore positions, the substrate-induced hinge-twist motion is predicted to move the attached chromophores further apart, causing a decrease in FRET. Nevertheless, type I PBPs such as GGBP have also been used to construct efficient FRET biosensors containing terminally fused donor and acceptor fluorophores (Fehr et al., 2003).

[0017] The present inventors have now surprisingly found that fusion of fluorescent domains to internal positions of a ligand binding protein, even within the same lobe of a PBP sensor, facilitates the design of an efficient biosensor that demonstrates a similar ligand affinity and a substantially larger delta ratio than its terminally fused counterpart. This is counterintuitive in view of the general model for intramolecular FRET sensors, wherein the donor and acceptor molecules are fused to separate termini on separate lobes of the protein in order to maximize the change in orientation and/or distance of the donor and acceptor chromophores upon ligand binding.

[0018] The improved signal from these sensors can be ascribed to increased rigidity and thus reduced rotational averaging. The invention thus leads to an alternative approach, also disclosed herein, to improve sensors by using more rigidly conjugated reporters. To increase the rigidity and reduce rotational averaging, we deleted portions of the fusion proteins corresponding to residues not belonging to the core structure of the three contributing partners, i.e. omitting linker sequences at the fusion sites and deleting N- or C-terminal portions of either of the three modules. Consistent with the observations made for sensors using fusion of fluorescent domains to internal positions of a ligand binding protein, enhanced terminally fused sensors also showed much increased FRET ratio changes.

SUMMARY OF THE INVENTION

[0019] The present invention therefore provides improved intramolecular biosensors and nanosensors for detecting and measuring changes in analyte concentrations, particularly transporter biosensors and biosensors constructed using bacterial periplasmic binding proteins (PBPs). In particular, the invention provides intramolecular biosensors containing at least one internally fused fluorophore moiety, as well as FRET fusion constructs encoding fluorophores with increased rigidity.

[0020] For instance, the invention provides an isolated nucleic acid encoding a ligand binding fluorescent indicator comprising a ligand binding protein moiety wherein the ligand binding protein moiety is genetically fused to a donor fluorophore moiety and an acceptor fluorophore moiety, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and the ligand binds to the ligand binding protein moiety, and wherein at least one of either said donor fluorophore moiety or said acceptor fluorophore moiety is fused to said ligand binding protein moiety at an internal site of said ligand binding protein moiety. In one embodiment, among others, the donor and acceptor fluorophore moieties are fluorescent proteins.

[0021] The invention also provides methods of improving the sensitivity of intramolecular biosensors, including terminally and internally fused biosensors. For instance, such methods may comprise the steps of (a) providing an intramolecular FRET biosensor comprising a ligand binding protein moiety, and donor and acceptor fluorescent protein moieties fused to said ligand binding protein moiety, respectively, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and said ligand binds to the ligand binding protein moiety; and (b) altering or modifying the fusion domain between the fluorophore and ligand binding moieties, wherein said alteration results in an intramolecular FRET biosensor with improved sensitivity as compared to said biosensor without said alteration. The alteration may be an amino acid deletion, insertion or mutation that increases the rigidity of the fluorophore linkage. The invention also encompasses nucleic acid constructs produced by such methods.

[0022] Vectors, including expression vectors, and host cells comprising the inventive nucleic acids are also provided, as well as biosensor proteins encoded by the nucleic acids. Such nucleic acids, vectors, host cells and proteins may be used in methods of detecting changes in analyte levels, and in methods of identifying compounds that modulate ligand binding.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A. YbeJ FLIP-E nanosensor construct used for expression in E. coli containing terminally fused fluorophores.

[0024] FIG. 1B. YbeJ FLIP-E nanosensor construct used for expression in E. coli containing terminally fused fluorophores.

[0025] FIG. 1C. YbeJ FLIP-E nanosensor construct used for expression in neuronal cell culture containing terminally fused fluorophores.

[0026] FIG. 1D. YbeJ FLIP-E nanosensor construct used for expression in neuronal cell culture containing terminally fused fluorophores.

[0027] FIG. 2. Spectra of FLIP-E 600n sensor (fluorescent glutamate nanosensor with a Kd for glutamate of 600 nM) at three different concentrations of glutamate: 0 mM, at the Kd, and at saturation. Curves share an isosbestic point at 520 nm.

[0028] FIG. 3. A hippocampal cell treated with 1 mg/ml trypsin. Images were taken at 10 second intervals. Note that signals on the cell surface largely disappear.

[0029] FIG. 4A. Emission intensity ratio change in a hippocampal cell expressing FLIP-E 600n sensor. The images are pseudo-colored to indicate the emission intensity ratio change. Open bars above the graph indicate the time point of treatment (stimulation/perfusion with glutamate).

[0030] FIG. 4B. Ratio images at the time points indicated by arrows are shown. The change in emission intensity ratio was both observed upon electrical stimulation and upon perfusion with glutamate. The ratio change was not observed when perfusing with low levels of substrate (10 nM glutamate).

[0031] FIG. 5A. Emission intensity ratio change in a hippocampal cell expressing FLIP-E 10.mu. sensor (fluorescent glutamate nanosensor with a Kd for glutamate of 10 .mu.M). Open bars above the graph indicate the time point of treatment (stimulation/perfusion with glutamate).

[0032] FIG. 5B. Ratio images at the time points indicated by arrow are shown. Electrical stimulation did not cause a large change in the emission intensity ratio, whereas perfusion with 100 .mu.M glutamate induces a reversible ratio change (c and e).

[0033] FIG. 6A. Internally fused pRSETB FLIP-E nanosensor construct showing insertion site for eCFP.

[0034] FIG. 6B. Internally fused pRSETB FLIP-E nanosensor construct showing insertion site for eCFP.

[0035] FIG. 7A. Graph comparing emission intensity of FLIP-E 600n with and without glutamate.

[0036] FIG. 7B. Graph comparing emission intensity of FLIP-E-internally-fused with and without glutamate.

[0037] FIG. 8A. Internally fused pRSETB FLIP-E 600n A216-cpVenus-K217 construct showing insertion site for cpVenus.

[0038] FIG. 8B. Internally fused pRSETB FLIP-E 600n A216-cpVenus-K217 construct showing insertion site for cpVenus.

[0039] FIG. 9. Emission intensity of internally fused FLIP-E 600n A216-cpVenus-K217 with and without glutamate.

[0040] FIG. 10A. Graph showing the ratio changes of internally fused glucose nanosensors.

[0041] FIG. 10B. Graph showing the normalized ratio changes of internally fused glucose nanosensors.

[0042] FIG. 11A. Graph showing titration curve of glucose nanosensors.

[0043] FIG. 11B. Graph showing spectra of glucose nanosensors.

[0044] FIG. 11C. Graph showing titration curve of glucose nanosensor.

[0045] FIG. 11D. Graph showing spectra of glucose nanosensors.

[0046] FIG. 11E. Graph showing titration curve of glucose nanosensor.

[0047] FIG. 11F. Graph showing spectra of glucose nanosensors.

[0048] FIG. 11G. Graph showing titration curve of glucose nanosensor.

[0049] FIG. 11H. Graph showing spectra of glucose nanosensors.

[0050] FIG. 11I. Graph showing titration curve of glucose nanosensor.

[0051] FIG. 11J. Graph showing spectra of glucose nanosensors.

[0052] FIG. 11K. Graph showing titration curve of glucose nanosensor.

[0053] FIG. 11L. Graph showing spectra of glucose nanosensors.

[0054] FIG. 11M. Graph showing titration curve of glucose nanosensor.

[0055] FIG. 11N. Graph showing spectra of glucose nanosensors.

[0056] FIG. 12. Figure showing the correlation between the starting ratio in the absence of glucose and the normalized ratio change.

[0057] FIG. 13. Diagram showing the various deletions constructed in the coding sequence of FLIPglu 600.mu. and the corresponding delta ratios obtained.

[0058] FIG. 14A. Construction of the FLII.sup.12Pglu-600.mu. and FLII.sup.275Pglu-4.6m deletion sensors. The N-terminal ECFP core is boxed blue. The dispensable C-terminal sequences of ECFP are underlined in blue. The flexible linker containing a KpnI restriction enzyme recognition site is shown in black. The mglB core is boxed red, while the dispensable C-terminal residues of mglB are underlined red. The EYFP core is boxed yellow, while the dispensable N-terminal residues are underlined yellow. Construct names are labeled on the left.

[0059] FIG. 14B. Construction of the FLII.sup.12Pglu-600.mu. and FLII.sup.275Pglu-4.6m deletion sensors.

[0060] FIG. 15. Correlation between .DELTA. ratio in MOPS buffer pH 7.0 (red), number of amino acid residues deleted and affinity (Kd, .mu.M) of the FLII.sup.12Pglu-600.mu. deletion constructs.

[0061] FIG. 16. Sensitivity of the FLII.sup.12Pglu-600.mu. deletion constructs to cell culture solution, synthetic cytosols and pH. Comparison of the A ratio FLII.sup.12Pglu-600.mu. deletion constructs in MOPS pH 7.0 (black), Hanks buffer pH 7.2 (red), Mammalian cytosol pH 7.4 (blue), plant cytosol pH 7.5 (green) and MOPS pH 5.0 (purple). FLII.sup.12Pglu-10aa, FLII.sup.12Pglu-14aa, FLII.sup.12Pglu-15aa and FLII.sup.12Pglu.delta.6 can be seen as the sensors least affected by different buffers and low pH.

[0062] FIG. 17. Diagram showing the constructs of three intramolecular glucose sensors: FLII.sup.12Pglu-600.mu.; FLII12Pglu.delta.4aa-593.mu., and FLII.sup.275Pglu-4600.mu..

[0063] FIG. 18. Diagram showing the FLIP constructs in pc DNA3.1.

[0064] FIG. 19A. FRET changes observed in NIH3T3 cells transformed with the improved glucose sensors. Perfusion of NIH3T3-L1 cells transiently cytosolic expressing by FLII.sup.12Pglu-600.mu., The bars indicate the presence of 10 mM glucose in the perfusion buffer.

[0065] FIG. 19B. FRET changes observed in NIH3T3 cells transformed with the improved glucose sensors. Perfusion of NIH3T3-L1 cells transiently cytosolic expressing by FLII.sup.12Pglu.delta.4aa-593.mu.. The bars indicate the presence of 10 mM glucose in the perfusion buffer.

[0066] FIG. 19C. FRET changes observed in NIH3T3 cells transformed with the improved glucose sensors. Perfusion of NIH3T3-L1 cells transiently cytosolic expressing by FLII.sup.275Pglu-4600.mu.. The bars indicate the presence of 10 mM glucose in the perfusion buffer.

DETAILED DESCRIPTION OF INVENTION

[0067] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0068] Other objects, advantages and features of the present invention become apparent to one skilled in the art upon reviewing the specification and the drawings provided herein. Thus, further objects and advantages of the present invention will be clear from the description that follows.

Internally Fused Intramolecular Biosensors

[0069] As described above, the present inventors have surprisingly found that fusion of fluorescent domains to internal positions of a ligand binding protein, even within the same lobe of a PBP sensor, facilitates the design of an efficient biosensor that demonstrates a similar ligand affinity and a substantially larger delta ratio than its terminally fused counterpart. This is counterintuitive in view of the general model for intramolecular FRET sensors, wherein the donor and acceptor molecules are typically fused to the termini on separate lobes of the protein in order to maximize the change in orientation and/or distance of the donor and acceptor chromophores upon ligand binding.

[0070] Without being bound to any particular theory, the present inventors believe that the data supports the prediction that rotational movements play a key role in FRET. The dipoles must be oriented in a certain position to each other for efficient resonance energy transfer. However, with terminally fused donor and acceptor moieties, one commonly assumes that the peptide bonds in the linker between the three moieties are freely rotating, thus randomizing this parameter, within a cone of steric compatibility. By inserting the fluorescent moiety into an internal position of the PBP, free or limited free rotation of the fluorophore around the peptide axis in the linker sequences is prevented, or greatly reduced. Thus, in an internal fusion, the fluorescent moiety is rigidly inserted at both ends, thereby reducing free rotation and possibly explaining the higher observed delta ratio. Alternatively, more rigidly fused chromophores enable enhanced allosteric coupling between the conformational change of the binding protein and the motion of the chromophore.

[0071] Thus, the biosensors of the present invention exhibit surprisingly enhanced activities over their terminally fused counterparts. Moreover, in some cases, internally fused donor and acceptor molecules permits the measurement of FRET increases upon ligand binding using sensors that typically operate by decreased FRET upon ligand binding, such as GGBP sensors. Thus, the direction of FRET alteration may be changed by using internally fused donor and/or acceptor moieties as compared to terminally fused counterparts.

[0072] The present invention encompasses isolated nucleic acids which encode ligand binding fluorescent indicator. An isolated nucleic acid according to the present invention encodes an indicator comprising a ligand binding protein moiety, a donor fluorophore moiety fused to the ligand binding protein moiety, and an acceptor fluorophore moiety fused to the ligand binding protein moiety, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and said ligand binds to the ligand binding protein moiety, and wherein at least one of either or both of said donor fluorophore moiety and/or said acceptor fluorophore moiety are fused to said ligand binding protein moiety at an internal site of said ligand binding protein moiety.

[0073] Either the donor fluorophore moiety or the acceptor fluorophore moiety or both may be fused to an internal site of said ligand binding protein moiety. Preferably, the donor and acceptor moieties are not fused in tandem, although the donor and acceptor moieties may be contained on the same protein domain or lobe. A domain is a portion of a protein that performs a particular function and is typically at least about 40 to about 50 amino acids in length. There may be several protein domains contained in a single protein. A "ligand binding protein moiety" according to the present invention can be a complete, naturally occurring protein sequence, or at least the ligand binding portion or portions thereof. In preferred embodiments, among others, a ligand binding moiety of the invention is at least about 40 to about 50 amino acids in length, or at least about 50 to about 100 amino acids in length, or more than about 100 amino acids in length.

Methods of Improving Sensitivity of FRET Biosensors

[0074] As described above, the invention also provides methods of improving the sensitivity of intramolecular biosensors, including terminally and internally fused biosensors. For instance, such methods may comprise the steps of (a) providing an intramolecular FRET biosensor comprising a ligand binding protein moiety, and donor and acceptor fluorescent protein moieties fused to the two termini of said ligand binding protein moiety, respectively, wherein fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety is altered when the donor moiety is excited and said ligand binds to the ligand binding protein moiety; and (b) altering or modifying the fusion domain between the fluorophore and ligand binding moieties, wherein said alteration results in an intramolecular FRET biosensor with improved sensitivity as compared to said biosensor without said alteration. The alteration may be a deletion, insertion or mutation of one or more amino acids from the linker, fluorophore or ligand binding domains that increases the rigidity of the fluorophore linkage.

[0075] The disclosed methods of improving FRET biosensor sensitivity stem from the present inventors' observations regarding internally fused FRET sensors. Having learned that the reduced rotational averaging in the intramolecular insertion of a fluorophores is a general strategy to generate sensors with high ratio changes, we hypothesized that one may obtain similar results by reducing the rotational freedom of the linkage between the analyte binding domain and the fluorophores. To test the hypothesis, we systematically removed sequences that connect the core protein structure of the binding domain and the fluorophore, i.e. by removing linker sequences and/or by deleting amino acids from the ends of the analyte binding moiety and/or the fluorophores. We found that the closer coupling achieved by such deletions also leads to higher ratio changes. This concept is exemplified herein for glucose binding constructs, but is applicable to any FRET-based biosensor.

[0076] Preferably, deletions are made by deleting at least one, or at least two, or at least three, or at least four, or at least five, or at least eight, or at least ten, or at least fifteen nucleotides in a nucleic acid construct encoding said intramolecular FRET biosensor that are located in the regions encoding the linker, or fluorophore, or ligand binding domains. Deletions in different regions may be combined in a single construct to create more than one region demonstrating increased rigidity. Amino acids may also be added or mutated to increase rigidity of the biosensor and improve sensitivity. For instance, by introducing a kink by adding a proline residue or other suitable amino acid. Improved sensitivity is measured by the ratio change in FRET fluorescence upon ligand binding, and preferably increases by at least a factor of 2 as a result of said deletion.

[0077] The invention also encompasses nucleic acid constructs produced by such methods, as well as vectors and cells containing the nucleic acids as described herein. The FRET biosensors encoded by the nucleic acid constructs are also included.

Ligand Binding Moieties

[0078] Preferred ligand binding protein moieties according to the present invention, among others, are transporter proteins and ligand binding sequences thereof, for instance transporters selected from the group consisting of channels, uniporters, coporters and antiporters. Also preferred are periplasmic binding proteins (PBP), such as any of the bacterial PBPs included in Table 1 below. As described above, bacterial PBPs comprise two globular domains or lobes and are convenient scaffolds for designing FRET sensors (Fehr et al., 2003). The binding site is located in the cleft between the domains, and upon binding, the two domains engulf the substrate and undergo a hinge-twist motion (Quiocho and Ledvina, 1996, Mol. Microbiol. 20: 17-25). In type I PBPs, such as GGBP (D-GalactoseD-Glucose Binding Protein), the termini are located at the proximal ends of the two lobes that move apart upon ligand binding (Fehr et al., 2004). In type II PBPs, such as Maltose Binding Protein (MBP), the termini are located at the distal ends of the lobes relative to the hinge region and come closer together upon ligand binding. Thus, depending on the type of PBP and/or the position of the internally fused donor or acceptor moiety, FRET may increase or decrease upon ligand binding and both instances are included in the present invention.

TABLE-US-00001 TABLE 1 Bacterial Periplasmic Binding Proteins Gene name Substrate Species 3D Reference AccA agrocinopine Agrobacterium sp. --/-- J. Bacteriol. (1997) 179, 7559-7572 AgpE alpha-glucosides (sucrose, maltose, Rhizobium meliloti --/-- J. Bacteriol. (1999) 181, 4176-4184 trehalose) AlgQ2 alginate Sphingomonas sp. --/c J. Biol. Chem. (2003) 278, 6552-6559 AlsB allose E. coli --/c J. Bacteriol. (1997) 179, 7631-7637 J. Mol. Biol. (1999) 286, 1519-1531 AraF arabinose E. coli --/c J. Mol. Biol. (1987) 197, 37-46 J. Biol. Chem. (1981) 256, 13213-13217 AraS arabinose/fructose/xylose Sulfolobus solfataricus --/-- Mol. Microbiol. (2001) 39, 1494-1503 ArgT lysine/arginine/ornithine Salmonella typhimurium o/c Proc. Natl. Acad. Sci. USA (1981) 78, 6038-6042 J. Biol. Chem. (1993) 268, 11348-11355 ArtI arginine E. coli Mol. Microbiol. (1995) 17, 675-686 ArtJ arginine E. coli Mol. Microbiol. (1995) 17, 675-686 b1310 (putative, multiple sugar) E. coli --/-- NCBI accession A64880 b1487 (putative, oligopeptide binding) E. coli --/-- NCBI accession B64902 b1516 (sugar binding protein homolog) E. coli --/-- NCBI accession G64905 BtuF vitamin B12 E. coli --/-- J. Bacteriol. (1986) 167, 928-934 CAC1474 proline/glycine/betaine Clostridium --/-- NCBI accession AAK79442 acetobutylicum cbt dicarboxylate E. coli --/-- J. Supramol. Struct. (1977) 7, 463-80 (succinate, malate, fumarat) J. Biol. Chem. (1978) 253, 7826-7831 J. Biol. Chem. (1975) 250, 1600-1602 CbtA cellobiose Sulfoblobus solfataricus --/-- Mol. Microbiol. (2001) 39, 1494-1503 ChvE sugar Agrobacterium --/-- J. Bacteriol. (1990) 172, 1814-1822 tumefaciens CysP thiosulfate E. coli --/-- J. Bacteriol. (1990) 172, 3358-3366 DctP C4-dicarboxylate Rhodobacter capsulatus --/-- Mol. Microbiol. (1991) 5, 3055-3062 DppA dipeptides E. coli o/c Biochemistry (1995) 34, 16585-16595 FbpA iron Neisseria gonorrhoeae --/c J. Bacteriol. (1996) 178, 2145-2149 FecB Fe(III)-dicitrate E. coli J. Bacteriol. (1989) 171, 2626-2633 FepB enterobactin-Fe E. coli --/-- J. Bacteriol. (1989) 171, 5443-5451 Microbiology (1995) 141, 1647-1654 FhuD ferrichydroxamate E. coli --/c Mol. Gen. Genet. (1987) 209, 49-55 Nat. Struct. Biol. (2000) 7, 287-291 Mol. Gen. Genet. (1987) 209, 49-55 FliY cystine E. coli --/-- J. Bacteriol. (1996) 178, 24-34 NCBI accession P39174 GlcS glucose/galactose/mannose Sulfolobus solfataricus --/-- Mol. Microbiol. (2001) 39, 1494-1503 GlnH glutamine E. coli o/-- Mol. Gen. Genet. (1986) 205, 260-9 (protein: J. Mol. Biol. (1996) 262, 225-242 GLNBP) J. Mol. Biol. (1998) 278, 219-229 GntX gluconate E. coli --/-- J. Basic. Microbiol. (1998) 38, 395-404 HemT haemin Yersinia enterocolitica --/-- Mol. Microbiol. (1994) 13, 719-732 HisJ histidine E. coli --/c Biochemistry (1994) 33, 4769-4779 (protein: HBP) HitA iron Haemophilus influenzae o/c Nat. Struct. Biol. (1997) 4, 919-924 Infect. Immun. (1994) 62, 4515-25 J. Biol. Chem. (195) 270, 25142-25149 LivJ leucine/valine/isoleucine E. coli --/c J. Biol. Chem. (1985) 260, 8257-8261 J. Mol. Biol. (1989) 206, 171-191 LivK leucine E. coli --/c J. Biol. Chem. (1985) 260, 8257-8261 (protein: L- J. Mol. Biol. (1989) 206, 193-207 BP) MalE maltodextrine/maltose E. coli o/c Structure (1997) 5, 997-1015 (protein: J. Bio.l Chem. (1984) 259, 10606-13 MBP) MglB glucose/galactose E. coli --/c J. Mol. Biol. (1979) 133, 181-184 (protein: Mol. Gen. Genet. (1991) 229, 453-459 GGBP) ModA molybdate E. coli --/c Nat. Struct. Biol. (1997) 4, 703-707 Microbiol. Res. (1995) 150, 347-361 MppA L-alanyl-gamma-D-glutamyl-meso- E. coli J. Bacteriol. (1998) 180, 1215-1223 diaminopimelate NasF nitrate/nitrite Klebsiella oxyloca --/-- J. Bacteriol. (1998) 180, 1311-1322 NikA nickel E. coli --/-- Mol. Microbiol. (1993) 9, 1181-1191 opBC choline Bacillus subtilis --/-- Mol. Microbiol. (1999) 32, 203-216 OppA oligopeptide Salmonella typhimurium o/c Biochemistry (1997) 36, 9747-9758 Eur. J. Biochem. (1986) 158, 561-567 PhnD alkylphosphonate E. coli --/-- J. Biol. Chem. (1990) 265, 4461-4471 PhoS (Psts) phosphate E. coli --/c J. Bacteriol. (1984) 157, 772-778 Nat. Struct. Biol. (1997) 4, 519-522 PotD putrescine/spermidine E. coli --/c J. Biol. Chem. (1996) 271, 9519-9525 PotF polyamines E. coli --/c J. Biol. Chem. (1998) 273, 17604-17609 ProX betaine E. coli J. Biol. Chem. (1987) 262, 11841-11846 rbsB ribose E. coli o/c J. Biol. Chem. (1983) 258, 12952-6 J. Mol. Biol. (1998) 279, 651-664 J. Mol. Biol. (1992) 225, 155-175 SapA peptides Salmonella typhimurium --/-- EMBO J. (1993) 12, 4053-4062 Sbp sulfate Salmonella typhimurium --/c J. Biol. Chem. (1980) 255, 4614-4618 Nature (1985) 314, 257-260 TauA taurin E. coli --/-- J. Bacteriol. (1996) 178, 5438-5446 TbpA thiamin E. coli --/-- J. Biol. Chem. (1998) 273, 8946-8950 TctC tricarboxylate Salmonella typhimurium --/-- ThuE trehalose/maltose/sucrose Sinorhizobium meliloti --/-- J. Bacteriol. (2002) 184, 2978-2986 TreS trehalose Sulfolobus solfataricus --/-- Mol. Microbiol. (2001) 39, 1494-1503 tTroA zinc Treponema pallidum --/c Gene (1997) 197, 47-64 Nat. Struct. Biol. (1999) 6, 628-633 UgpB sn-glycerol-3-phosphate E. coli --/-- Mol. Microbiol. (1988) 2, 767-775 XylF xylose E. coli --/-- Receptors Channels (1995) 3, 117-128 YaeC unknown E. coli --/-- J Bacteriol (1992) 174, 8016-22 NCBI accession P28635 YbeJ(GltI) glutamate/aspartate (putative, E. coli --/-- NCBI accession E64800 superfamily: lysine-arginine-ornithine- binding protein) YdcS (putative, spermidine) E. coli --/-- NCBI accession P76108 (b1440) YehZ unknown E. coli --/-- NCBI accession AE000302 YejA (putative, homology to periplasmic E. coli --/-- NCBI accession AAA16375 oligopeptide-binding protein - Helicobacter pylori) YgiS oligopeptides E. coli --/-- NCBI accession Q46863 (b3020) YhbN unknown E. coli --/-- NCBI accession P38685 YhdW (putative, amino acids) E. coli --/-- NCBI accession AAC76300 YliB (b0830) (putative, peptides) E. coli --/-- NCBI accession P75797 YphF (putative sugars) E. coli --/-- NCBI accession P77269 Ytrf acetoin B. subtilis --/-- J. Bacteriol. (2000) 182, 5454-5461 ZnuA zinc Synechocystis --/-- J. Mol. Biol. (2003) 333, 1061-1069

[0079] Bacterial PBPs have the ability to bind a variety of different molecules and nutrients, including sugars, amino acids, vitamins, minerals, ions, metals and peptides, as shown in Table 1. Thus, PBP-based ligand binding sensors may be designed to permit detection and quantitation of any of these molecules according to the methods of the present invention. Naturally occurring species variants of the PBPs listed in Table 1 may also be used, in addition to artificially engineered variants comprising site-specific mutations, deletions or insertions that maintain measurable ligand binding function. Variant nucleic acid sequences suitable for use in the nucleic acid constructs of the present invention will preferably have at least 70, 75, 80, 85, 90, 95, or 99% similarity or identity to the native gene sequence for a given PBP.

[0080] Suitable variant nucleic acid sequences may also hybridize to the gene for a PBP under highly stringent hybridization conditions. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10.degree. C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0081] Preferred biosensors of the present invention, among others, include glutamate sensors constructed using YbeJ binding domains, and other amino acid biosensors. Such proteins may be used as neurotransmitter biosensors for detecting and measuring changes in neurotransmitter concentrations using Fluorescence Resonance Energy Transfer (FRET) (see U.S. provisional applications 60/618,179, herein incorporated by reference in their entirety). The three major categories of substances that act as neurotransmitters are (1) amino acids (primarily glutamic acid or glutamate, GABA, aspartic acid & glycine), (2) peptides (vasopressin, somatostatin, neurotensin, etc.) and (3) monoamines (norepinephrine, dopamine & serotonin) plus acetylcholine. In particular, the invention provides glutamate binding fluorescent indicators, particularly indicators comprising a glutamate binding protein moiety from the Escherichia coli glutamate/aspartate receptor, YbeJ. Additional neurotransmitter biosensors for the neurotransmitters listed above may also be prepared using the constructs and methods provided herein.

[0082] YbeJ is also known in the art as YzzK and GltI, and its DNA sequence (SEQ ID No. 27) and protein sequence (YbeJ, protein accession no. NP_415188, SEQ ID No. 28) are known. SEQ ID Nos. 1 and 2 provide alternative nucleic acid and protein sequences for YbeJ, respectively, and include additional upstream material that may be part of the full length protein. Naturally occurring homologues from other bacterial species may also be used, for instance the PA5082 gene from Pseudomonas aeruginosa, whose gene product is 70% similar to the YbeJ protein from E. coli. Any portion of the YbeJ DNA sequence which encodes a glutamate binding region may be used in the nucleic acids of the present invention. Glutamate binding portions of YbeJ or any of its homologues may be cloned into the vectors described herein and screened for activity according to the disclosed assays.

[0083] For instance, one region that is suitable for use in the nucleic acids of the present invention is provided by SEQ ID No. 3, which encodes a truncated glutamate-aspartate binding protein sequence (SEQ ID No. 4), encoding mature protein without signal peptide. A preferred internally fused intramolecular sensor according to the present invention comprises a fluorescent protein moiety inserted between amino acids corresponding to amino acids 58 and 59, and amino acid 216 and 217 of SEQ ID No. 28. In preferred embodiments, the donor fluorescent protein moiety is eCFP, however any of the donor moieties described herein may be used. In such sensors, the acceptor fluorescent protein moiety is preferably YFP VENUS or cpVenus, inserted at the C-terminus of said glutamate binding protein moiety or internally fused to said glutamate binding protein. Further, other acceptor moieties may be used, as described herein.

[0084] Preferred artificial variants of the sensors of the present invention may exhibit increased or decreased affinity for ligands, in order to expand the range of ligand concentration that can be measured. For instance, preferred artificial variants for YbeJ sensors include, among others, glutamate binding regions comprising the mutations A207G, A207P, A207K, A207M, A2075, A207C, A207R, A207V, A207L, A207Q, A207T, A207F, A207Y, A207N, A207W, A207H, A207D, and S95W. Additional artificial variants showing decreased or increased binding affinity for glutamate may be constructed by random or site-directed mutagenesis and other known mutagenesis techniques, and cloned into the vectors described herein and screened for activity according to the disclosed assays.

[0085] The sensors of the invention may also be designed with a reporter element different from a donor/acceptor pair of FRET-compatible fluorescent proteins. For instance, the ligand-binding moiety of the sensor may be fused with an enzyme in such a manner to create an allosterically regulated enzyme whose activity is regulated by a specified ligand (Guntas and Ostermeier, 2004, J. Mol. Biol. 336(1): 263-73). In addition, such an allosterically-regulated reporter domain may be divided into two or more separate and complementing halves, e.g. complementing fragments of .beta.-lactamase (Galarneau et al., 2002, Nature Biotechnol. 20: 619-622) or of GFP (Cabantous et al., 2005, Nature Biotechnol. 23: 102-107). Any and all reporter element fragments may be fused with the ligand-binding moiety in either an end-to-end fashion (e.g. a typical fusion protein) or inserted internally into the sequence of the ligand-binding moiety (e.g. an internally-fused fluorescent protein as described herein).

[0086] Other preferred PBPs to be used in the present invention include sugar binding proteins, such as maltose binding protein (MBP) and galactose/glucose binding protein (GGBP). Glucose sensors, such as GGBP sensors of the present invention, may be used for measuring blood glucose levels, for instance in diabetes or pregnancy. Other preferred ligand-binding moieties which provide a global conformational change in response to ligand binding include, but are not limited to, nuclear hormone receptors, lipocalins, fatty acid-binding proteins, and antibodies. Also possible are inactivated enzymes, including but not limited to, hexokinase, glucokinase, ribokinase, and any other conformationally responsive enzyme or enzyme domain.

General Materials and Methods

[0087] The isolated nucleic acids of the invention may incorporate any suitable donor and acceptor fluorescent protein moieties that are capable in combination of serving as donor and acceptor moieties in FRET. Preferred donor and acceptor moieties are selected from the group consisting of GFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), and enhanced variants thereof, with a particularly preferred embodiment provided by the donor/acceptor pair CFP/YFP-Venus, a variant of YFP with improved pH tolerance and maturation time (Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87-90). An alternative is the MiCy/mKO pair with higher pH stability and a larger spectral separation (Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J. 2004 381:307-12). Also suitable as either a donor or acceptor is native DsRed from a Discosoma species, an ortholog of DsRed from another genus, or a variant of a native DsRed with optimized properties (e.g. a K83M variant or DsRed2 (available from Clontech)). Criteria to consider when selecting donor and acceptor fluorescent moieties are known in the art, for instance as disclosed in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

[0088] As used herein, the term "variant" is intended to refer to polypeptides with at least about 70%, more preferably at least 75% identity, including at least 80%, 90%, 95% or greater identity to native fluorescent molecules. Many such variants are known in the art, or can be readily prepared by random or directed mutagenesis of native fluorescent molecules (see, for example, Fradkov et al., FEBS Lett. 479:127-130 (2000)).

[0089] When the fluorophores of the biosensor contain stretches of similar or related sequence(s), the present inventors have recently discovered that gene silencing may adversely affect expression of the biosensor in certain cells and particularly whole organisms. In such instances, it is possible to modify the fluorophore coding sequences at one or more degenerate or wobble positions of the codons of each fluorophore, such that the nucleic acid sequences of the fluorophores are modified but not the encoded amino acid sequences. Alternative, one or more conservative substitutions that do not adversely affect the function of the fluorophores may also be incorporated. See PCT application [PCT/US2005/036953, "Methods of Reducing Repeat-Induced Silencing of Transgene Expression and Improved Fluorescent Biosensors], which is herein incorporated by reference in its entirety.

[0090] It is also possible to use dyes for FRET, alone or in combination with one or more of the fluorophores listed above, including but not limited to TOTO dyes (Laib and Seeger, 2004, J Fluoresc. 14(2):187-91), Cy3 and Cy5 (Churchman et al., 2005, Proc Natl Acad Sci US A. 102(5): 1419-23), Texas Red, fluorescein, and tetramethylrhodamine (TAMRA) (Unruh et al., Photochem Photobiol. 2004 Oct. 1), AlexaFluor 488, to name a few, as well as fluorescent tags (see, for example, Hoffman et al., 2005, Nat. Methods 2(3): 171-76).

[0091] It is also possible to use luminescent quantum dots (QD) or pebble-coupled approaches for FRET (Clapp et al., 2005, J. Am. Chem. Soc. 127(4): 1242-50; Medintz et al., 2004, Proc. Natl. Acad. Sci. USA 101(26): 9612-17; Buck et al., 2004, Curr. Opin. Chem. Biol. 8(5): 540-6), including Surface-Enhanced Raman Scattering, where sensors are bound to the surface of nanoparticles and detection is achieved by Raman spectroscopy (Haes and Van Duyne, 2004, Expert Rev. Mol. Diagn. 4(4): 527-37).

[0092] Bioluminescence resonance energy transfer (BRET) may also be used for both in vitro and in vivo measurements, and offers the advantages of FRET without the consequences of fluorescence excitation. BRET is a naturally occurring phenomenon. For instance, when the photoprotein aequorin is purified from the jellyfish, Aequorea, it emits blue light in the absence of GFP, but when GFP and aequorin are associated as they are in vivo, GFP accepts the energy from aequorin and emits green light. In BRET, the donor fluorophore of the FRET technique is replaced by a luciferase. In the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore through the same Forster resonance energy transfer mechanisms described above. Thus, by using a luciferase/GFP mutant or other fluorophore combination, BRET can be used to measure protein interactions both in vivo and in vitro (see Xu et al, 1999, Proc. Natl. Acad. Sci. USA 96: 151-56, which is herein incorporated by reference).

[0093] The invention further provides vectors containing isolated nucleic acid molecules encoding improved and internally fused biosensor polypeptides as disclosed herein. Exemplary vectors include vectors derived from a virus, such as a bacteriophage, a baculovirus or a retrovirus, and vectors derived from bacteria or a combination of bacterial sequences and sequences from other organisms, such as a cosmid or a plasmid. Such vectors include expression vectors containing expression control sequences operatively linked to the nucleic acid sequence coding for the neurotransmitter biosensor. Vectors may be adapted for function in a prokaryotic cell, such as E. coli or other bacteria, or a eukaryotic cell, including yeast and animal cells. For instance, the vectors of the invention will generally contain elements such as an origin of replication compatible with the intended host cells, one or more selectable markers compatible with the intended host cells and one or more multiple cloning sites. The choice of particular elements to include in a vector will depend on factors such as the intended host cells, the insert size, whether regulated expression of the inserted sequence is desired, i.e., for instance through the use of an inducible or regulatable promoter, the desired copy number of the vector, the desired selection system, and the like. The factors involved in ensuring compatibility between a host cell and a vector for different applications are well known in the art.

[0094] Preferred vectors for use in the present invention will permit cloning of the ligand binding domain or receptor genetically fused to nucleic acids encoding donor and acceptor fluorescent molecules, resulting in expression of a chimeric or fusion protein comprising the ligand binding domain genetically fused to donor and acceptor fluorescent molecules. Exemplary vectors include the bacterial pRSET-FLIP derivatives disclosed in Fehr et al. (2002) (Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851), which is herein incorporated by reference in its entirety. Methods of cloning nucleic acids into vectors in the correct frame so as to express fusion proteins are well known in the art.

[0095] The chimeric internally fused nucleic acids of the present invention are preferably constructed such that either or both the donor and acceptor fluorescent moiety coding sequences are fused to internal positions of the ligand binding protein sequence upon expression in a manner such that changes in FRET between donor and acceptor may be detected upon ligand binding. Fluorescent domains can optionally be separated from the ligand binding domain by one or more flexible linker sequences. Such linker moieties are preferably between about 1 and 50 amino acid residues in length, and more preferably between about 1 and 30 amino acid residues. Linker moieties and their applications are well known in the art and described, for example, in U.S. Pat. Nos. 5,998,204 and 5,981,200, and Newton et al., Biochemistry 35:545-553 (1996). Alternatively, shortened versions of the fluorophores or the binding proteins described herein may be used.

[0096] The invention also includes host cells transfected with a vector or an expression vector of the invention, including prokaryotic cells, such as E. coli or other bacteria, or eukaryotic cells, such as yeast cells or animal cells. In another aspect, the invention features a transgenic non-human animal having a phenotype characterized by expression of the nucleic acid sequence coding for the expression of the biosensor. The phenotype is conferred by a transgene contained in the somatic and germ cells of the animal, which may be produced by (a) introducing a transgene into a zygote of an animal, the transgene comprising a DNA construct encoding the biosensor; (b) transplanting the zygote into a pseudopregnant animal; (c) allowing the zygote to develop to term; and (d) identifying at least one transgenic offspring containing the transgene. The step of introducing of the transgene into the embryo can be by introducing an embryonic stem cell containing the transgene into the embryo, or infecting the embryo with a retrovirus containing the transgene. Transgenic animals of the invention include transgenic C. elegans and transgenic mice and other animals.

[0097] The present invention also encompasses isolated improved and internally fused biosensor molecules having the properties described herein, particularly PBP-based fluorescent indicators. Such polypeptides are preferably recombinantly expressed using the nucleic acid constructs described herein. The expressed polypeptides can optionally be produced in and/or isolated from a transcription-translation system or from a recombinant cell, by biochemical and/or immunological purification methods known in the art. The polypeptides of the invention can be introduced into a lipid bilayer, such as a cellular membrane extract, or an artificial lipid bilayer (e.g. a liposome vesicle) or nanoparticle.

[0098] The present invention includes methods of detecting changes in the levels of ligands in samples, comprising (a) providing a cell expressing a nucleic acid encoding an improved or internally fused sensor according to the present invention and a sample comprising said ligand; and (b) detecting a change in FRET between said donor fluorescent protein moiety and said acceptor fluorescent protein moiety, wherein a change in FRET between said donor moiety and said acceptor moiety indicates a change in the level of said ligand in the sample. The ligand may be any suitable ligand for which a fused FRET biosensor may be constructed, including any of the ligands described herein. Preferably the ligand is one recognized by a PBP, and more preferably a bacterial PBP, such as those included in Table 1 and homologues and natural and artificial variants thereof.

[0099] The amino acid binding sensors of the present invention are useful for detecting and measuring changes in the levels of neurotransmitters in the brain or nervous system of an animal, particularly changes in the level of extracellular glutamate, which can be a signal of a disorder or disease associated with glutamate excitotoxicity. In one embodiment, the invention comprises a method of detecting changes in the level of extracellular glutamate in a sample of neurons, comprising (a) providing a cell expressing a nucleic acid encoding a glutamate binding biosensor as described herein and a sample of neurons; and (b) detecting a change in FRET between a donor fluorescent protein moiety and an acceptor fluorescent protein moiety, each covalently attached to the glutamate binding domain, wherein a change in FRET between said donor moiety and said acceptor moiety indicates a change in the level of extracellular glutamate in the sample of neurons. Alternatively, the protein may be produced in a heterologous host, e.g. in bacteria, purified and injected into organs directly or into the intercellular spaces. The protein or derivatives thereof may also be coupled to particles including quantum dots and introduced into cells or compartments.

[0100] FRET may be measured using a variety of techniques known in the art. For instance, the step of determining FRET may comprise measuring light emitted from the acceptor fluorescent protein moiety. Alternatively, the step of determining FRET may comprise measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety. The step of determining FRET may also comprise measuring the excited state lifetime of the donor moiety or anisotropy changes (Squire A, Verveer P J, Rocks O, Bastiaens P I. J Struct Biol. 2004 July; 147(1):62-9. Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells.). Such methods are known in the art and described generally in U.S. Pat. No. 6,197,928, which is herein incorporated by reference in its entirety.

[0101] The amount of ligand in a sample can be determined by determining the degree of FRET. First the sensor must be introduced into the sample. Changes in ligand concentration can be determined by monitoring FRET changes at time intervals. The amount of ligand in the sample can be quantified for example by using a calibration curve established by titration in vivo.

[0102] The sample to be analyzed by the methods of the invention may be contained in vivo, for instance in the measurement of ligand transport on the surface of cells, or in vitro, wherein ligand efflux may be measured in cell culture. Alternatively, a fluid extract from cells or tissues may be used as a sample from which ligands are detected or measured. With amino acid sensors such as glutamate sensors, such measurements may be used to detect extracellular glutamate associated with traumatic injury to said neurons, or as a possible indicator of a neurological disorder associated with glutamate excitotoxicity, including stroke, epilepsy, Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis, among others.

[0103] Methods for detecting ligands as disclosed herein may be used to screen and identify compounds that may be used to modulate ligand receptor binding. In one embodiment, among others, the invention comprises a method of identifying a compound that modulates binding of a ligand to a receptor, comprising (a) contacting a mixture comprising a cell expressing a biosensor nucleic acid of the present invention and said ligand with one or more test compounds; and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates ligand binding. The term "modulate" generally means that such compounds may increase or decrease or inhibit the interaction of a ligand with the ligand binding domain.

[0104] The methods of the present invention may also be used as a tool for high throughput and high content drug screening. For instance, a solid support or multiwell dish comprising the biosensors of the present invention may be used to screen multiple potential drug candidates simultaneously. Thus, the invention comprises a high throughput method of identifying compounds that modulate binding of a ligand to a receptor, comprising (a) contacting a solid support comprising at least one biosensor of the present invention, or at least one cell expressing a biosensor nucleic acid of the present invention, with said ligand and a plurality of test compounds; and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that a particular test compound is a compound that modulates ligand binding.

[0105] In one preferred embodiment, among others, the invention provides a method of identifying a compound that modulates glutamate excitotoxicity comprising (a) contacting a glutamate biosensor or a cell expressing a glutamate biosensor as disclosed herein and a sample of neurons with one or more test compounds, and (b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting, wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates glutamate excitotoxicity. The term "modulate" in this embodiment means that such compounds may increase or decrease glutamate excitotoxicity. Compounds that increase glutamate levels are targets for therapeutic intervention and treatment of disorders associated with glutamate excitotoxicity, as described above. Compounds that decrease glutamate levels may be developed into therapeutic products for the treatment of disorders associated with glutamate excitotoxicity.

[0106] The targeting of the sensor to the outer leaflet of the plasma membrane is only one embodiment of the potential applications. It demonstrates that the nanosensor can be targeted to a specific compartment. Alternatively, other targeting sequences may be used to express the sensors in other compartments such as vesicles, ER, vacuole, etc.

[0107] Expression systems comprise not only rat neurons, but also human cell lines, animal cells and organs, fungi and plant cells. The sensors can also be used to monitor levels of glutamate in fungal and plant organisms where glutamate serves as an important nitrogen compound, but potentially also a signaling molecule. Expression in bacteria may be used to monitor glutamate levels at sites of infection or in compartments in which the bacteria reside or are introduced. Specifically, bacteria or fungi expressing the sensors may serve as biosensors or as tools to identify new pesticides using a similar scheme as outlined for drug screening above.

[0108] The biosensors of the present invention can also be expressed on the surface of animal cells to determine the function of neurons. For example, in C. elegans, many of the neurons present have not been assigned a specific function. Expression of the biosensors on the surface permits visualization of neuron activity in living worms in response to stimuli, permitting assignment of function and analysis of neuronal networks. Similarly, the introduction of multiphoton probes into the brain of living mice or rats permits imaging these processes. Finally, expression in specific neurons or glia will allow the study of phenomena such as stroke or Alzheimers Disease and the effect of such disorders on glutamate levels inside neuronal cells or on their surface. Moreover, the effect of medication on localized brain areas or neuronal networks can be studied in vivo.

[0109] Finally, it is possible to use the sensors as tools to modify ligand binding, and particularly glutamate fluxes, by introducing them as artificial ligand scavengers, for instance presented on membrane or artificial lipid complexes. Artificial glutamate scavengers may be used to manipulate brain or neuron function.

[0110] The following examples are provided to describe and illustrate the present invention. As such, they should not be construed to limit the scope of the invention. Those in the art will well appreciate that many other embodiments also fall within the scope of the invention, as it is described hereinabove and in the claims.

Examples

Example 1

Construction of Nucleic Acids and Vectors

[0111] A truncated glutamate-aspartate binding protein sequence (SEQ ID No. 4), encoding mature protein without signal peptide, was amplified by PCR using E. coli K12 genomic DNA as a template. The primers used were 5'-ggtaccggaggcgccgcaggcagcacgctggacaaaatc-3' (SEQ ID No. 5) and 5'-accggtaccggcgccgttcagtgccttgtcattcggttc-3' (SEQ ID No. 6). The PCR fragment was cloned into the KpnI site of digested FLIPmal-25.mu. (Fehr et al. 2002) in pRSET vector (Invitrogen), exchanging the maltose binding protein sequence with the YbeJ sequence. The resulting plasmid was named pRSET-FLIP-E-600n (SEQ ID NO: 9).

[0112] To improve the pH and chloride tolerance and maturation of the sensor protein, the fragment containing the enhanced YFP (EYFP, CLONTECH) sequence in pRSET-FLIP-E-600n was replaced with the coding sequence of Venus, a variant of YFP with improved pH tolerance and maturation time (Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87-90). Affinity mutants carrying substitutions A207G, A207P, A207K, A207M, A2075, A207C, A207R, A207V, A207L, A207Q, A207T, A207F, A207Y, A207N, A207W, A207H, A207D, and S95W were created by site-directed mutagenesis (Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367-382).

[0113] pRSET-FLIP-E constructs (SEQ ID NOs: 9 and 10) were transferred to E. coli BL21(DE3)Gold (Stratagene) using electroporation (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning. A laboratory manual. (Cold Spring Harbor N.Y.: Cold Spring Harbor Laboratory Press). FLIP-E proteins expressed in BL21(DE3)Gold strain were extracted and purified as previously described (Fehr et al. 2002). For expression in rat primary neuronal cell culture and PC12 cell culture, FLIP-E 600n (SEQ ID NO: 13) and -10.mu. cassettes (SEQ ID NO: 14) were cloned into pDisplay (Invitrogen) as follows: XmaI site and SalI site were introduced on the 5'- and 3'-ends of FLIP-E cassette, respectively, by PCR. The primers used were 5'-gagcccgggatggtgagcaagggcgaggag-3' (SEQ ID No. 7) and 5'-gaggtcgaccttgtacagctcgtccatgccgag-3' (SEQ ID No. 8). The PCR fragments were sequenced to confirm that there was no additional PCR error, digested with XmaI/SalI, and cloned into the XmaI/SalI sites of the pDisplay vector. Cell cultures were transfected using a modified calcium phosphate transfection protocol (Xia, Z., Dudek, H., Miranti, C. K., and Greenberg, M. E. (1996). Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425-5436) or Lipofectamine (Invitrogen).

Example 2

In Vitro Characterization of FLIP-E Nanosensors

[0114] A DNA fragment encoding the mature YBEJ protein was fused to ECFP and the Venus sequence at the N- and C-termini, respectively (FIG. 1). Emission spectra and substrate titration curves were obtained by using monochromator microplate reader Safire (Tecan, Austria). Excitation filter was 433.+-.12 nm, emission filters for CFP and YFP emission were 485.+-.12, 528 nm.+-.12 nm, respectively. All analyses were done in 20 mM sodium phosphate buffer, pH 7.0.

[0115] Addition of glutamate resulted in an increase in CFP emission and a decrease in YFP emission, suggesting that binding of glutamate to YBEJ results in a conformational change of the chimeric protein potentially due to a relative change in the orientation of the dipoles of the fluorophores (FIG. 2). Since CFP and YFP moieties are assumed to be attached to the same lobe, we speculate that glutamate binding causes the change in dipole-dipole angle of two fluorophores. Interestingly, the ratio and ratio change were in a similar range as compared to other sensors generated so far (Fehr et al., 2002; Fehr et al., 2003; Lager et al., 2003), suggesting that distance changes may not be the primary factor in underlying the mechanisms for FRET changes. Spectra at three different glutamate concentrations (zero, Kd, saturation) reveals an isosbestic point at 520 nm (FIG. 2). The binding constant (Kd) for glutamate was determined to be 600 nM, consistent with data obtained by other methods (de Lorimier et al., 2002). Binding constants for aspartate, glutamine, asparagine were determined to be 6 .mu.M, 100 .mu.M, 300 .mu.M, respectively (see Table 1, below).

[0116] In order to expand the range of concentration that can be measured by YBEJ-based glutamate nanosensors, the YBEJ moiety was mutagenized to create nanosensors with lower affinity for glutamate. It has previously been shown that conjugating various fluorophores to sites located at the perimeter of the interdomain cleft that forms the ligand binding site (named "peristeric") changes the ligand-binding affinity in periplasmic binding proteins (de Lorimier et al., 2002). Among the residues tested, mutation of alanine 207 to lysine, methionine, serine, cysteine, arginine, valine, leucine, glutamine, threonine, phenylalanine, tyrosine, aspargine, tryptophan, histidine, aspartate (based on the YbeJ sequence provided in SEQ ID No. 28) lowered the binding affinity significantly (Table 2). In addition, the mutation of serine 95 to tryptophan, which is suggested to interact with the nitrogen of glutamate, was found to decrease the affinity of the protein. Thus, mutations introduced into the FLIPE nanosensor can yield affinity mutants suitable to cover a wide range of physiological glutamate concentrations.

TABLE-US-00002 TABLE 2 Kd (M) YbeJ Kd (M) Kd (M) Kd (M) Aspar- Vector moiety Glutamate Aspartate Glutamine agine FLIPE-600n-1 WT 6 .times. 10-7 6 .times. 10-6 1 .times. 10-4 3 .times. 10-4 FLIPE-600n-2 A207G 6 .times. 10-7 4 .times. 10-6 2 .times. 10-4 n.d. FLIPE-600n-3 A207P 6 .times. 10-7 4 .times. 10-6 2 .times. 10-4 n.d. FLIPE-3.mu. A207K 3 .times. 10-6 2 .times. 10-5 7 .times. 10-4 n.d. FLIPE-5.mu. A207M 5 .times. 10-6 3 .times. 10-5 1 .times. 10-3 n.d. FLIPE-5.mu.-2 A207S 5 .times. 10-6 3 .times. 10-5 1 .times. 10-3 n.d. FLIPE-6.mu. A207C 6 .times. 10-6 5 .times. 10-5 n.d. n.d. FLIPE-10.mu.-1 A207R 1 .times. 10-5 6 .times. 10-5 1 .times. 10-3 n.d. FLIPE-10.mu.-2 A207V 1 .times. 10-5 8 .times. 10-5 6 .times. 10-3 n.d. FLIPE-30.mu. A207L 3 .times. 10-5 n.d. n.d. n.d. FLIPE-40.mu.-1 A207Q 4 .times. 10-5 2 .times. 10-4 7 .times. 10-3 n.d. FLIPE-40.mu.-1 A207T 4 .times. 10-5 1 .times. 10-4 7 .times. 10-3 n.d. FLIPE-100.mu.-1 S95W 1 .times. 10-4 n.d. n.d. n.d. FLIPE-100.mu.-2 A207F 1 .times. 10-4 6 .times. 10-4 n.d. n.d. FLIPE-300.mu. A207Y 3 .times. 10-4 5 .times. 10-4 n.d. n.d. FLIPE-400.mu. A207N 4 .times. 10-4 1 .times. 10-3 n.d. n.d. FLIPE-1m A207W 1 .times. 10-3 n.d. n.d. n.d. FLIPE-2m-1 A207H 2 .times. 10-3 2 .times. 10-3 n.d. n.d. FLIPE-2m-2 A207D 2 .times. 10-3 9 .times. 10-4 n.d. n.d.

Example 3

In Vivo Characterization of FLIP-E

[0117] For the in vivo characterization of FLIP-E nanosensors, FLIPE-600n and FLIPE-10.mu. were cloned into the mammalian expression vector pDisplay (Invitrogen, USA). The pDisplay vector carries a leader sequence which directs the protein to the secretory pathway, and the transmembrane domain which anchors the protein to the plasma membrane, displaying the protein on the extracellular face. Rat hippocampal cells and PC12 cells were transfected with pDisplay FLIPE-600n (SEQ ID NO: 11) and -10.mu. (SEQ ID NO: 12) constructs. FRET was imaged 24-48 hours after transfection on a fluorescent microscope (DM IRE2, Leica) with a cooled CoolSnap HQ digital camera (Photometrics). Dual emission intensity ratios were simultaneously recorded following excitation at 436 nm and splitting CFP and Venus emission by Dual View with the OI-5-EM filter set (Optical Insights) and Metafluor 6.1r1 software (Universal Imaging).

[0118] The expression of FLIP-E was observed on the plasma membrane of rat hippocampal cell culture, and to some extent also in intracellular compartments, probably in compartments involved in plasma membrane targeting of plasma membrane proteins. When treated with Tyrode's buffer containing 1 mg/mL of trypsin, the majority of fluorescence on the cell surface was eliminated, demonstrating that the FLIPE protein was indeed displayed on the extracellular face of the plasma membrane as expected from the properties of the pDisplay construct (FIG. 3). The nanosensors should thus measure extracellular glutamate levels close to the cell's surface.

[0119] To quantify the intensity of CFP and Venus emission, the fluorescence intensity in the two channels in the periphery of the cell was integrated on a pixel-by-pixel basis, and the CFP/Venus ratio was calculated. When the hippocampal cells displaying FLIPE-600n (SEQ ID NO: 13) on the surface were electrically stimulated by passing current pulse, a decrease in CFP/Venus emission ratio was observed (FIG. 4 a-c), suggesting that the glutamate is released from hippocampal cells by membrane depolarization. To confirm that the ratio change is due to changes in the extracellular concentration of glutamate, the cell was perfused with increasing concentrations of glutamate. The emission intensity ratio changed in a concentration dependent manner, (FIG. 4 d-h), indicating that the FLIPE-600n (SEQ ID NO: 13) displayed on the cell surface recognizes the extracellular glutamate. The working range of the FLIP-E 600n (SEQ ID NO: 13) sensor was between 100 nM to 1 .mu.M, which is consistent with the in vitro working range of FLIPE-600n nanosensor (SEQ ID NO: 13). The CFP/Venus ratio increased when the external medium was washed away by perfusion, suggesting that the change in FRET intensity in vivo is reversible.

[0120] In contrast to the cells expressing FLIPE/600n sensor, the CFP/Venus emission intensity change was not observed in cells expressing FLIPE-10.mu. (SEQ ID NO: 14) upon electro-stimulation (FIG. 5). However, a ratio change was observed when the cells were perfused with higher concentrations of glutamate, (FIG. 5 c and e), suggesting that the glutamate concentration change induced by depolarization of the cell was below the working range of FLIP-E 10.mu. sensor.

[0121] The novel nanosensors are thus able to measure glutamate on the surface of neuronal cells and to follow the glutamate secretion of presynaptic neurons directly.

Example 4

Internally Fused YbeJ Sensor

[0122] There is currently no crystal structure for YbeJ. We homology-modeled a potential structure on the basis of existing structures of related amino acid biding proteins (His and Gln). We then predicted positions which might be permissive, i.e., sites where an insertion would not affect the overall structure of the protein. We then introduced restriction sites by site directed mutagenesis in these positions (see Table 3 below). Then the coding region for eCFP was inserted into these sites. We then looked for bacterial colonies that showed fluorescence. Only N58V-Q59N with eCFP inserted was fluorescent (based on the YbeJ sequence provided in SEQ ID No. 28). We then attached Venus at the C-terminus (FLIP-E intermol) (see FIG. 6). The affinity was tested and we saw a much larger delta ratio change and an affinity of approximately which is only slightly higher than the 600n version of YbeJ carrying the fluorophores at the ends (see FIG. 7).

[0123] Attempts to insert the eCFP molecule in the Ybej protein were, except for the case of N58V-Q59N, unsuccessful. We speculated that the N-terminus and C-terminus of the eCFP molecules were too far apart, resulting in destabilizing the chimera molecule by making too wide a gap in the Ybej peptide sequence. Circular permutated GFP variants, on the other hand, had N- and C-termini that were next to each other in the original protein. Therefore, we speculated that inserting permutated fluorescent protein instead of eCFP might be less harmful for protein stability. Therefore, we inserted circular permutated Venus (Nagai T., Yamada S. Tominaga T., Ichikawa M., Miyawaki A (2004) Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA. 101:10554-9) between A216 and K217 with linker sequences GNNSAG (SEQ ID NO: 30) and GSADDG (SEQ ID NO: 31). Then eCFP was fused at the N-terminus (see FIG. 8). The affinity was tested and we saw a much larger delta ratio change and an affinity of approximately 600 nM, which is unchanged from the 600n version of YbeJ carrying the fluorophores at the ends (see FIG. 9).

[0124] Without being bound to any particular theory, we believe that the data supports the prediction that rotational movements play a role in FRET. The dipoles have to be oriented in a certain position to each other for efficient resonance energy transfer. However, with terminally fused donor and acceptor moieties, commonly one assumes that the peptide bonds in the linker between the three moieties are freely rotating, thus randomizing this parameter.

By inserting the fluorescent moiety into an internal position of the PBP, we prevent free or limited free rotation of the fluorophore around the peptide axis in the linker sequences. Thus, the fluorescent moiety is now rigidly inserted at both ends, thereby reducing free wiggling and possibly explaining the higher observed delta ratio.

TABLE-US-00003 TABLE 3 YbeJ Positions Original Altered Sequence eCFP Mutated Sequence (restriction site) Fluorescence N58V-Q59N aatcag gttaac (HpaI) + G142-G143A ggcggc ggcgcc (NarI) - G143-D144A ggcgat ggcgcc (NarI) - D144-I145 gatatc gatatc (native EcoRV - site) A149V-N150 gccgac gttaac (HpaI) - N150D-L151 gacctg gagctc (Ecl136II) - M177-N178H atgaat atgcat (BfrBI) -

Example 5

Internally Fused GGBP Sensors

[0125] To demonstrate that internally fused FRET biosensors could be constructed using other proteins, we constructed nanosensors comprising the Escherichia coli glucose/galactose binding protein (GGBP) as a binding domain and the Aequorea Victoria green fluorescent protein variants CFP and YFP as reporter domains. Whereas YFP was either fused to the C- or N-terminus of the binding protein, CFP was inserted into various positions of the binding protein yielding a set of internally fused sensors. Each of these sensors is characterized by different relative spatial orientations of the chromophores.

Step 1: Selection of Insertion Sites in GGBP

[0126] To scan for permissive sites inside GGBP that tolerate chromophore insertions a total of 13 different sites were selected. Those sites were preferentially located on loops or at the ends of secondary structure elements that are protruding from the core protein and which show a high B-factor in the crystal structure. Sites on both lobes of GGBP were selected. To enable CFP insertions the Nru I restriction recognition sequence was introduced by site directed mutagenesis into the respective positions in the GGBP coding sequence using Kunkel's method. Table 4 depicts the selected sites in GGBP and the mutations introduced by the Nru I recognition sequence.

TABLE-US-00004 TABLE 4 Insertion sites and mutations in GGBP. Numbering starts with first amino acid of the mature protein lacking the 23 amino acid signal sequence. mutation secondary structure insertion sites in N-terminal domain of GGBP Y12S D13R Loop P32S D33R loop at end of helix S46S K47R Helix K58S G59R loop at end of helix Q83S N84R Loop Y102S D103R Loop G275S K276R Loop T282S N283R Loop insertion sites in C-terminal domain of GGBP N130S Q131R loop at end of helix N136S K137R Loop P150S G151R Loop G198S P199R Loop N226S K227R loop at end of helix

Step 2: Insertion of CFP and Screening for Fluorescent Colonies

[0127] The CFP coding sequence was inserted into the Nru I site in GGBP by molecular cloning. The constructs were designed to permit expression of the unfinished sensors at all stages of development. Two sets of constructs were engineered that bear the same insertion sites. One set was designed to enable the N-terminal fusion with YFP, the other to enable the C-terminal fusion with YFP.

[0128] The ligation reactions were transferred into the E. coli expression strain BL21(DE3)gold. After transformation, the bacteria were spread on plates using selective conditions for the presence of the vector. Cells were allowed to form colonies over night at 37 degree Celsius. Subsequently, the plates were transferred to 4 degree Celsius for about 10 days to facilitate chromophore maturation. Fluorescent colonies were selected for further cloning using a UV lamp or the fluorescence module of a dissecting microscope. The screening approach permits the effective and time-saving construction of a larger number of insertions in parallel. Furthermore, it offers the opportunity to identify insertions that do not fold correctly leading to very dim fluorescence of the colonies. Table 5 reflects the relative fluorescence intensity of the colonies.

TABLE-US-00005 TABLE 5 Relative fluorescence of bacterial colonies. Fluorescence intensities range from microscope visible only < low < normal < high fluorescence on plate Fluorescence of colonies after insertion of CFP Set for C-terminal YFP fusion pRSETB-BamHI-mgIBF16A/Y12S-CFP-D13R-kpnI 3a/1 low pRSETB-BamHI-mgIBF16A/P32S-CFP-D33R-kpnI 6a/3 low pRSETB-BamHI-mgIBF16A/S46S-CFP-K47R-kpnI 11a/5 microscope visible only pRSETB-BamHI-mgIBF16A/K58S-CFP-G59R-kpnI 1d/7 microscope visible only pRSETB-BamHI-mgIBF16A/Q83S-CFP-N84R-kpnI 6d/57 microscope visible only pRSETB-BamHI-mgIBF16A/Y102S-CFP-D103R-kpnI 17a/13 microscope visible only pRSETB-BamHI-mgIBF16A/G275S-CFP-K276R-kpnI 14c/15 microscope visible only pRSETB-BamHI-mgIBF16A/T282S-CFP-N283R-kpnI 13/73 microscope visible only pRSETB-BamHI-mgIBF16A/N130S-CFP-Q131R-kpnI 17/65 microscope visible only pRSETB-BamHI-mgIBF16A/N136S-CFP-K137R-kpnI 23/69 microscope visible only pRSETB-BamHI-mgIBF16A/P150S-CFP-G151R-kpnI 25/23 microscope visible only pRSETB-BamHI-mgIBF16A/G198S-CFP-P199R-kpnI 30/26 microscope visible only pRSETB-BamHI-mgIBF16A/N226S-CFP-K227R-kpnI 33/27 microscope visible only Set for N-terminal YFP fusion pRSETB-kpnI-mgIBF16A/Y12S-CFP-D13R-HindIII 3/29 normal pRSETB-kpnI-mgIBF16A/P32S-CFP-D33R-HindIII 3b/32 low pRSETB-kpnI-mgIBF16A/S46S-CFP-K47R-HindIII 6/34 normal pRSETB-kpnI-mgIBF16A/K58S-CFP-G59R-HindIII 5b/35 low pRSETB-kpnI-mgIBF16AQ83S-CFP-N84R-HindIII 11/37 normal pRSETB-kpnI-mgIBF16A/Y102S-CFP-D103R-HindIII 14/41 low pRSETB-kpnI-mgIBF16A/G275S-CFP-K276R-HindIII 18/43 high pRSETB-kpnI-mgIBF16A/T282S-CFP-N283R-HindIII 22/46 normal pRSETB-kpnI-mgIBF16A/N130S-CFP-Q131R-HindIII 25/47 norm pRSETB-kpnI-mgIBF16A/N136S-CFP-K137R-HindIII 29/49 low pRSETB-kpnI-mgIBF16A/P150S-CFP-G151R-HindIII 17b/51 microscope visible only pRSETB-kpnI-mgIBF16A/G198S-CFP-P199R-HindIII 33/54 microscope visible only pRSETB-kpnI-mgIBF16A/N226S-CFP-K227R/-HindIII 37/55 low

Step 3: Fusion to YFP and Screening for Colonies Expressing Both Chromophores

[0129] The coding sequence of YFP was inserted into the expression cassettes containing the CFP insertions of step 2 by molecular cloning. Using the two sets of CFP insertions two sets of fluorescent nanosensors were obtained that bear the same insertion of CFP but have YFP attached either to their N- or C-terminus. The ligation reactions were transferred into the expression strain BL21(DE3)gold. Following growth under selective conditions the resulting colonies were used to start 200 .mu.l cultures in a microtiter plate to screen for clones expressing both chromophores. The cultures were grown for two days at room temperature and allowed to rest for two days at 4 degrees Celsius to facilitate chromophore maturation. Subsequently the cultures were excited at the CFP excitation wavelength (433 nm) and emission intensities were recorded from 460 nm to 560 nm covering the emission peaks of CFP and YFP. Two to three clones of each nanosensor expression cassette that showed the presence of both chromophores were selected for further analysis. Small scale cultures were started to harvest the protein by Ni-NTA affinity chromatography. To analyze the ratio changes of the new nanosensors, spectra of the purified proteins were recorded in the absence and presence of 10 mM glucose and the difference in YFP/CFP emission intensity ratios were calculated. Table 6 depicts the measured ratio changes.

Step 4: Analysis of Selected Nanosensors

[0130] Nanosensors with a ratio change greater 0.2 (depicted in bold letters in table 6) were selected for further analysis. Protein was purified from larger scale cultures using Ni-NTA affinity chromatography. The resulting protein extracts were titrated with increasing concentrations of glucose in a microplate based FRET assay. The affinity of the nanosensors was determined by non-linear regression of the titration curves. Furthermore, spectra were recorded in the absence, at half-saturation and saturating glucose concentrations. As a control the original nanosensor, FLIPmglBF16A, where GGBP is sandwiched between CFP and YFP, is included. To normalize the ratio change (delta ratio), the ratio change was divided by the ratio in the absence of glucose (Table 7) (see FIGS. 8 and 9).

TABLE-US-00006 TABLE 7 Properties of nanosensors. ratio .DELTA./ ab- satu- ab- Kd sensor sence ration .DELTA. sence (mM) FLIP-mgIBF16A/Y12S-CFP-D13R- 4.55 7.21 2.66 0.58 0.6 YFP (SEQ ID NOs: 15 and 16) FLIP-YFP-mgIBF16A/G275S-CFP- 1.63 2.32 0.69 0.42 4.6 K276R (SEQ ID NOs: 23 and 24) FLIP-YFP-mgIBF16A/T282S-CFP- 2.11 2.55 0.44 0.21 4 N283R (SEQ ID NOs: 25 and 26) FLIP-YFP-mgIBF16A/P32S-CFP- 3.4 3.84 0.44 0.13 2.2 D33R (SEQ ID NOs: 21 and 22) FLIP-YFP-mgIBF16A/Y12S-CFP- 2.6 2.33 -0.27 -0.10 1.8 D13R (SEQ ID NOs: 19 and 20) FLIPmgIBF16A 2.95 2.6 -0.35 -0.12 0.6 FLIP-mgIBF16A/G275S-CFP- 1.93 1.6 -0.33 -0.17 13.8 K276R-YFP (SEQ ID NO: 17 and 18)

Absence depicts the ratio at the absence of glucose, saturation at saturating concentrations of glucose. .DELTA. shows the delta ratio between saturation and absence of glucose. .DELTA./absence is the normalized delta ratio.

SUMMARY AND DISCUSSION

[0131] Among 22 insertions, six functional glucose sensors were identified. Four sensors showed positive ratio changes upon addition of glucose. Only two displayed negative ratio changes as the original sensor FLIPmglBF16A. Four sensors had greater relative ratio changes as compared to FLIPmglBF16A. Two sensors showed relative ratio changes similar to FLIPmglBF16A. Hence, the insertion of a chromophore into the binding protein proved to be an efficient strategy to design and improve the nanosensors. Moreover, the chromophores do not have to be located on different lobes of the binding protein to yield functional sensors.

[0132] The direction and extent of a sensor's ratio change depend on the relative spatial orientation of the chromophores before and after binding of glucose. The change in spatial orientation can be a change in distance, a change in angular orientation or both. The contribution of the change in angular orientation increases, when the chromophores are fixed and cannot freely randomize prior to the transfer of energy.

[0133] Inserting CFP into the binding protein stiffens the connection between these two components of the sensor as compared to simple C- or N-terminal fusions of CFP. This has a major impact on the sensor. The stiffer connection improves the allosteric coupling between the hinge-twist motion of the binding protein and the change in spatial orientation of the chromophores. Particularly, the change in angular orientation of the chromophores is intensified, since the wobbling of CFP is reduced. Because under this condition the direction of the ratio change cannot be predicted from the change in chromophore distance alone, it follows that sensors with ratio changes in both directions were engineered by inserting CFP.

[0134] However, due to the nature of FRET, not every relative change in chromophore orientation can translate into a change in ratio. Certain combinations of relative spatial chromophore orientations exist that are completely different but lead to a similar degree of FRET. Thus despite a large spatial reorientation of the chromophores, no significant ratio change might be observed. Moreover, insertion of CFP might abolish glucose binding by GGBP and some insertions might not even fold correctly.

[0135] The chart in FIG. 10 shows the correlation between the starting ratio in the absence of glucose and the normalized ratio change and assesses the overall success rate of the insertions. Sector 1 depicts the insertions that do not fold properly. For two insertions, both the N-terminal and C-terminal YFP fusion display a low ratio and a negligible ratio change. Sector 2 harbors 8 insertions which fold correctly but do no show a significant ratio change. This can be attributed to similar degrees of FRET before and after binding of glucose or to the fact that glucose binding is abolished. The fact that at least some functional sensors show a decreased affinity towards glucose supports the assumption that by reverting mutation F16A a number of these insertions can be turned into functional sensors. Sector 3 depicts 5 sensors based on 4 different insertions that possess higher ratio changes than the original sensor FLIPmglBF16A, which is shown as a reference point.

[0136] Thus, despite the above limitations, scanning different insertion sites for CFP in GGBP appears be an efficient method to improve the sensors. Further, the fact that the chromophores can be located on the same lobe to yield a functional sensor potentially enables us to use chromophore insertions to turn virtually each binding protein or enzyme into a sensor. It may be imagined given the above data that a further increase in signal response may be obtained by internally fusing both chromophores into the ligand-binding moiety sequence. We are creating these constructs, and expect them to show further improved properties.

Example 6

Design of FRET Biosensors with Improved Sensitivity

[0137] Having learned that the reduced rotational averaging in the internal insertion of a fluorophores is a general strategy to generate sensors with high ratio changes, we hypothesized that one may obtain similar results by reducing the rotational freedom of the linkage between the analyte binding domain and the fluorophores. We thus systematically removed sequences that connect the core protein structure of the binding domain and the fluorophore, i.e. by removing linker sequences and by deleting both amino acids from the ends of the analyte binding moiety and the fluorophores. We found that close coupling also leads to higher ratio changes. This concept is exemplified for FLIPglu.

[0138] To perform the comparison, thirteen different shortened sensor proteins were generated. Deletions of up to 8 amino acids of the linker regions between the fluorophores and the analyte binding domain did not result in a marked increase of the ratio change (see FIG. 13). Further deletions were done on the C-terminus of the ECFP (6 or 9 amino acids), on the C-terminus of the mglB analyte binding domain (5 amino acids) and on the N-terminus of the EYFP (1, 2 or 6 amino acids), which resulted in an overall increase of the change in ratio in 5 of the proteins (see FIG. 12). In all cases, the core of the fluorophore determined necessary for fluorescence (amino acid 7 to 229, Li et al., 1997, JBC 272 pp. 28545) was included.

Example 7

Testing of FRET Biosensors with Improved Sensitivity In Vitro

Materials and Methods:

Linker Deletions for FLIPglu Internally Fused Sensors

[0139] Two internally fused glucose sensors were chosen on the basis of their .DELTA. ratio and affinities, FLII.sup.12Pglu-600.mu. and FLIIP.sup.275Pglu-4.6m. For FLII.sup.12Pglu-600.mu., the linker and less well-structured domains at the termini of mglB and Citrine (together comprising the 17 amino acid "composite linker") was systematically deleted starting at the mglB using Kunkel mutagenesis (Kunkel et al.). 17 primers were used designed to delete increasing number of amino acid residues from FLII.sup.12Pglu-600.mu. creating FLII.sup.12Pglu-1aa through FLII.sup.12Pglu-17aa. In addition, deletion of 16 amino acids, FLII.sup.12Pglu.DELTA.16 was also created by adding a XhoI site at residue 305 of mglB and cloning a shortened Citrine (amino acids 7-238) using XhoI and HindIII. FLII.sup.12Pglu-16aa and FLII.sup.12Pglu.DELTA.16 thus differ in a single amino acid residue at position 305 of mglB (Ala for FLII.sup.12Pglu-16aa and Leu for FLII.sup.12Pglu .delta.16). Two more primers were used to delete 4 and 6 amino acid residues Gly-Gly-Thr-Gly-Gly-Ala (SEQ ID NO: 32) (GGTGGTACCGGAGGCGCC (SEQ ID NO: 33)) of the plasmid derived linker between the mglB and Citrine keeping the mglB and Citrine intact (FLII.sup.12Pglu .delta.4 and FLII.sup.12Pglu .delta.6). In case of FLIIP.sup.275Pglu-4.6m, where Citrine is at the N-terminus, a single primer was used to delete 15 amino acid residues (9 from the C-terminus dispensable portion of Citrine and 6 of the plasmid derived linker connecting the Citrine and mglB) (FIG. 14).

In Vitro Analysis of Sensors

[0140] Constructs were transferred to E. coli BL21(DE3)Gold (Stratagene, USA) using electroporation, extracted and purified as previously described (Fehr et al., 2002, Proc. Natl. Acad. Sci. USA 99: 9846-9851). Emission spectra and ligand titration curves were obtained by using a monochromator microplate reader (Safire, Tecan, Austria). The excitation filter was 433/12 nm; emission filters for ECFP and EYFP (also Citrine and Venus) emission was 485/12 and 528/12 nm, respectively. All analyses for FLIPE constructs and linearly-fused FLIPglu constructs were performed in 20 mM sodium phosphate buffer, pH 7.0; analyses of FLII.sup.XPglu were done in 20 mM MOPS buffer, pH 7.0. In order to compare the FLII.sup.12Pglu-600.mu. and FLIIP.sup.275Pglu-4.6m deletions better, the Citrine emission values for each was kept constant at about 20000 and the emission gain was kept constant at 80. The sensors were also analysed in Hanks buffer (pH 7.2), synthetic mammalian cytosol (pH 7.2), synthetic plant cytosol (pH 7.2) and MOPS pH 5.0 using the same amount of protein as used for assay in MOPS pH 7.0. The K.sub.d of each sensor was determined by fitting to a single site binding isotherm: S=(r-r.sub.apo)/(r.sub.sat-r.sub.apo)=[L]/(K.sub.d+[L]), where S is saturation; [L], ligand concentration; r, ratio; r.sub.apo, ratio in the absence of ligand; and r.sub.sat, ratio at saturation with ligand. Measurements were performed with at least three independent protein extracts. ECFP emission is characterized by two peaks at 485 and 502 nm; the ratio was defined here as the uncorrected fluorescence intensity at 528 nm divided by the intensity at 485 nm.

Analysis in Different Buffers

[0141] In order to see the effect of environmental conditions on the sensors, they were analysed under various conditions, in mammalian cell culture solution (Hanks buffer: 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na.sub.2HPO.sub.4, 0.4 mM KH.sub.2PO.sub.4, 4.2 mM NaHCO.sub.3, 0.6 MgSO.sub.4, 10 mM Lactate, 1 mM Pyruvate pH 7.4), synthetic mammalian cytosol (135 mM K(gluconate), 4 mM KCl, 12 mM NaHCO.sub.3, 0.8 mM MgCl.sub.2, 0.2 .mu.M CaCl.sub.2 pH 7.4), synthetic plant cytosol (10 mM NaCl, 150 mM K(gluconate), 1 mM MgCl.sub.2, 100 mg/mL BSA, 10 mM HEPES pH 7.5 with BTP) and MOPS buffer pH 5.0. The protein amount was kept constant as for the analysis in MOPS buffer pH 7.0. The spectrum was measured with no glucose, 10 mM glucose and 100 mM glucose in triplicate and the analysis was done with 2 independent protein preps for each sensor.

Results:

FLIPglu Linker Variation

[0142] To further improve the signal to noise ratio and to develop environmentally stable sensors, a systematic deletion analysis of the linkers in the intramolecular FRET sensor FLII.sup.12Pglu-600.mu. (Deuschle et al., 2005, Protein Science 14:2304-2314) was carried out. The glucose nanosensor FLII.sup.12Pglu-600.mu. consists of the mature glucose/galactose-binding protein mglB from Escherichia coli into which CFP had been inserted at position 12 and a linearly fused EYFP via a 6-amino acid linker to the C-terminus (Deuschle 2005). The linker and less well-structured domains at the termini of mglB and EYFP variants (together comprising the "composite linker") would be assumed to allow flexible (if not free) rotation of the fluorophores relative to the binding protein and one another. The composite linker was systematically truncated in an attempt to decrease rotational averaging and to enhance the allosteric coupling. FPs possess terminal regions not absolutely required for folding and fluorescence (an N-terminal helix and a C-terminal coil) (Li et al., 1997, J. Biol. Chem. 272: 28545-28549). Furthermore, five amino acids may be deleted from the C-terminal region of the mglB binding protein without affecting binding. These together yield 17 amino acids, the removal of which might a priori be expected to preserve binding and fluorescence (FIG. 14). Composite linker regions were deleted from FLII.sup.12Pglu-600.mu. in a stepwise manner.

Effect of Deletions on Ratio and Kd

[0143] Most of the FLII.sup.12Pglu-600.mu. deletions showed a decreased FRET compared to the full length sensor. The .DELTA. ratio of the deletion constructs varying between 0.52 (FLII.sup.12Pglu-17aa, 78% decrease) to 2.26 (FLII.sup.12Pglu-12aa, 5% decrease). Out of the 20 deletion constructs 14 still had a .DELTA. ratio of above 1 of which 5 constructs had .DELTA. ratio of 1.3 or more (FLII.sup.12Pglu-6aa 1.32, FLII.sup.12Pglu-7aa 1.31, FLII.sup.12Pglu-10aa 1.3, FLII.sup.12Pglu-12aa 2.26, FLII.sup.12Pglu-16aa-1.40). FLII.sup.12Pglu .delta.4 and FLII.sup.12Pglu .delta.6 had a slightly improved .DELTA. ratio (4% increase) as compared to FLII.sup.12Pglu-600.mu.. Interestingly, FLII.sup.12Pglu-16aa and FLII.sup.12Pglu .delta.16, showed a decrease in ratio upon ligand binding whereas the FLII.sup.12Pglu-600.mu. and all of the other deletions show increased ratio upon ligand binding. The affinity of each of the sensors was determined by titrating with glucose (Table 8). The affinity to glucose decreased after deletion of 2 amino acids FLII.sup.12Pglu-2aa through FLII.sup.12Pglu-13aa have binding constants ranging between 1.5-2.0 mM, deletion of more than 13 amino acids further decreased the affinity (FLII.sup.12Pglu-14aa 3.4 mM, FLII.sup.12Pglu-15aa 2.6 mM). FLII.sup.12Pglu-17aa has a dramatically decreased affinity of 6.8 mM. FLII.sup.12Pglu .delta.4 and FLII.sup.12Pglu .delta.6 however have Kd comparable to FLII.sup.12Pglu-600.mu. (FIG. 15, Table 8).

[0144] FLIIP.sup.275Pglu-15aa showed an increased .DELTA. ratio of 1.14 (73% increase) as compared to FLIIP.sup.275Pglu-4.6m, which has a .DELTA. ratio of 0.66. However the deletion affected affinity dramatically, decreasing it to a point where the sensor was no longer measurable (data not shown). So, in order to make a usable sensor, the alanine-16 in the mglB, was mutated back to wild-type phenylalanine which is involved in glucose binding (Fehr et al. 2003), thus decreasing the affinity of FLIIP.sup.275Pglu-15aa to 1.5 mM and an increased .DELTA. ratio.

[0145] The FLII.sup.12Pglu-600.mu. loop-inserted sensor shows a significantly higher ratio change than the linear-fusion FLIPglu-600.mu. sensor; with little effect on ligand affinity. Upon deletion of up to eleven residues from the sensor (first from the C-terminal helix of the mglB domain: 5 residues, then from the synthetic linker connecting the mglB and YFP domains: 6 residues), there is a slight decrease in ligand affinity, and a decrease in ligand-dependent signal change. Molecular modeling suggests that up to this point, there is still a good degree of separation between the YFP and both the N- and C-terminal lobes of mglB (the CFP is not modeled to be highly sterically regulated by any of the other domains). The N-terminal domain of mglB is modeled to be in closer proximity to the YFP in the open versus the closed conformation (modeled by overlaying the open and closed structures of the E. coli ribose-binding protein rbsB). Thus it appears that the YFP domain is coming into closer contact with the N-terminal mglB domain, perhaps making some favorable contacts, thus driving the equilibrium slightly towards the open state, and slightly decreasing affinity. Up to the -11 aa deletion, signal change and ligand-binding affinity appear to be positively correlated, with higher-affinity sensors also having a higher signal change. This is consistent with the YFP domain having some sort of interaction with the N-terminal domain of mglB in the open state, with the result that affinity is decreased by shifting the equilibrium, and the ratio change is adversely affected, perhaps through quenching. After this amount of deletion, molecular modeling suggests that the YFP is coming into very close proximity to the mglB N- and C-terminal domains, and indeed the -12aa deletion appears as if it may be conformationally restricted by this proximity, resulting in decreased rotational averaging and a higher signal change. Beyond this point, signal change and ligand-binding affinity become negatively correlated, with higher-affinity sensors yielding a lower ratio change. This is consistent with the molecular modeling, and suggests that after this point, the YFP and the mglB open-form N-terminal domain come in sufficient proximity as to give rise to energetically-unfavorable clashes, thus making the closed-form more favorable and increasing affinity.

[0146] Deletions beyond 15 amino acids were most sensitive to small deletions, consistent with an overall "tightening" of the allosteric linkage between domains. In this regime, even deletion of a single amino acid reversed the sign of the fluorescence signal change. This is somewhat surprising since similar deletions in the linearly-fused FLIPglu-600.DELTA.13 sensor did not show these dramatic effects. This suggests that perhaps there is some degree of allosteric cross-regulation between the YFP and the loop-inserted CFP, which is modeled to be about 20 .ANG. away, giving rise to the high sensitivity to small deletions.

[0147] Effects of deletions targeted solely to the center of the synthetic linker were assayed independently (right section of FIG. 15), and had minimal effect on affinity, as would be expected (the linker is still quite long, and inter-domain contacts are not affected), and a slight increase in signal change, consistent with a slight decrease in the rotational average caused by the likely-unstructured synthetic linker, without any quenching due to deletions of the highly-structured terminal helices of the mglB and YFP domains.

Sensitivity to Environmental Conditions

[0148] It has been noted before that buffers can affect ratio change. Moreover, in vivo the ratio change is always dampened owing to various factors such as pH, presence of ions, sugars etc. Therefore, to identify the sensors best suited for in vivo applications, various buffers mimicking cell medium (Hank's), mammalian cytosol, plant cytosol and low pH similar to that inside vesicles, vacuoles or cell wall were tested (Table 8). FLII.sup.12Pglu-600.mu. shows a 57% to 74% decrease in ratio change in MOPS pH 5.0 and plant cytosol. Most of the deletion constructs have a 20-70% decreased .DELTA. ratio in various buffers. Of the 5 constructs having a .DELTA. ratio of 1.3 or more, FLII.sup.12Pglu-6aa and FLII.sup.12Pglu-7aa are greatly affected by all the buffers tested showing a decreased .DELTA. ratio of 20-61%. FLII.sup.12Pglu-10aa is unaffected by Hanks buffer and very slightly affected in mammalian cytosol (10% decrease), it shows a 28% decrease in .DELTA. ratio in plant cytosol and a 66% decrease in low pH. FLII.sup.12Pglu-12aa shows a decrease of 52-59% in all buffers but still has a .DELTA. ratio of 1.0. FLII.sup.12Pglu-16aa shows a decrease of about 30% in Hanks buffer and mammalian cytosol and is unaffected in plant cytosol and MOPS pH 5.0 but, it completely changes orientation in response to different ions. It shows increase in ratio in Hanks buffer and mammalian cytosol and decrease in ratio in plant cytosol and MOPS pH 5.0 (same as FLII.sup.12Pglu .delta.16). FLII.sup.12Pglu-15aa however is the least affected in all the buffers and even has an improved .DELTA. ratio in Hanks buffer and mammalian cytosol (FIG. 16).

[0149] FLIIP.sup.275Pglu-4.6m is unaffected in Hanks buffer and mammalian cytosol but shows a decreased .DELTA. ratio in plant cytosol (28%) and MOPS pH 5.0 (82%). FLIIP.sup.275Pglu-15aa showed a 40 and 45% decrease in Hanks buffer and mammalian cytosol respectively, and a 75 and 88% decrease in plant cytosol and low pH (Table 8).

Sensors with the Highest Ratio and Resistance to Environmental Conditions

[0150] Though most of the FLII.sup.12Pglu-600.mu. deletion constructs have a decreased .DELTA. ratio than the original sensor, they showed more resistance to the environmental conditions tested. The deletion of residues most likely rearranges the sensor in a way that residues most sensitive to ions are no longer exposed thus making the sensor more resistant to environmental conditions.

TABLE-US-00007 TABLE 8 Ratio change and affinity of the FLII.sup.12Pglu-600.mu. and FLII.sup.275Pglu-4.6m in MOPS buffer pH 7.0, Hanks buffer, mammalian cytosol, plant cytosol and MOPS buffer pH 5.0 HANKS Mamm MOPS pH 7.0 Kd BUFFER cytosol Plant cytosol MOPS pH 5.0 Sensor Name Ratio Stdev (.mu.M) Stdev Ratio stdev Ratio stdev Ratio stdev Ratio stdev FLIP glu 600.mu. -0.29 0.0208 583 8.49 FLII.sup.12Pglu-600.mu. 2.37 0.0764 675 45.25 0.84 0.2056 0.64 0.0306 0.62 0.2723 1.02 0.0354 FLII.sup.12Pglu-1aa 1.24 0.0624 796 277.89 0.64 0.0566 0.42 0.0283 0.73 0.0354 0.87 0.0707 FLII.sup.12Pglu-2aa 1.13 0.0907 1580 108.19 0.35 0.0058 0.36 0.1021 0.31 0.0569 0.94 0.1250 FLII.sup.12Pglu-3aa 0.81 0.0306 1904 376.18 0.41 0.1079 0.39 0.1380 0.24 0.0361 0.89 0.2121 FLII.sup.12Pglu-4aa 0.97 0.0586 1562 451.84 0.48 0.1002 0.48 0.1380 0.38 0.0577 0.99 0.1061 FLII.sup.12Pglu-5aa 1.15 0.0862 1465 53.03 0.55 0.0850 0.48 0.1450 0.46 0.1222 0.79 0.1273 FLII.sup.12Pglu-6aa 1.32 0.1041 1474 118.09 0.70 0.1914 0.52 0.0557 1.07 0.0751 0.78 0.1531 FLII.sup.12Pglu-7aa 1.31 0.1212 1580 113.14 0.75 0.1217 0.58 0.1997 0.53 0.0200 0.95 0.1415 FLII.sup.12Pglu-8aa 1.14 0.0306 1924 217.79 0.57 0.0854 0.58 0.0551 0.51 0.0495 0.80 0.2108 FLII.sup.12Pglu-9aa 1.03 0.0300 1600 229.81 0.59 0.0700 0.58 0.0252 0.44 0.0141 0.83 0.0757 FLII.sup.12Pglu-10aa 1.29 0.0265 1473 41.72 1.27 0.1137 1.16 0.1159 0.94 0.0212 0.44 0.0100 FLII.sup.12Pglu-11aa 0.72 0.2397 1733 333.05 0.49 0.2030 0.39 0.1701 0.26 0.2829 0.22 0.1935 FLII.sup.12Pglu-12aa 2.26 0.0493 1953 192.33 1.05 0.0700 1.07 0.0874 0.95 0.1670 0.92 0.0707 FLII.sup.12Pglu-13aa 0.58 0.1484 2007 292.04 0.50 0.1838 0.57 0.1061 0.17 0.0071 0.29 0.0778 FLII.sup.12Pglu-14aa 1.08 0.0346 3423 74.95 0.83 0.1609 0.86 0.1758 1.02 0.0379 0.43 0.1768 FLII.sup.12Pglu-15aa 1.04 0.0321 2642 260.22 1.37 0.0778 1.36 0.1626 0.99 0.2828 0.95 0.0566 FLII.sup.12Pglu-16aa -1.40 0.0929 1235 157.68 0.46 0.0458 0.29 0.1858 -1.37 0.0495 -1.15 0.0990 FLII.sup.12Pglu-17aa 0.52 0.0513 6800 424.26 0.50 0.1365 0.51 0.1332 -0.04 0.1931 -0.09 0.0071 FLII.sup.12Pglu .delta. 4 2.45 0.1767 594 64.35 1.38 0.2401 1.00 0.2060 2.53 0.1290 1.29 0.1819 FLII.sup.12Pglu .delta. 6 2.46 0.1890 659 156.98 2.31 0.0707 2.06 0.1697 2.12 0.0495 0.93 0.0105 FLII.sup.12Pglu .delta. 16 -0.79 0.0100 1766 221 0.48 0.0636 0.21 0.0424 -0.61 0.0707 -1.07 0.0586 FLII.sup.275Pglu 4.6m 0.66 0.0112 5200 520 0.73 0.04 0.68 0.0707 0.62 0.1484 0.16 0.0565 FLII.sup.275Pglu-15aa 1.14 0.0909 1500 251 0.76 0.10 0.60 0.0494 0.53 0.1838 0.19 0.0777

SUMMARY AND DISCUSSION

[0151] We have accumulated a large data set following the effect residue-by-residue of a series of deletions from the binding protein (BP)-to-fluorescent protein (FP) boundary in a high-signal change loop-inserted glucose sensor FLII.sup.12Pglu-600.mu.. Deletions have concomitant effects both on the signal change and glucose-binding affinity of the nanosensor family, consistent with predictions from crude molecular modeling. Of all the sensor modifications, only deletions of one or two amino acids from the center of the synthetic linker connecting the mglB C-terminus with the YFP N-terminus give rise to sensors with higher signal change or higher ligand-binding affinity (in this case, both). All other deletions decrease affinity for glucose, and the glucose-dependent signal change. Some sensors give a higher signal change than the original sensor in different buffer conditions, however, which will be useful for in vivo sensing. Perhaps most importantly, the family of linker-deleted sensors provides a robust data set for the rationalization and design of further linker variants, which may allow high-response sensors to be created out of non-functional ones.

[0152] Taken together, the data set supports a model in which local allosteric regulation, particularly of reporter element orientation, plays a significant role in the resonance energy transfer of a family of genetically-encoded nanosensor proteins. Testing of this hypothesis by rational protein design produced sensors with greatly-improved signal-to-noise, enabling a wide array of in vivo applications. Molecular modeling may provide a route to further sensor improvement, and may prove useful in the optimization of other signal transduction mechanisms, such as allosteric enzymatic switches. These findings may be relevant for the optimization of other types of FRET sensors as well as the generation of novel sensors.

Example 8

Testing of FRET Biosensors with Improved Sensitivity In Vivo

[0153] To test the improved sensors for glucose detection in living cells and to test whether the sensors can be used also in other cell types, three intramolecular sensors (FLII.sup.12Pglu-600.mu.; FLII.sup.12Pglu .delta.4aa-593.mu.; FLII.sup.275Pglu-4600.mu.; FIG. 17) were cloned into pcDNA3.1 (-) (FIG. 18). FIG. 19 shows FRET changes observed in NIH3T3 cells transformed with the improved glucose sensors.

[0154] All publications, patents and patent applications discussed herein are incorporated herein by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 59 <210> SEQ ID NO 1 <211> LENGTH: 978 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(978) <400> SEQUENCE: 1 atg ata aca aca caa aca ctc aca acg ggt atc cat gcg ttc tta acg 48 Met Ile Thr Thr Gln Thr Leu Thr Thr Gly Ile His Ala Phe Leu Thr 1 5 10 15 cag aag ata aag gag ttg gat atg caa tta cgt aaa cct gcc aca gca 96 Gln Lys Ile Lys Glu Leu Asp Met Gln Leu Arg Lys Pro Ala Thr Ala 20 25 30 atc ctc gcc ctg gcg ctt tcc gca gga ctg gca cag gca gat gac gcc 144 Ile Leu Ala Leu Ala Leu Ser Ala Gly Leu Ala Gln Ala Asp Asp Ala 35 40 45 gcc ccg gca gcg ggc agt act ctg gac aaa atc gcc aaa aac ggt gtg 192 Ala Pro Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn Gly Val 50 55 60 att gtc gtc ggt cac cgt gaa tct tca gtg cct ttc tct tat tac gac 240 Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr Tyr Asp 65 70 75 80 aat cag caa aaa gtg gtg ggt tac tcg cag gat tac tcc aac gcc att 288 Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn Ala Ile 85 90 95 gtt gaa gca gtg aaa aag aaa ctc aac aaa ccg gac ttg cag gta aaa 336 Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln Val Lys 100 105 110 ctg att ccg att acc tca caa aac cgt att cca ctg ctg caa aac ggc 384 Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln Asn Gly 115 120 125 act ttc gat ttt gaa tgt ggt tct acc acc aac aac gtc gaa cgc caa 432 Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu Arg Gln 130 135 140 aaa cag gcg gct ttc tct gac act att ttc gtg gtc ggt acg cgc ctg 480 Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr Arg Leu 145 150 155 160 ttg acc aaa aag ggt ggc gat atc aaa gat ttt gcc aac ctg aaa gac 528 Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu Lys Asp 165 170 175 aaa gcc gta gtc gtc act tcc ggc act acc tct gaa gtt ttg ctc aac 576 Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu Leu Asn 180 185 190 aaa ctg aat gaa gag caa aaa atg aat atg cgc atc atc agc gcc aaa 624 Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser Ala Lys 195 200 205 gat cac ggt gac tct ttc cgc acc ctg gaa agc ggt cgt gcc gtt gcc 672 Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala Val Ala 210 215 220 ttt atg atg gat gac gct ctg ctg gcc ggt gaa cgt gcg aaa gcg aag 720 Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg Ala Lys Ala Lys 225 230 235 240 aaa cca gac aac tgg gaa atc gtc ggc aag ccg cag tct cag gag gcc 768 Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln Glu Ala 245 250 255 tac ggt tgt atg ttg cgt aaa gat gat ccg cag ttc aaa aag ctg atg 816 Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys Leu Met 260 265 270 gat gac acc atc gct cag gtg cag acc tcc ggt gaa gcg gaa aaa tgg 864 Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu Lys Trp 275 280 285 ttt gat aag tgg ttc aaa aat cca att ccg ccg aaa aac ctg aac atg 912 Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu Asn Met 290 295 300 aat ttc gaa ctg tca gac gaa atg aaa gca ctg ttc aaa gaa ccg aat 960 Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu Pro Asn 305 310 315 320 gac aag gca ctg aac taa 978 Asp Lys Ala Leu Asn 325 <210> SEQ ID NO 2 <211> LENGTH: 325 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 2 Met Ile Thr Thr Gln Thr Leu Thr Thr Gly Ile His Ala Phe Leu Thr 1 5 10 15 Gln Lys Ile Lys Glu Leu Asp Met Gln Leu Arg Lys Pro Ala Thr Ala 20 25 30 Ile Leu Ala Leu Ala Leu Ser Ala Gly Leu Ala Gln Ala Asp Asp Ala 35 40 45 Ala Pro Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn Gly Val 50 55 60 Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr Tyr Asp 65 70 75 80 Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn Ala Ile 85 90 95 Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln Val Lys 100 105 110 Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln Asn Gly 115 120 125 Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu Arg Gln 130 135 140 Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr Arg Leu 145 150 155 160 Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu Lys Asp 165 170 175 Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu Leu Asn 180 185 190 Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser Ala Lys 195 200 205 Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala Val Ala 210 215 220 Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg Ala Lys Ala Lys 225 230 235 240 Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln Glu Ala 245 250 255 Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys Leu Met 260 265 270 Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu Lys Trp 275 280 285 Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu Asn Met 290 295 300 Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu Pro Asn 305 310 315 320 Asp Lys Ala Leu Asn 325 <210> SEQ ID NO 3 <211> LENGTH: 810 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(810) <400> SEQUENCE: 3 ctg gac aaa atc gcc aaa aac ggt gtg att gtc gtc ggt cac cgt gaa 48 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 1 5 10 15 tct tca gtg cct ttc tct tat tac gac aat cag caa aaa gtg gtg ggt 96 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 20 25 30 tac tcg cag gat tac tcc aac gcc att gtt gaa gca gtg aaa aag aaa 144 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 35 40 45 ctc aac aaa ccg gac ttg cag gta aaa ctg att ccg att acc tca caa 192 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 50 55 60 aac cgt att cca ctg ctg caa aac ggc act ttc gat ttt gaa tgt ggt 240 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 65 70 75 80 tct acc acc aac aac gtc gaa cgc caa aaa cag gcg gct ttc tct gac 288 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 85 90 95 act att ttc gtg gtc ggt acg cgc ctg ttg acc aaa aag ggt ggc gat 336 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 100 105 110 atc aaa gat ttt gcc aac ctg aaa gac aaa gcc gta gtc gtc act tcc 384 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 115 120 125 ggc act acc tct gaa gtt ttg ctc aac aaa ctg aat gaa gag caa aaa 432 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 130 135 140 atg aat atg cgc atc atc agc gcc aaa gat cac ggt gac tct ttc cgc 480 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 145 150 155 160 acc ctg gaa agc ggt cgt gcc gtt gcc ttt atg atg gat gac gct ctg 528 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 165 170 175 ctg gcc ggt gaa cgt gcg aaa gcg aag aaa cca gac aac tgg gaa atc 576 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 180 185 190 gtc ggc aag ccg cag tct cag gag gcc tac ggt tgt atg ttg cgt aaa 624 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 195 200 205 gat gat ccg cag ttc aaa aag ctg atg gat gac acc atc gct cag gtg 672 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 210 215 220 cag acc tcc ggt gaa gcg gaa aaa tgg ttt gat aag tgg ttc aaa aat 720 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 225 230 235 240 cca att ccg ccg aaa aac ctg aac atg aat ttc gaa ctg tca gac gaa 768 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 245 250 255 atg aaa gca ctg ttc aaa gaa ccg aat gac aag gca ctg aac 810 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 260 265 270 <210> SEQ ID NO 4 <211> LENGTH: 270 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 4 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 1 5 10 15 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 20 25 30 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 35 40 45 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 50 55 60 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 65 70 75 80 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 85 90 95 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 100 105 110 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 115 120 125 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 130 135 140 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 145 150 155 160 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 165 170 175 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 180 185 190 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 195 200 205 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 210 215 220 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 225 230 235 240 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 245 250 255 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 260 265 270 <210> SEQ ID NO 5 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 5 ggtaccggag gcgccgcagg cagcacgctg gacaaaatc 39 <210> SEQ ID NO 6 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 6 accggtaccg gcgccgttca gtgccttgtc attcggttc 39 <210> SEQ ID NO 7 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 7 gagcccggga tggtgagcaa gggcgaggag 30 <210> SEQ ID NO 8 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 8 gaggtcgacc ttgtacagct cgtccatgcc gag 33 <210> SEQ ID NO 9 <211> LENGTH: 5206 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pRSETB FLIP-E 600n vector <400> SEQUENCE: 9 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 60 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat 120 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat 180 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg 240 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 300 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 360 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc 420 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 480 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 540 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 600 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc 660 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag 720 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 780 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 840 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 900 atggacgagc tgtacaaggg tggtaccgga ggcgccgcag gcagcacgct ggacaaaatc 960 gccaaaaacg gtgtgattgt cgtcggtcac cgtgaatctt cagtgccttt ctcttattac 1020 gacaatcagc aaaaagtggt gggttactcg caggattact ccaacgccat tgttgaagca 1080 gtgaaaaaga aactcaacaa accggacttg caggtaaaac tgattccgat tacctcacaa 1140 aaccgtattc cactgctgca aaacggcact ttcgattttg aatgtggttc taccaccaac 1200 aacgtcgaac gccaaaaaca ggcggctttc tctgacacta ttttcgtggt cggtacgcgc 1260 ctgttgacca aaaagggtgg cgatatcaaa gattttgcca acctgaaaga caaagccgta 1320 gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca aactgaatga agagcaaaaa 1380 atgaatatgc gcatcatcag cgccaaagat cacggtgact ctttccgcac cctggaaagc 1440 ggtcgtgccg ttgcctttat gatggatgac gctctgctgg ccggtgaacg tgcgaaagcg 1500 aagaaaccag acaactggga aatcgtcggc aagccgcagt ctcaggaggc ctacggttgt 1560 atgttgcgta aagatgatcc gcagttcaaa aagctgatgg atgacaccat cgctcaggtg 1620 cagacctccg gtgaagcgga aaaatggttt gataagtggt tcaaaaatcc aattccgccg 1680 aaaaacctga acatgaattt cgaactgtca gacgaaatga aagcactgtt caaagaaccg 1740 aatgacaagg cactgaacgg cgccggtacc ggtggaatgg tgagcaaggg cgaggagctg 1800 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1860 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1920 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc 1980 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2040 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2100 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2160 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2220 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2280 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2340 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg 2400 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 2460 gggatcactc tcggcatgga cgagctgtac aagtaaaagc ttgatccggc tgctaacaaa 2520 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 2580 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct 2640 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 2700 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2760 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2820 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2880 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2940 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3000 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatcgcg gtctattctt 3060 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3120 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3180 tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa 3240 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3300 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3360 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3420 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3480 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3540 ttccaatgat gagcactttt aaagttctgc tatgtgatac actattatcc cgtattgacg 3600 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3660 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 3720 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 3780 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 3840 aaccggagct gaatgaagcc ataccaaacg acgagagtga caccacgatg cctgtagcaa 3900 tgccaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3960 aattaataga ctgaatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4020 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4080 ttgcagcact ggggccagat ggtaagcgct cccgtatcgt agttatctac acgacgggga 4140 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4200 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4260 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4320 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4380 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4440 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4500 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4560 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4620 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4680 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4740 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4800 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 4860 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 4920 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 4980 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg 5040 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5100 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5160 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcag 5206 <210> SEQ ID NO 10 <211> LENGTH: 5206 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pRSETB FLIP-E 10u vector <400> SEQUENCE: 10 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 60 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat 120 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat 180 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg 240 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 300 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 360 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc 420 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 480 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 540 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 600 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc 660 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag 720 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 780 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 840 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 900 atggacgagc tgtacaaggg tggtaccgga ggcgccgcag gcagcacgct ggacaaaatc 960 gccaaaaacg gtgtgattgt cgtcggtcac cgtgaatctt cagtgccttt ctcttattac 1020 gacaatcagc aaaaagtggt gggttactcg caggattact ccaacgccat tgttgaagca 1080 gtgaaaaaga aactcaacaa accggacttg caggtaaaac tgattccgat tacctcacaa 1140 aaccgtattc cactgctgca aaacggcact ttcgattttg aatgtggttc taccaccaac 1200 aacgtcgaac gccaaaaaca ggcggctttc tctgacacta ttttcgtggt cggtacgcgc 1260 ctgttgacca aaaagggtgg cgatatcaaa gattttgcca acctgaaaga caaagccgta 1320 gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca aactgaatga agagcaaaaa 1380 atgaatatgc gcatcatcag cgccaaagat cacggtgact ctttccgcac cctggaaagc 1440 ggtcgtgccg ttgcctttat gatggatgac cggctgctgg ccggtgaacg tgcgaaagcg 1500 aagaaaccag acaactggga aatcgtcggc aagccgcagt ctcaggaggc ctacggttgt 1560 atgttgcgta aagatgatcc gcagttcaaa aagctgatgg atgacaccat cgctcaggtg 1620 cagacctccg gtgaagcgga aaaatggttt gataagtggt tcaaaaatcc aattccgccg 1680 aaaaacctga acatgaattt cgaactgtca gacgaaatga aagcactgtt caaagaaccg 1740 aatgacaagg cactgaacgg cgccggtacc ggtggaatgg tgagcaaggg cgaggagctg 1800 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1860 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1920 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc 1980 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2040 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2100 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2160 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2220 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2280 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2340 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg 2400 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 2460 gggatcactc tcggcatgga cgagctgtac aagtaaaagc ttgatccggc tgctaacaaa 2520 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 2580 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct 2640 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 2700 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2760 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2820 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2880 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2940 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3000 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatcgcg gtctattctt 3060 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3120 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3180 tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa 3240 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3300 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3360 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3420 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3480 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3540 ttccaatgat gagcactttt aaagttctgc tatgtgatac actattatcc cgtattgacg 3600 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3660 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 3720 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 3780 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 3840 aaccggagct gaatgaagcc ataccaaacg acgagagtga caccacgatg cctgtagcaa 3900 tgccaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3960 aattaataga ctgaatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4020 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4080 ttgcagcact ggggccagat ggtaagcgct cccgtatcgt agttatctac acgacgggga 4140 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4200 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4260 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4320 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4380 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4440 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4500 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4560 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4620 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4680 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4740 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4800 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 4860 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 4920 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 4980 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg 5040 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5100 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5160 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcag 5206 <210> SEQ ID NO 11 <211> LENGTH: 7603 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pDisplay FLIP-E 600n <400> SEQUENCE: 11 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg 720 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 780 tccaggttcc actggtgact atccatatga tgttccagat tatgctgggg cccagccggc 840 cagatctccc gggatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 900 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 960 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1020 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 1080 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1140 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1200 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1260 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 1320 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 1380 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 1440 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 1500 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 1560 gctgtacaag ggtggtaccg gaggcgccgc aggcagcacg ctggacaaaa tcgccaaaaa 1620 cggtgtgatt gtcgtcggtc accgtgaatc ttcagtgcct ttctcttatt acgacaatca 1680 gcaaaaagtg gtgggttact cgcaggatta ctccaacgcc attgttgaag cagtgaaaaa 1740 gaaactcaac aaaccggact tgcaggtaaa actgattccg attacctcac aaaaccgtat 1800 tccactgctg caaaacggca ctttcgattt tgaatgtggt tctaccacca acaacgtcga 1860 acgccaaaaa caggcggctt tctctgacac tattttcgtg gtcggtacgc gcctgttgac 1920 caaaaagggt ggcgatatca aagattttgc caacctgaaa gacaaagccg tagtcgtcac 1980 ttccggcact acctctgaag ttttgctcaa caaactgaat gaagagcaaa aaatgaatat 2040 gcgcatcatc agcgccaaag atcacggtga ctctttccgc accctggaaa gcggtcgtgc 2100 cgttgccttt atgatggatg acgctctgct ggccggtgaa cgtgcgaaag cgaagaaacc 2160 agacaactgg gaaatcgtcg gcaagccgca gtctcaggag gcctacggtt gtatgttgcg 2220 taaagatgat ccgcagttca aaaagctgat ggatgacacc atcgctcagg tgcagacctc 2280 cggtgaagcg gaaaaatggt ttgataagtg gttcaaaaat ccaattccgc cgaaaaacct 2340 gaacatgaat ttcgaactgt cagacgaaat gaaagcactg ttcaaagaac cgaatgacaa 2400 ggcactgaac ggcgccggta ccggtggaat ggtgagcaag ggcgaggagc tgttcaccgg 2460 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 2520 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 2580 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg 2640 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 2700 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 2760 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 2820 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 2880 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 2940 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3000 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga 3060 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3120 tctcggcatg gacgagctgt acaaggtcga cgaacaaaaa ctcatctcag aagaggatct 3180 gaatgctgtg ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa 3240 ggtggtggtg atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat 3300 cctcatcatg ctttggcaga agaagccacg ttaggcggcc gctcgagatc agcctcgact 3360 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3420 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3480 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3540 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3600 accagtggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3660 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3720 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3780 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3840 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3900 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3960 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4020 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4080 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4140 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4200 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4260 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4320 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4380 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4440 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc 4500 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc 4560 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac 4620 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt 4680 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg 4740 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg 4800 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga 4860 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac 4920 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 4980 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 5040 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 5100 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 5160 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 5220 tgctcttgat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 5280 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 5340 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 5400 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 5460 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc 5520 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 5580 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 5640 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 5700 cgaaacgatc ctcatcctgt ctcttgatcg atctttgcaa aagcctaggc ctccaaaaaa 5760 gcctcctcac tacttctgga atagctcaga ggccgaggag gcggcctcgg cctctgcata 5820 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg 5880 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg 5940 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag 6000 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg 6060 acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca ataaatcaat 6120 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6180 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6240 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6300 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6360 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6420 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6480 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6540 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6600 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6660 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6720 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6780 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6840 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6900 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6960 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7020 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7080 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7140 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 7200 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 7260 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 7320 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 7380 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7440 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 7500 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7560 atttaacgcg aattttaaca aaatattaac gcttacaatt tac 7603 <210> SEQ ID NO 12 <211> LENGTH: 7603 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pDisplay FLIP-E 10u <400> SEQUENCE: 12 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg 720 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 780 tccaggttcc actggtgact atccatatga tgttccagat tatgctgggg cccagccggc 840 cagatctccc gggatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 900 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 960 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1020 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 1080 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1140 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1200 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1260 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 1320 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 1380 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 1440 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 1500 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 1560 gctgtacaag ggtggtaccg gaggcgccgc aggcagcacg ctggacaaaa tcgccaaaaa 1620 cggtgtgatt gtcgtcggtc accgtgaatc ttcagtgcct ttctcttatt acgacaatca 1680 gcaaaaagtg gtgggttact cgcaggatta ctccaacgcc attgttgaag cagtgaaaaa 1740 gaaactcaac aaaccggact tgcaggtaaa actgattccg attacctcac aaaaccgtat 1800 tccactgctg caaaacggca ctttcgattt tgaatgtggt tctaccacca acaacgtcga 1860 acgccaaaaa caggcggctt tctctgacac tattttcgtg gtcggtacgc gcctgttgac 1920 caaaaagggt ggcgatatca aagattttgc caacctgaaa gacaaagccg tagtcgtcac 1980 ttccggcact acctctgaag ttttgctcaa caaactgaat gaagagcaaa aaatgaatat 2040 gcgcatcatc agcgccaaag atcacggtga ctctttccgc accctggaaa gcggtcgtgc 2100 cgttgccttt atgatggatg accggctgct ggccggtgaa cgtgcgaaag cgaagaaacc 2160 agacaactgg gaaatcgtcg gcaagccgca gtctcaggag gcctacggtt gtatgttgcg 2220 taaagatgat ccgcagttca aaaagctgat ggatgacacc atcgctcagg tgcagacctc 2280 cggtgaagcg gaaaaatggt ttgataagtg gttcaaaaat ccaattccgc cgaaaaacct 2340 gaacatgaat ttcgaactgt cagacgaaat gaaagcactg ttcaaagaac cgaatgacaa 2400 ggcactgaac ggcgccggta ccggtggaat ggtgagcaag ggcgaggagc tgttcaccgg 2460 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 2520 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 2580 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg 2640 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 2700 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 2760 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 2820 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 2880 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 2940 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3000 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga 3060 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3120 tctcggcatg gacgagctgt acaaggtcga cgaacaaaaa ctcatctcag aagaggatct 3180 gaatgctgtg ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa 3240 ggtggtggtg atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat 3300 cctcatcatg ctttggcaga agaagccacg ttaggcggcc gctcgagatc agcctcgact 3360 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3420 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3480 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3540 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3600 accagtggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3660 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3720 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3780 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3840 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3900 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3960 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4020 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4080 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4140 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4200 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4260 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4320 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4380 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4440 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc 4500 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc 4560 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac 4620 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt 4680 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg 4740 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg 4800 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga 4860 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac 4920 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 4980 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 5040 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 5100 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 5160 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 5220 tgctcttgat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 5280 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 5340 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 5400 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 5460 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc 5520 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 5580 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 5640 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 5700 cgaaacgatc ctcatcctgt ctcttgatcg atctttgcaa aagcctaggc ctccaaaaaa 5760 gcctcctcac tacttctgga atagctcaga ggccgaggag gcggcctcgg cctctgcata 5820 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg 5880 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg 5940 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag 6000 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg 6060 acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca ataaatcaat 6120 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6180 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6240 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6300 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6360 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6420 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6480 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6540 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6600 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6660 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6720 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6780 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6840 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6900 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6960 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7020 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7080 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7140 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 7200 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 7260 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 7320 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 7380 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7440 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 7500 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7560 atttaacgcg aattttaaca aaatattaac gcttacaatt tac 7603 <210> SEQ ID NO 13 <211> LENGTH: 799 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Amino acid sequence of FLIP-E 600n (expressed as 6xHis fusion in pRSET FLIP-E 600n) <400> SEQUENCE: 13 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Gly Ser Thr Leu Asp Lys Ile 275 280 285 Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu Ser Ser Val Pro 290 295 300 Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp 305 310 315 320 Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro 325 330 335 Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro 340 345 350 Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn 355 360 365 Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val 370 375 380 Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe 385 390 395 400 Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser 405 410 415 Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg 420 425 430 Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser 435 440 445 Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu 450 455 460 Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro 465 470 475 480 Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln 485 490 495 Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly 500 505 510 Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro 515 520 525 Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu 530 535 540 Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn Gly Ala Gly Thr Gly Gly 545 550 555 560 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 565 570 575 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 580 585 590 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 595 600 605 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 610 615 620 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 625 630 635 640 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 645 650 655 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 660 665 670 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 675 680 685 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 690 695 700 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 705 710 715 720 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 725 730 735 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 740 745 750 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 755 760 765 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 770 775 780 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 785 790 795 <210> SEQ ID NO 14 <211> LENGTH: 764 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Amino acid sequence of FLIP-E 10u (expressed as 6xHis fusion in pRSET FLIP-E 10u) <400> SEQUENCE: 14 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 225 230 235 240 Gly Thr Gly Gly Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn 245 250 255 Gly Val Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr 260 265 270 Tyr Asp Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn 275 280 285 Ala Ile Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln 290 295 300 Val Lys Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln 305 310 315 320 Asn Gly Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu 325 330 335 Arg Gln Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr 340 345 350 Arg Leu Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu 355 360 365 Lys Asp Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu 370 375 380 Leu Asn Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser 385 390 395 400 Ala Lys Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala 405 410 415 Val Ala Phe Met Met Asp Asp Arg Leu Leu Ala Gly Glu Arg Ala Lys 420 425 430 Ala Lys Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln 435 440 445 Glu Ala Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys 450 455 460 Leu Met Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu 465 470 475 480 Lys Trp Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu 485 490 495 Asn Met Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu 500 505 510 Pro Asn Asp Lys Ala Leu Asn Gly Ala Gly Thr Gly Gly Met Val Ser 515 520 525 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 530 535 540 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 545 550 555 560 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 565 570 575 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr 580 585 590 Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp 595 600 605 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 610 615 620 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 625 630 635 640 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 645 650 655 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn 660 665 670 Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys 675 680 685 Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 690 695 700 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 705 710 715 720 Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp 725 730 735 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 740 745 750 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> SEQ ID NO 15 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-Y12S-CFP-D13R-YFP Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 15 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc gct gat act cgc att ggt gta aca atc tat aag tcg gct 144 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Ser Ala 35 40 45 ggt atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc 192 Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 50 55 60 ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc 240 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 65 70 75 80 ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc 288 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 85 90 95 atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc 336 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 100 105 110 acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg 384 Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 115 120 125 aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag 432 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 130 135 140 gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc 480 Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 145 150 155 160 gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag 528 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 165 170 175 ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag 576 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 180 185 190 tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag cag aag 624 Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 195 200 205 aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag gac ggc 672 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 210 215 220 agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac 720 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 225 230 235 240 ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gcc 768 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 245 250 255 ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag 816 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 260 265 270 ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac ggt 864 Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly 275 280 285 agc cga gat aac gcg atg tct gta gtg cgc aag gct att gag caa gat 912 Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp 290 295 300 gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat tct cag aat 960 Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn 305 310 315 320 gac cag tcc aag cag aac gat cag atc gac gta ttg ctg gcg aaa ggg 1008 Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly 325 330 335 gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct gcg ggt acg 1056 Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr 340 345 350 gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt ttc ttc aac 1104 Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn 355 360 365 aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa gcc tac tac 1152 Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr 370 375 380 gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc gat ttg att 1200 Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile 385 390 395 400 gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac aaa gac ggt 1248 Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly 405 410 415 cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat ccg gat gca 1296 Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala 420 425 430 gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat aaa ggc atc 1344 Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile 435 440 445 aaa act gaa cag tta cag tta gat acc gca atg tgg gac acc gct cag 1392 Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln 450 455 460 gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac gcc aac aaa 1440 Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys 465 470 475 480 atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg ggc gcg gtt 1488 Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val 485 490 495 gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg ttt ggc gtc 1536 Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val 500 505 510 gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt gca ctg gcg 1584 Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala 515 520 525 ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg acc ttt gat 1632 Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp 530 535 540 ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg gct gat ggc acc aac 1680 Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 tgg aaa atc gac aac aaa gtg gtc cgc gta cct tat gtt ggc gta gat 1728 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 aaa gac aac ctg gct gaa ttc agc aag aaa ggc gcc ggt acc ggt gga 1776 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 1824 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 1872 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 1920 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 1968 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 ttc ggc tac ggc ctg cag tgc ttc gcc cgc tac ccc gac cac atg aag 2016 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 2064 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 2112 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 2160 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 2208 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac 2256 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 2304 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 2352 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 ccc gtg ctg ctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 2400 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 2448 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 2496 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 16 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-Y12S-CFP-D13R-YFP Vector <400> SEQUENCE: 16 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Ser Ala 35 40 45 Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 50 55 60 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 65 70 75 80 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 85 90 95 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 100 105 110 Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 115 120 125 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 130 135 140 Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 145 150 155 160 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 165 170 175 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 180 185 190 Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 195 200 205 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 210 215 220 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 225 230 235 240 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 245 250 255 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 260 265 270 Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly 275 280 285 Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp 290 295 300 Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn 305 310 315 320 Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly 325 330 335 Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr 340 345 350 Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn 355 360 365 Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr 370 375 380 Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile 385 390 395 400 Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly 405 410 415 Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala 420 425 430 Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile 435 440 445 Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln 450 455 460 Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys 465 470 475 480 Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val 485 490 495 Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val 500 505 510 Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala 515 520 525 Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp 530 535 540 Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 17 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-G275S-CFP-K276R-YFP Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 17 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc gct gat act cgc att ggt gta aca atc tat aag tac gac 144 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Tyr Asp 35 40 45 gat aac gcg atg tct gta gtg cgc aag gct att gag caa gat gcg aaa 192 Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp Ala Lys 50 55 60 gcc gcg cca gat gtt cag ctg ctg atg aat gat tct cag aat gac cag 240 Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn Asp Gln 65 70 75 80 tcc aag cag aac gat cag atc gac gta ttg ctg gcg aaa ggg gtg aag 288 Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly Val Lys 85 90 95 gca ctg gca atc aac ctg gtt gac ccg gca gct gcg ggt acg gtg att 336 Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr Val Ile 100 105 110 gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt ttc ttc aac aaa gaa 384 Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn Lys Glu 115 120 125 ccg tct cgt aag gcg ctg gat agc tac gac aaa gcc tac tac gtt ggc 432 Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr Val Gly 130 135 140 act gac tcc aaa gag tcc ggc att att caa ggc gat ttg att gct aaa 480 Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile Ala Lys 145 150 155 160 cac tgg gcg gcg aat cag ggt tgg gat ctg aac aaa gac ggt cag att 528 His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly Gln Ile 165 170 175 cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat ccg gat gca gaa gca 576 Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala Glu Ala 180 185 190 cgt acc act tac gtg att aaa gaa ttg aac gat aaa ggc atc aaa act 624 Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile Lys Thr 195 200 205 gaa cag tta cag tta gat acc gca atg tgg gac acc gct cag gcg aaa 672 Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln Ala Lys 210 215 220 gat aag atg gac gcc tgg ctg tct ggc ccg aac gcc aac aaa atc gaa 720 Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys Ile Glu 225 230 235 240 gtg gtt atc gcc aac aac gat gcg atg gca atg ggc gcg gtt gaa gcg 768 Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val Glu Ala 245 250 255 ctg aaa gca cac aac aag tcc agc att ccg gtg ttt ggc gtc gat gcg 816 Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val Asp Ala 260 265 270 ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt gca ctg gcg ggc acc 864 Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala Gly Thr 275 280 285 gta ctg aac gat gct aac aac cag gcg aaa gcg acc ttt gat ctg gcg 912 Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp Leu Ala 290 295 300 aaa aac ctg gcc gat tcg gct ggt atg gtg agc aag ggc gag gag ctg 960 Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu 305 310 315 320 ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac 1008 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 325 330 335 ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac 1056 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 340 345 350 ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg 1104 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 355 360 365 ccc tgg ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc 1152 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe 370 375 380 agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc 1200 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 385 390 395 400 atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac 1248 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 405 410 415 ggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg 1296 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 420 425 430 gtg aac cgc atc gag ctg aag ggc atc gac ttc aag gag gac ggc aac 1344 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 435 440 445 atc ctg ggg cac aag ctg gag tac aac tac atc agc cac aac gtc tat 1392 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr 450 455 460 atc acc gcc gac aag cag aag aac ggc atc aag gcc aac ttc aag atc 1440 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 465 470 475 480 cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag 1488 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 485 490 495 cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac 1536 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 500 505 510 tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc 1584 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 515 520 525 gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc 1632 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 530 535 540 ggc atg gac gag ctg tac ggt agc cga ggt gcg gct gat ggc acc aac 1680 Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 tgg aaa atc gac aac aaa gtg gtc cgc gta cct tat gtt ggc gta gat 1728 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 aaa gac aac ctg gct gaa ttc agc aag aaa ggc gcc ggt acc ggt gga 1776 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 1824 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 1872 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 1920 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 1968 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 ttc ggc tac ggc ctg cag tgc ttc gcc cgc tac ccc gac cac atg aag 2016 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 2064 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 2112 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 2160 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 2208 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac 2256 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 2304 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 2352 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 ccc gtg ctg ctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 2400 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 2448 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 2496 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 18 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-G275S-CFP-K276R-YFP Vector <400> SEQUENCE: 18 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Tyr Asp 35 40 45 Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp Ala Lys 50 55 60 Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn Asp Gln 65 70 75 80 Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly Val Lys 85 90 95 Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr Val Ile 100 105 110 Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn Lys Glu 115 120 125 Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr Val Gly 130 135 140 Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile Ala Lys 145 150 155 160 His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly Gln Ile 165 170 175 Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala Glu Ala 180 185 190 Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile Lys Thr 195 200 205 Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln Ala Lys 210 215 220 Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys Ile Glu 225 230 235 240 Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val Glu Ala 245 250 255 Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val Asp Ala 260 265 270 Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala Gly Thr 275 280 285 Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp Leu Ala 290 295 300 Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu 305 310 315 320 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 325 330 335 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 340 345 350 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 355 360 365 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe 370 375 380 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 385 390 395 400 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 405 410 415 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 420 425 430 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 435 440 445 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr 450 455 460 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 465 470 475 480 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 485 490 495 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 500 505 510 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 515 520 525 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 530 535 540 Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 19 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-Y12S-CFP-D13R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 19 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tcg gct ggt atg gtg agc aag ggc gag gag ctg ttc acc 912 Ile Tyr Lys Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr 290 295 300 ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac 960 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His 305 310 315 320 aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag 1008 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys 325 330 335 ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg 1056 Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp 340 345 350 ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc 1104 Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg 355 360 365 tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc 1152 Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro 370 375 380 gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac 1200 Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 385 390 395 400 tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac 1248 Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 405 410 415 cgc atc gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg 1296 Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu 420 425 430 ggg cac aag ctg gag tac aac tac atc agc cac aac gtc tat atc acc 1344 Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr 435 440 445 gcc gac aag cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac 1392 Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His 450 455 460 aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac 1440 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 465 470 475 480 acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg 1488 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 485 490 495 agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 1536 Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 500 505 510 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg 1584 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 515 520 525 gac gag ctg tac ggt agc cga gat aac gcg atg tct gta gtg cgc aag 1632 Asp Glu Leu Tyr Gly Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys 530 535 540 gct att gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg 1680 Ala Ile Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met 545 550 555 560 aat gat tct cag aat gac cag tcc aag cag aac gat cag atc gac gta 1728 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 ttg ctg gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg 1776 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 gca gct gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg 1824 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 gtg gtt ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac 1872 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 gac aaa gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att 1920 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 caa ggc gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat 1968 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 ctg aac aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg 2016 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 ggc cat ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg 2064 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 aac gat aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg 2112 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 tgg gac acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc 2160 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 ccg aac gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg 2208 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 gca atg ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att 2256 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 ccg gtg ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa 2304 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 tcc ggt gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg 2352 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 aaa gcg acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg 2400 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 20 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-Y12S-CFP-D13R Vector <400> SEQUENCE: 20 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr 290 295 300 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His 305 310 315 320 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys 325 330 335 Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp 340 345 350 Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg 355 360 365 Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro 370 375 380 Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 385 390 395 400 Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 405 410 415 Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu 420 425 430 Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr 435 440 445 Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His 450 455 460 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 465 470 475 480 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 485 490 495 Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 500 505 510 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 515 520 525 Asp Glu Leu Tyr Gly Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys 530 535 540 Ala Ile Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met 545 550 555 560 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 21 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-P32S-CFP-D33R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 21 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg tcg gct ggt atg gtg agc aag ggc gag 960 Glu Gln Asp Ala Lys Ala Ala Ser Ala Gly Met Val Ser Lys Gly Glu 305 310 315 320 gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac 1008 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 325 330 335 gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc 1056 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 340 345 350 acc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg 1104 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 355 360 365 ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag 1152 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln 370 375 380 tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag 1200 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 385 390 395 400 tcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag 1248 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 405 410 415 gac gac ggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac 1296 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 420 425 430 acc ctg gtg aac cgc atc gag ctg aag ggc atc gac ttc aag gag gac 1344 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 435 440 445 ggc aac atc ctg ggg cac aag ctg gag tac aac tac atc agc cac aac 1392 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn 450 455 460 gtc tat atc acc gcc gac aag cag aag aac ggc atc aag gcc aac ttc 1440 Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe 465 470 475 480 aag atc cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac 1488 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 485 490 495 tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac 1536 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 500 505 510 aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag 1584 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 515 520 525 aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc 1632 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 530 535 540 act ctc ggc atg gac gag ctg tac ggt agc cga gtt cag ctg ctg atg 1680 Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Val Gln Leu Leu Met 545 550 555 560 aat gat tct cag aat gac cag tcc aag cag aac gat cag atc gac gta 1728 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 ttg ctg gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg 1776 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 gca gct gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg 1824 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 gtg gtt ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac 1872 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 gac aaa gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att 1920 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 caa ggc gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat 1968 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 ctg aac aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg 2016 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 ggc cat ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg 2064 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 aac gat aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg 2112 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 tgg gac acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc 2160 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 ccg aac gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg 2208 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 gca atg ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att 2256 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 ccg gtg ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa 2304 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 tcc ggt gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg 2352 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 aaa gcg acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg 2400 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 22 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-P32S-CFP-D33R Vector <400> SEQUENCE: 22 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Ser Ala Gly Met Val Ser Lys Gly Glu 305 310 315 320 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 325 330 335 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 340 345 350 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 355 360 365 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln 370 375 380 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 385 390 395 400 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 405 410 415 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 420 425 430 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 435 440 445 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn 450 455 460 Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe 465 470 475 480 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 485 490 495 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 500 505 510 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 515 520 525 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 530 535 540 Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Val Gln Leu Leu Met 545 550 555 560 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 23 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-G275S-CFP-K276R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 23 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat 960 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 tct cag aat gac cag tcc aag cag aac gat cag atc gac gta ttg ctg 1008 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct 1056 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt 1104 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa 1152 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc 1200 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac 1248 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat 1296 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat 1344 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg tgg gac 1392 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac 1440 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg 1488 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg 1536 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt 1584 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg 1632 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 acc ttt gat ctg gcg aaa aac ctg gcc gat tcg gct ggt atg gtg agc 1680 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser 545 550 555 560 aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg 1728 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 565 570 575 gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag 1776 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 580 585 590 ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc 1824 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 595 600 605 ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tgg 1872 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp 610 615 620 ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac 1920 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp 625 630 635 640 ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atc 1968 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 645 650 655 ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag gtg aag ttc 2016 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 660 665 670 gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc atc gac ttc 2064 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 675 680 685 aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac aac tac atc 2112 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile 690 695 700 agc cac aac gtc tat atc acc gcc gac aag cag aag aac ggc atc aag 2160 Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys 705 710 715 720 gcc aac ttc aag atc cgc cac aac atc gag gac ggc agc gtg cag ctc 2208 Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 725 730 735 gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg 2256 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 740 745 750 ctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac 2304 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp 755 760 765 ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc 2352 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 770 775 780 gcc ggg atc act ctc ggc atg gac gag ctg tac ggt agc cga ggt gcg 2400 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 24 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-G275S-CFP-K276R Vector <400> SEQUENCE: 24 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser 545 550 555 560 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 565 570 575 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 580 585 590 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 595 600 605 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp 610 615 620 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp 625 630 635 640 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 645 650 655 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 660 665 670 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 675 680 685 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile 690 695 700 Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys 705 710 715 720 Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 725 730 735 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 740 745 750 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp 755 760 765 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 770 775 780 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 25 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-T282S-CFP-N283R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 25 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat 960 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 tct cag aat gac cag tcc aag cag aac gat cag atc gac gta ttg ctg 1008 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct 1056 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt 1104 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa 1152 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc 1200 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac 1248 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat 1296 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat 1344 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg tgg gac 1392 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac 1440 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg 1488 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg 1536 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt 1584 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg 1632 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg gct gat 1680 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp 545 550 555 560 ggc tcg gct ggt atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg 1728 Gly Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 565 570 575 gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc 1776 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 580 585 590 agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc 1824 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 595 600 605 ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc 1872 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 610 615 620 ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc 1920 Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro 625 630 635 640 gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc 1968 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 645 650 655 tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag 2016 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 660 665 670 acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc 2064 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 675 680 685 gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac 2112 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 690 695 700 aag ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac 2160 Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp 705 710 715 720 aag cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc 2208 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile 725 730 735 gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc 2256 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 740 745 750 atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc 2304 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 755 760 765 cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc 2352 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 770 775 780 ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag 2400 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 785 790 795 800 ctg tac ggt agc cga tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Leu Tyr Gly Ser Arg Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 26 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-T282S-CFP-N283R Vector <400> SEQUENCE: 26 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp 545 550 555 560 Gly Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 565 570 575 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 580 585 590 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 595 600 605 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 610 615 620 Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro 625 630 635 640 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 645 650 655 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 660 665 670 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 675 680 685 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 690 695 700 Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp 705 710 715 720 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile 725 730 735 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 740 745 750 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 755 760 765 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 770 775 780 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 785 790 795 800 Leu Tyr Gly Ser Arg Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 27 <211> LENGTH: 909 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(909) <400> SEQUENCE: 27 atg caa tta cgt aaa cct gcc aca gca atc ctc gcc ctg gcg ctt tcc 48 Met Gln Leu Arg Lys Pro Ala Thr Ala Ile Leu Ala Leu Ala Leu Ser 1 5 10 15 gca gga ctg gca cag gca gat gac gcc gcc ccg gca gcg ggc agt act 96 Ala Gly Leu Ala Gln Ala Asp Asp Ala Ala Pro Ala Ala Gly Ser Thr 20 25 30 ctg gac aaa atc gcc aaa aac ggt gtg att gtc gtc ggt cac cgt gaa 144 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 35 40 45 tct tca gtg cct ttc tct tat tac gac aat cag caa aaa gtg gtg ggt 192 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 50 55 60 tac tcg cag gat tac tcc aac gcc att gtt gaa gca gtg aaa aag aaa 240 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 65 70 75 80 ctc aac aaa ccg gac ttg cag gta aaa ctg att ccg att acc tca caa 288 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 85 90 95 aac cgt att cca ctg ctg caa aac ggc act ttc gat ttt gaa tgt ggt 336 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 100 105 110 tct acc acc aac aac gtc gaa cgc caa aaa cag gcg gct ttc tct gac 384 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 115 120 125 act att ttc gtg gtc ggt acg cgc ctg ttg acc aaa aag ggt ggc gat 432 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 130 135 140 atc aaa gat ttt gcc aac ctg aaa gac aaa gcc gta gtc gtc act tcc 480 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 145 150 155 160 ggc act acc tct gaa gtt ttg ctc aac aaa ctg aat gaa gag caa aaa 528 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 165 170 175 atg aat atg cgc atc atc agc gcc aaa gat cac ggt gac tct ttc cgc 576 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 180 185 190 acc ctg gaa agc ggt cgt gcc gtt gcc ttt atg atg gat gac gct ctg 624 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 195 200 205 ctg gcc ggt gaa cgt gcg aaa gcg aag aaa cca gac aac tgg gaa atc 672 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 210 215 220 gtc ggc aag ccg cag tct cag gag gcc tac ggt tgt atg ttg cgt aaa 720 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 225 230 235 240 gat gat ccg cag ttc aaa aag ctg atg gat gac acc atc gct cag gtg 768 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 245 250 255 cag acc tcc ggt gaa gcg gaa aaa tgg ttt gat aag tgg ttc aaa aat 816 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 260 265 270 cca att ccg ccg aaa aac ctg aac atg aat ttc gaa ctg tca gac gaa 864 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 275 280 285 atg aaa gca ctg ttc aaa gaa ccg aat gac aag gca ctg aac taa 909 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 290 295 300 <210> SEQ ID NO 28 <211> LENGTH: 302 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 28 Met Gln Leu Arg Lys Pro Ala Thr Ala Ile Leu Ala Leu Ala Leu Ser 1 5 10 15 Ala Gly Leu Ala Gln Ala Asp Asp Ala Ala Pro Ala Ala Gly Ser Thr 20 25 30 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 35 40 45 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 50 55 60 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 65 70 75 80 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 85 90 95 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 100 105 110 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 115 120 125 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 130 135 140 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 145 150 155 160 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 165 170 175 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 180 185 190 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 195 200 205 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 210 215 220 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 225 230 235 240 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 245 250 255 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 260 265 270 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 275 280 285 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 290 295 300 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 30 Gly Asn Asn Ser Ala Gly 1 5 <210> SEQ ID NO 31 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 31 Gly Ser Ala Asp Asp Gly 1 5 <210> SEQ ID NO 32 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 32 Gly Gly Thr Gly Gly Ala 1 5 <210> SEQ ID NO 33 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 33 ggtggtaccg gaggcgcc 18 <210> SEQ ID NO 34 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 34 agcgctggta tggtg 15 <210> SEQ ID NO 35 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 35 actctcggta gcgct 15 <210> SEQ ID NO 36 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 36 Ala Gly Met Val 1 <210> SEQ ID NO 37 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 37 Thr Leu Gly Ser 1 <210> SEQ ID NO 38 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 38 aaagcgggca acaacagcgc tggtgacggc 30 <210> SEQ ID NO 39 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 39 atcgagggta gcgctgacga cggaaagaaa 30 <210> SEQ ID NO 40 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 40 Lys Ala Gly Asn Asn Ser Ala Gly Asn Gly 1 5 10 <210> SEQ ID NO 41 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 41 Ile Glu Gly Ser Ala Asp Asp Gly Lys Lys 1 5 10 <210> SEQ ID NO 42 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII275 Pglu plasmid <400> SEQUENCE: 42 actctcggca tggacgagct gtacaagggt ggtaccggag gcgcc 45 <210> SEQ ID NO 43 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 43 actctcggca tggacgagct gtacggtagc cga 33 <210> SEQ ID NO 44 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 44 gaattcagca agaaaggcgc cggtaccggt ggaatggtga gcaagggcga g 51 <210> SEQ ID NO 45 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 45 gaattcagca agaaaggtac catggtgagc aagggcgag 39 <210> SEQ ID NO 46 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 46 gaattcagca agaaaatggt gagcaagggc gag 33 <210> SEQ ID NO 47 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 47 ttcagcaaga aaggcgccgg taccggtgga atggtgagca agggcgag 48 <210> SEQ ID NO 48 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 48 agcaagaaag gcgccggtac cggtggaatg gtgagcaagg gcgag 45 <210> SEQ ID NO 49 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 49 aagaaaggcg ccggtaccgg tggaatggtg agcaagggcg ag 42 <210> SEQ ID NO 50 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 50 aaaggcgccg gtaccggtgg aatggtgagc aagggcgag 39 <210> SEQ ID NO 51 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 51 ggcgccggta ccggtggaat ggtgagcaag ggcgag 36 <210> SEQ ID NO 52 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 52 gccggtaccg gtggaatggt gagcaagggc gag 33 <210> SEQ ID NO 53 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 53 ggtaccggtg gaatggtgag caagggcgag 30 <210> SEQ ID NO 54 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 54 accggtggaa tggtgagcaa gggcgag 27 <210> SEQ ID NO 55 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 55 ggtggaatgg tgagcaaggg cgag 24 <210> SEQ ID NO 56 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 56 ggaatggtga gcaagggcga g 21 <210> SEQ ID NO 57 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 57 atggtgagca agggcgag 18 <210> SEQ ID NO 58 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 58 gtgagcaagg gcgag 15 <210> SEQ ID NO 59 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 59 agcaagggcg ag 12

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 59 <210> SEQ ID NO 1 <211> LENGTH: 978 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(978) <400> SEQUENCE: 1 atg ata aca aca caa aca ctc aca acg ggt atc cat gcg ttc tta acg 48 Met Ile Thr Thr Gln Thr Leu Thr Thr Gly Ile His Ala Phe Leu Thr 1 5 10 15 cag aag ata aag gag ttg gat atg caa tta cgt aaa cct gcc aca gca 96 Gln Lys Ile Lys Glu Leu Asp Met Gln Leu Arg Lys Pro Ala Thr Ala 20 25 30 atc ctc gcc ctg gcg ctt tcc gca gga ctg gca cag gca gat gac gcc 144 Ile Leu Ala Leu Ala Leu Ser Ala Gly Leu Ala Gln Ala Asp Asp Ala 35 40 45 gcc ccg gca gcg ggc agt act ctg gac aaa atc gcc aaa aac ggt gtg 192 Ala Pro Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn Gly Val 50 55 60 att gtc gtc ggt cac cgt gaa tct tca gtg cct ttc tct tat tac gac 240 Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr Tyr Asp 65 70 75 80 aat cag caa aaa gtg gtg ggt tac tcg cag gat tac tcc aac gcc att 288 Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn Ala Ile 85 90 95 gtt gaa gca gtg aaa aag aaa ctc aac aaa ccg gac ttg cag gta aaa 336 Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln Val Lys 100 105 110 ctg att ccg att acc tca caa aac cgt att cca ctg ctg caa aac ggc 384 Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln Asn Gly 115 120 125 act ttc gat ttt gaa tgt ggt tct acc acc aac aac gtc gaa cgc caa 432 Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu Arg Gln 130 135 140 aaa cag gcg gct ttc tct gac act att ttc gtg gtc ggt acg cgc ctg 480 Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr Arg Leu 145 150 155 160 ttg acc aaa aag ggt ggc gat atc aaa gat ttt gcc aac ctg aaa gac 528 Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu Lys Asp 165 170 175 aaa gcc gta gtc gtc act tcc ggc act acc tct gaa gtt ttg ctc aac 576 Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu Leu Asn 180 185 190 aaa ctg aat gaa gag caa aaa atg aat atg cgc atc atc agc gcc aaa 624 Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser Ala Lys 195 200 205 gat cac ggt gac tct ttc cgc acc ctg gaa agc ggt cgt gcc gtt gcc 672 Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala Val Ala 210 215 220 ttt atg atg gat gac gct ctg ctg gcc ggt gaa cgt gcg aaa gcg aag 720 Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg Ala Lys Ala Lys 225 230 235 240 aaa cca gac aac tgg gaa atc gtc ggc aag ccg cag tct cag gag gcc 768 Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln Glu Ala 245 250 255 tac ggt tgt atg ttg cgt aaa gat gat ccg cag ttc aaa aag ctg atg 816 Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys Leu Met 260 265 270 gat gac acc atc gct cag gtg cag acc tcc ggt gaa gcg gaa aaa tgg 864 Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu Lys Trp 275 280 285 ttt gat aag tgg ttc aaa aat cca att ccg ccg aaa aac ctg aac atg 912 Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu Asn Met 290 295 300 aat ttc gaa ctg tca gac gaa atg aaa gca ctg ttc aaa gaa ccg aat 960 Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu Pro Asn 305 310 315 320 gac aag gca ctg aac taa 978 Asp Lys Ala Leu Asn 325 <210> SEQ ID NO 2 <211> LENGTH: 325 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 2 Met Ile Thr Thr Gln Thr Leu Thr Thr Gly Ile His Ala Phe Leu Thr 1 5 10 15 Gln Lys Ile Lys Glu Leu Asp Met Gln Leu Arg Lys Pro Ala Thr Ala 20 25 30 Ile Leu Ala Leu Ala Leu Ser Ala Gly Leu Ala Gln Ala Asp Asp Ala 35 40 45 Ala Pro Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn Gly Val 50 55 60 Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr Tyr Asp 65 70 75 80 Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn Ala Ile 85 90 95 Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln Val Lys 100 105 110 Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln Asn Gly 115 120 125 Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu Arg Gln 130 135 140 Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr Arg Leu 145 150 155 160 Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu Lys Asp 165 170 175 Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu Leu Asn 180 185 190 Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser Ala Lys 195 200 205 Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala Val Ala 210 215 220 Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu Arg Ala Lys Ala Lys 225 230 235 240 Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln Glu Ala 245 250 255 Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys Leu Met 260 265 270 Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu Lys Trp 275 280 285 Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu Asn Met 290 295 300 Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu Pro Asn 305 310 315 320 Asp Lys Ala Leu Asn 325 <210> SEQ ID NO 3 <211> LENGTH: 810 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(810) <400> SEQUENCE: 3 ctg gac aaa atc gcc aaa aac ggt gtg att gtc gtc ggt cac cgt gaa 48 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 1 5 10 15 tct tca gtg cct ttc tct tat tac gac aat cag caa aaa gtg gtg ggt 96 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 20 25 30 tac tcg cag gat tac tcc aac gcc att gtt gaa gca gtg aaa aag aaa 144 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 35 40 45 ctc aac aaa ccg gac ttg cag gta aaa ctg att ccg att acc tca caa 192 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 50 55 60 aac cgt att cca ctg ctg caa aac ggc act ttc gat ttt gaa tgt ggt 240 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 65 70 75 80 tct acc acc aac aac gtc gaa cgc caa aaa cag gcg gct ttc tct gac 288 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 85 90 95 act att ttc gtg gtc ggt acg cgc ctg ttg acc aaa aag ggt ggc gat 336 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 100 105 110 atc aaa gat ttt gcc aac ctg aaa gac aaa gcc gta gtc gtc act tcc 384 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 115 120 125 ggc act acc tct gaa gtt ttg ctc aac aaa ctg aat gaa gag caa aaa 432 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 130 135 140 atg aat atg cgc atc atc agc gcc aaa gat cac ggt gac tct ttc cgc 480 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 145 150 155 160 acc ctg gaa agc ggt cgt gcc gtt gcc ttt atg atg gat gac gct ctg 528 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 165 170 175 ctg gcc ggt gaa cgt gcg aaa gcg aag aaa cca gac aac tgg gaa atc 576 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 180 185 190 gtc ggc aag ccg cag tct cag gag gcc tac ggt tgt atg ttg cgt aaa 624 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 195 200 205 gat gat ccg cag ttc aaa aag ctg atg gat gac acc atc gct cag gtg 672 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 210 215 220 cag acc tcc ggt gaa gcg gaa aaa tgg ttt gat aag tgg ttc aaa aat 720 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 225 230 235 240 cca att ccg ccg aaa aac ctg aac atg aat ttc gaa ctg tca gac gaa 768 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 245 250 255 atg aaa gca ctg ttc aaa gaa ccg aat gac aag gca ctg aac 810 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 260 265 270

<210> SEQ ID NO 4 <211> LENGTH: 270 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 4 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 1 5 10 15 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 20 25 30 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 35 40 45 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 50 55 60 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 65 70 75 80 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 85 90 95 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 100 105 110 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 115 120 125 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 130 135 140 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 145 150 155 160 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 165 170 175 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 180 185 190 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 195 200 205 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 210 215 220 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 225 230 235 240 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 245 250 255 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 260 265 270 <210> SEQ ID NO 5 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 5 ggtaccggag gcgccgcagg cagcacgctg gacaaaatc 39 <210> SEQ ID NO 6 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 6 accggtaccg gcgccgttca gtgccttgtc attcggttc 39 <210> SEQ ID NO 7 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 7 gagcccggga tggtgagcaa gggcgaggag 30 <210> SEQ ID NO 8 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 8 gaggtcgacc ttgtacagct cgtccatgcc gag 33 <210> SEQ ID NO 9 <211> LENGTH: 5206 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pRSETB FLIP-E 600n vector <400> SEQUENCE: 9 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 60 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat 120 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat 180 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg 240 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 300 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 360 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc 420 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 480 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 540 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 600 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc 660 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag 720 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 780 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 840 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 900 atggacgagc tgtacaaggg tggtaccgga ggcgccgcag gcagcacgct ggacaaaatc 960 gccaaaaacg gtgtgattgt cgtcggtcac cgtgaatctt cagtgccttt ctcttattac 1020 gacaatcagc aaaaagtggt gggttactcg caggattact ccaacgccat tgttgaagca 1080 gtgaaaaaga aactcaacaa accggacttg caggtaaaac tgattccgat tacctcacaa 1140 aaccgtattc cactgctgca aaacggcact ttcgattttg aatgtggttc taccaccaac 1200 aacgtcgaac gccaaaaaca ggcggctttc tctgacacta ttttcgtggt cggtacgcgc 1260 ctgttgacca aaaagggtgg cgatatcaaa gattttgcca acctgaaaga caaagccgta 1320 gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca aactgaatga agagcaaaaa 1380 atgaatatgc gcatcatcag cgccaaagat cacggtgact ctttccgcac cctggaaagc 1440 ggtcgtgccg ttgcctttat gatggatgac gctctgctgg ccggtgaacg tgcgaaagcg 1500 aagaaaccag acaactggga aatcgtcggc aagccgcagt ctcaggaggc ctacggttgt 1560 atgttgcgta aagatgatcc gcagttcaaa aagctgatgg atgacaccat cgctcaggtg 1620 cagacctccg gtgaagcgga aaaatggttt gataagtggt tcaaaaatcc aattccgccg 1680 aaaaacctga acatgaattt cgaactgtca gacgaaatga aagcactgtt caaagaaccg 1740 aatgacaagg cactgaacgg cgccggtacc ggtggaatgg tgagcaaggg cgaggagctg 1800 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1860 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1920 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc 1980 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2040 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2100 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2160 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2220 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2280 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2340 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg 2400 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 2460 gggatcactc tcggcatgga cgagctgtac aagtaaaagc ttgatccggc tgctaacaaa 2520 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 2580 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct 2640 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 2700 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2760 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2820 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2880 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2940 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3000 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatcgcg gtctattctt 3060 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3120 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3180 tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa 3240 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3300 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3360 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3420 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3480 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3540 ttccaatgat gagcactttt aaagttctgc tatgtgatac actattatcc cgtattgacg 3600 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3660 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 3720 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 3780 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 3840 aaccggagct gaatgaagcc ataccaaacg acgagagtga caccacgatg cctgtagcaa 3900 tgccaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3960 aattaataga ctgaatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4020

cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4080 ttgcagcact ggggccagat ggtaagcgct cccgtatcgt agttatctac acgacgggga 4140 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4200 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4260 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4320 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4380 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4440 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4500 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4560 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4620 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4680 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4740 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4800 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 4860 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 4920 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 4980 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg 5040 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5100 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5160 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcag 5206 <210> SEQ ID NO 10 <211> LENGTH: 5206 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pRSETB FLIP-E 10u vector <400> SEQUENCE: 10 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 60 gataattttg tttaacttta agaaggagat atacatatgc ggggttctca tcatcatcat 120 catcatggta tggctagcat gactggtgga cagcaaatgg gtcgggatct gtacgacgat 180 gacgataagg atccgggccg catggtgagc aagggcgagg agctgttcac cggggtggtg 240 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 300 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 360 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc 420 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 480 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 540 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 600 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc 660 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag 720 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 780 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 840 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 900 atggacgagc tgtacaaggg tggtaccgga ggcgccgcag gcagcacgct ggacaaaatc 960 gccaaaaacg gtgtgattgt cgtcggtcac cgtgaatctt cagtgccttt ctcttattac 1020 gacaatcagc aaaaagtggt gggttactcg caggattact ccaacgccat tgttgaagca 1080 gtgaaaaaga aactcaacaa accggacttg caggtaaaac tgattccgat tacctcacaa 1140 aaccgtattc cactgctgca aaacggcact ttcgattttg aatgtggttc taccaccaac 1200 aacgtcgaac gccaaaaaca ggcggctttc tctgacacta ttttcgtggt cggtacgcgc 1260 ctgttgacca aaaagggtgg cgatatcaaa gattttgcca acctgaaaga caaagccgta 1320 gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca aactgaatga agagcaaaaa 1380 atgaatatgc gcatcatcag cgccaaagat cacggtgact ctttccgcac cctggaaagc 1440 ggtcgtgccg ttgcctttat gatggatgac cggctgctgg ccggtgaacg tgcgaaagcg 1500 aagaaaccag acaactggga aatcgtcggc aagccgcagt ctcaggaggc ctacggttgt 1560 atgttgcgta aagatgatcc gcagttcaaa aagctgatgg atgacaccat cgctcaggtg 1620 cagacctccg gtgaagcgga aaaatggttt gataagtggt tcaaaaatcc aattccgccg 1680 aaaaacctga acatgaattt cgaactgtca gacgaaatga aagcactgtt caaagaaccg 1740 aatgacaagg cactgaacgg cgccggtacc ggtggaatgg tgagcaaggg cgaggagctg 1800 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1860 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1920 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc 1980 ctgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2040 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2100 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2160 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2220 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2280 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2340 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg 2400 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 2460 gggatcactc tcggcatgga cgagctgtac aagtaaaagc ttgatccggc tgctaacaaa 2520 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 2580 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatct 2640 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 2700 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2760 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2820 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2880 tccgatttag agctttacgg cacctcgacc gcaaaaaact tgatttgggt gatggttcac 2940 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3000 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatcgcg gtctattctt 3060 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3120 aaatatttaa cgcgaatttt aacaaaatat taacgtttac aatttcgcct gatgcggtat 3180 tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact tttcggggaa 3240 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 3300 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 3360 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 3420 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 3480 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 3540 ttccaatgat gagcactttt aaagttctgc tatgtgatac actattatcc cgtattgacg 3600 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 3660 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 3720 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 3780 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 3840 aaccggagct gaatgaagcc ataccaaacg acgagagtga caccacgatg cctgtagcaa 3900 tgccaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3960 aattaataga ctgaatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 4020 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 4080 ttgcagcact ggggccagat ggtaagcgct cccgtatcgt agttatctac acgacgggga 4140 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 4200 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 4260 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 4320 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 4380 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 4440 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 4500 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 4560 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 4620 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 4680 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 4740 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 4800 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 4860 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 4920 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 4980 acgcggcctt tttacggttc ctgggctttt gctggccttt tgctcacatg ttctttcctg 5040 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 5100 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 5160 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcag 5206 <210> SEQ ID NO 11 <211> LENGTH: 7603 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pDisplay FLIP-E 600n <400> SEQUENCE: 11 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg 720 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 780 tccaggttcc actggtgact atccatatga tgttccagat tatgctgggg cccagccggc 840 cagatctccc gggatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 900 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 960 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1020 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 1080 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1140 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1200 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1260 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 1320 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 1380 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 1440 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 1500 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 1560 gctgtacaag ggtggtaccg gaggcgccgc aggcagcacg ctggacaaaa tcgccaaaaa 1620 cggtgtgatt gtcgtcggtc accgtgaatc ttcagtgcct ttctcttatt acgacaatca 1680 gcaaaaagtg gtgggttact cgcaggatta ctccaacgcc attgttgaag cagtgaaaaa 1740 gaaactcaac aaaccggact tgcaggtaaa actgattccg attacctcac aaaaccgtat 1800 tccactgctg caaaacggca ctttcgattt tgaatgtggt tctaccacca acaacgtcga 1860 acgccaaaaa caggcggctt tctctgacac tattttcgtg gtcggtacgc gcctgttgac 1920 caaaaagggt ggcgatatca aagattttgc caacctgaaa gacaaagccg tagtcgtcac 1980 ttccggcact acctctgaag ttttgctcaa caaactgaat gaagagcaaa aaatgaatat 2040 gcgcatcatc agcgccaaag atcacggtga ctctttccgc accctggaaa gcggtcgtgc 2100 cgttgccttt atgatggatg acgctctgct ggccggtgaa cgtgcgaaag cgaagaaacc 2160 agacaactgg gaaatcgtcg gcaagccgca gtctcaggag gcctacggtt gtatgttgcg 2220 taaagatgat ccgcagttca aaaagctgat ggatgacacc atcgctcagg tgcagacctc 2280 cggtgaagcg gaaaaatggt ttgataagtg gttcaaaaat ccaattccgc cgaaaaacct 2340 gaacatgaat ttcgaactgt cagacgaaat gaaagcactg ttcaaagaac cgaatgacaa 2400 ggcactgaac ggcgccggta ccggtggaat ggtgagcaag ggcgaggagc tgttcaccgg 2460 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 2520 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 2580 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg 2640 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 2700 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 2760 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 2820 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 2880 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 2940 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3000 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga 3060 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3120 tctcggcatg gacgagctgt acaaggtcga cgaacaaaaa ctcatctcag aagaggatct 3180 gaatgctgtg ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa 3240 ggtggtggtg atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat 3300 cctcatcatg ctttggcaga agaagccacg ttaggcggcc gctcgagatc agcctcgact 3360 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3420 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3480 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3540 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3600 accagtggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3660 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3720 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3780 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3840 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3900 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3960 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4020 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4080 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4140 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4200 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4260 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4320 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4380 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4440 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc 4500 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc 4560 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac 4620 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt 4680 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg 4740 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg 4800 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga 4860 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac 4920 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 4980 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 5040 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 5100 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 5160 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 5220 tgctcttgat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 5280 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 5340 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 5400 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 5460 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc 5520 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 5580 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 5640 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 5700 cgaaacgatc ctcatcctgt ctcttgatcg atctttgcaa aagcctaggc ctccaaaaaa 5760 gcctcctcac tacttctgga atagctcaga ggccgaggag gcggcctcgg cctctgcata 5820 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg 5880 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg 5940 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag 6000 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg 6060 acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca ataaatcaat 6120 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6180 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6240 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6300 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6360 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6420 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6480 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6540 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6600 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6660 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6720 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6780 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6840 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6900 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6960 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7020 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7080 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7140 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 7200 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 7260 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 7320 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 7380 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7440 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 7500 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7560 atttaacgcg aattttaaca aaatattaac gcttacaatt tac 7603 <210> SEQ ID NO 12 <211> LENGTH: 7603 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA sequence of pDisplay FLIP-E 10u <400> SEQUENCE: 12 gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180 caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600 taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660 cccaagcttg gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgg 720 cttggggata tccaccatgg agacagacac actcctgcta tgggtactgc tgctctgggt 780 tccaggttcc actggtgact atccatatga tgttccagat tatgctgggg cccagccggc 840 cagatctccc gggatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 900 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 960 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1020 gccctggccc accctcgtga ccaccctgac ctggggcgtg cagtgcttca gccgctaccc 1080 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1140 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1200 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1260 catcctgggg cacaagctgg agtacaacta catcagccac aacgtctata tcaccgccga 1320 caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcag 1380 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 1440 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 1500 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 1560 gctgtacaag ggtggtaccg gaggcgccgc aggcagcacg ctggacaaaa tcgccaaaaa 1620 cggtgtgatt gtcgtcggtc accgtgaatc ttcagtgcct ttctcttatt acgacaatca 1680 gcaaaaagtg gtgggttact cgcaggatta ctccaacgcc attgttgaag cagtgaaaaa 1740 gaaactcaac aaaccggact tgcaggtaaa actgattccg attacctcac aaaaccgtat 1800 tccactgctg caaaacggca ctttcgattt tgaatgtggt tctaccacca acaacgtcga 1860 acgccaaaaa caggcggctt tctctgacac tattttcgtg gtcggtacgc gcctgttgac 1920 caaaaagggt ggcgatatca aagattttgc caacctgaaa gacaaagccg tagtcgtcac 1980 ttccggcact acctctgaag ttttgctcaa caaactgaat gaagagcaaa aaatgaatat 2040 gcgcatcatc agcgccaaag atcacggtga ctctttccgc accctggaaa gcggtcgtgc 2100 cgttgccttt atgatggatg accggctgct ggccggtgaa cgtgcgaaag cgaagaaacc 2160 agacaactgg gaaatcgtcg gcaagccgca gtctcaggag gcctacggtt gtatgttgcg 2220 taaagatgat ccgcagttca aaaagctgat ggatgacacc atcgctcagg tgcagacctc 2280 cggtgaagcg gaaaaatggt ttgataagtg gttcaaaaat ccaattccgc cgaaaaacct 2340 gaacatgaat ttcgaactgt cagacgaaat gaaagcactg ttcaaagaac cgaatgacaa 2400 ggcactgaac ggcgccggta ccggtggaat ggtgagcaag ggcgaggagc tgttcaccgg 2460 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 2520 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 2580 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgcagtg 2640 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 2700 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 2760 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 2820 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 2880 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 2940 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 3000 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga 3060 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 3120 tctcggcatg gacgagctgt acaaggtcga cgaacaaaaa ctcatctcag aagaggatct 3180 gaatgctgtg ggccaggaca cgcaggaggt catcgtggtg ccacactcct tgccctttaa 3240 ggtggtggtg atctcagcca tcctggccct ggtggtgctc accatcatct cccttatcat 3300 cctcatcatg ctttggcaga agaagccacg ttaggcggcc gctcgagatc agcctcgact 3360 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3420 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3480 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3540 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3600 accagtggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3660 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 3720 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 3780 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 3840 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 3900 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 3960 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4020 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4080 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4140 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4200 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4260 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4320 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4380 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4440 ctaaagtata tatgagtaac ctgaggctat ggcagggcct gccgccccga cgttggctgc 4500 gagccctggg ccttcacccg aacttggggg gtggggtggg gaaaaggaag aaacgcgggc 4560 gtattggccc caatggggtc tcggtggggt atcgacagag tgccagccct gggaccgaac 4620 cccgcgttta tgaacaaacg acccaacacc gtgcgtttta ttctgtcttt ttattgccgt 4680 catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc tccccctagg 4740 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg 4800 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga 4860 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac 4920 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc 4980 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac 5040 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag 5100 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc 5160 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga 5220 tgctcttgat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg 5280 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc 5340 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc 5400 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc 5460 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc 5520 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct 5580 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg 5640 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg 5700 cgaaacgatc ctcatcctgt ctcttgatcg atctttgcaa aagcctaggc ctccaaaaaa 5760 gcctcctcac tacttctgga atagctcaga ggccgaggag gcggcctcgg cctctgcata 5820 aataaaaaaa attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg 5880 cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg 5940 catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag 6000 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg 6060 acacacattc cacagctggt tctttccgcc tcaggactct tcctttttca ataaatcaat 6120 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 6180 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 6240 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 6300 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6360 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6420 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 6480 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 6540 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 6600 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 6660 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 6720 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 6780 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 6840 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 6900 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 6960 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 7020 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 7080 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7140 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 7200 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 7260 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 7320 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 7380 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 7440 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 7500 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 7560 atttaacgcg aattttaaca aaatattaac gcttacaatt tac 7603

<210> SEQ ID NO 13 <211> LENGTH: 799 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Amino acid sequence of FLIP-E 600n (expressed as 6xHis fusion in pRSET FLIP-E 600n) <400> SEQUENCE: 13 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Gly Ser Thr Leu Asp Lys Ile 275 280 285 Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu Ser Ser Val Pro 290 295 300 Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp 305 310 315 320 Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro 325 330 335 Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro 340 345 350 Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn 355 360 365 Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val 370 375 380 Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe 385 390 395 400 Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser 405 410 415 Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg 420 425 430 Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser 435 440 445 Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu Leu Ala Gly Glu 450 455 460 Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro 465 470 475 480 Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln 485 490 495 Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly 500 505 510 Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro 515 520 525 Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu 530 535 540 Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn Gly Ala Gly Thr Gly Gly 545 550 555 560 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 565 570 575 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 580 585 590 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 595 600 605 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 610 615 620 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 625 630 635 640 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 645 650 655 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 660 665 670 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 675 680 685 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 690 695 700 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 705 710 715 720 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 725 730 735 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 740 745 750 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 755 760 765 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 770 775 780 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 785 790 795 <210> SEQ ID NO 14 <211> LENGTH: 764 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Amino acid sequence of FLIP-E 10u (expressed as 6xHis fusion in pRSET FLIP-E 10u) <400> SEQUENCE: 14 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly 225 230 235 240 Gly Thr Gly Gly Ala Ala Gly Ser Thr Leu Asp Lys Ile Ala Lys Asn 245 250 255 Gly Val Ile Val Val Gly His Arg Glu Ser Ser Val Pro Phe Ser Tyr 260 265 270 Tyr Asp Asn Gln Gln Lys Val Val Gly Tyr Ser Gln Asp Tyr Ser Asn 275 280 285 Ala Ile Val Glu Ala Val Lys Lys Lys Leu Asn Lys Pro Asp Leu Gln 290 295 300 Val Lys Leu Ile Pro Ile Thr Ser Gln Asn Arg Ile Pro Leu Leu Gln 305 310 315 320 Asn Gly Thr Phe Asp Phe Glu Cys Gly Ser Thr Thr Asn Asn Val Glu 325 330 335 Arg Gln Lys Gln Ala Ala Phe Ser Asp Thr Ile Phe Val Val Gly Thr 340 345 350 Arg Leu Leu Thr Lys Lys Gly Gly Asp Ile Lys Asp Phe Ala Asn Leu 355 360 365 Lys Asp Lys Ala Val Val Val Thr Ser Gly Thr Thr Ser Glu Val Leu 370 375 380 Leu Asn Lys Leu Asn Glu Glu Gln Lys Met Asn Met Arg Ile Ile Ser 385 390 395 400 Ala Lys Asp His Gly Asp Ser Phe Arg Thr Leu Glu Ser Gly Arg Ala 405 410 415

Val Ala Phe Met Met Asp Asp Arg Leu Leu Ala Gly Glu Arg Ala Lys 420 425 430 Ala Lys Lys Pro Asp Asn Trp Glu Ile Val Gly Lys Pro Gln Ser Gln 435 440 445 Glu Ala Tyr Gly Cys Met Leu Arg Lys Asp Asp Pro Gln Phe Lys Lys 450 455 460 Leu Met Asp Asp Thr Ile Ala Gln Val Gln Thr Ser Gly Glu Ala Glu 465 470 475 480 Lys Trp Phe Asp Lys Trp Phe Lys Asn Pro Ile Pro Pro Lys Asn Leu 485 490 495 Asn Met Asn Phe Glu Leu Ser Asp Glu Met Lys Ala Leu Phe Lys Glu 500 505 510 Pro Asn Asp Lys Ala Leu Asn Gly Ala Gly Thr Gly Gly Met Val Ser 515 520 525 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 530 535 540 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 545 550 555 560 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 565 570 575 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr 580 585 590 Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp 595 600 605 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 610 615 620 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 625 630 635 640 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 645 650 655 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn 660 665 670 Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys 675 680 685 Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 690 695 700 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 705 710 715 720 Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp 725 730 735 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 740 745 750 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 755 760 <210> SEQ ID NO 15 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-Y12S-CFP-D13R-YFP Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 15 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc gct gat act cgc att ggt gta aca atc tat aag tcg gct 144 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Ser Ala 35 40 45 ggt atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc 192 Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 50 55 60 ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc 240 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 65 70 75 80 ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc 288 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 85 90 95 atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc 336 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 100 105 110 acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg 384 Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 115 120 125 aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag 432 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 130 135 140 gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc 480 Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 145 150 155 160 gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag 528 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 165 170 175 ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag 576 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 180 185 190 tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag cag aag 624 Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 195 200 205 aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag gac ggc 672 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 210 215 220 agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac 720 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 225 230 235 240 ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gcc 768 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 245 250 255 ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag 816 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 260 265 270 ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac ggt 864 Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly 275 280 285 agc cga gat aac gcg atg tct gta gtg cgc aag gct att gag caa gat 912 Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp 290 295 300 gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat tct cag aat 960 Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn 305 310 315 320 gac cag tcc aag cag aac gat cag atc gac gta ttg ctg gcg aaa ggg 1008 Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly 325 330 335 gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct gcg ggt acg 1056 Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr 340 345 350 gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt ttc ttc aac 1104 Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn 355 360 365 aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa gcc tac tac 1152 Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr 370 375 380 gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc gat ttg att 1200 Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile 385 390 395 400 gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac aaa gac ggt 1248 Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly 405 410 415 cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat ccg gat gca 1296 Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala 420 425 430 gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat aaa ggc atc 1344 Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile 435 440 445 aaa act gaa cag tta cag tta gat acc gca atg tgg gac acc gct cag 1392 Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln 450 455 460 gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac gcc aac aaa 1440 Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys 465 470 475 480 atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg ggc gcg gtt 1488 Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val 485 490 495 gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg ttt ggc gtc 1536 Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val 500 505 510 gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt gca ctg gcg 1584 Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala 515 520 525 ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg acc ttt gat 1632 Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp 530 535 540 ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg gct gat ggc acc aac 1680 Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 tgg aaa atc gac aac aaa gtg gtc cgc gta cct tat gtt ggc gta gat 1728 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 aaa gac aac ctg gct gaa ttc agc aag aaa ggc gcc ggt acc ggt gga 1776 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 1824 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 1872 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 1920 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 1968 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 ttc ggc tac ggc ctg cag tgc ttc gcc cgc tac ccc gac cac atg aag 2016 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 2064 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685

cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 2112 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 2160 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 2208 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac 2256 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 2304 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 2352 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 ccc gtg ctg ctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 2400 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 2448 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 2496 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 16 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-Y12S-CFP-D13R-YFP Vector <400> SEQUENCE: 16 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Ser Ala 35 40 45 Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 50 55 60 Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser 65 70 75 80 Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 85 90 95 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 100 105 110 Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 115 120 125 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 130 135 140 Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 145 150 155 160 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 165 170 175 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu 180 185 190 Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys 195 200 205 Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly 210 215 220 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 225 230 235 240 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 245 250 255 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu 260 265 270 Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly 275 280 285 Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp 290 295 300 Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn 305 310 315 320 Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly 325 330 335 Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr 340 345 350 Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn 355 360 365 Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr 370 375 380 Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile 385 390 395 400 Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly 405 410 415 Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala 420 425 430 Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile 435 440 445 Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln 450 455 460 Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys 465 470 475 480 Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val 485 490 495 Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val 500 505 510 Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala 515 520 525 Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp 530 535 540 Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 17 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-G275S-CFP-K276R-YFP Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 17 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc gct gat act cgc att ggt gta aca atc tat aag tac gac 144 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Tyr Asp 35 40 45 gat aac gcg atg tct gta gtg cgc aag gct att gag caa gat gcg aaa 192 Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp Ala Lys 50 55 60 gcc gcg cca gat gtt cag ctg ctg atg aat gat tct cag aat gac cag 240 Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn Asp Gln 65 70 75 80 tcc aag cag aac gat cag atc gac gta ttg ctg gcg aaa ggg gtg aag 288 Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly Val Lys 85 90 95 gca ctg gca atc aac ctg gtt gac ccg gca gct gcg ggt acg gtg att 336 Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr Val Ile 100 105 110 gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt ttc ttc aac aaa gaa 384 Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn Lys Glu 115 120 125 ccg tct cgt aag gcg ctg gat agc tac gac aaa gcc tac tac gtt ggc 432 Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr Val Gly 130 135 140

act gac tcc aaa gag tcc ggc att att caa ggc gat ttg att gct aaa 480 Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile Ala Lys 145 150 155 160 cac tgg gcg gcg aat cag ggt tgg gat ctg aac aaa gac ggt cag att 528 His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly Gln Ile 165 170 175 cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat ccg gat gca gaa gca 576 Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala Glu Ala 180 185 190 cgt acc act tac gtg att aaa gaa ttg aac gat aaa ggc atc aaa act 624 Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile Lys Thr 195 200 205 gaa cag tta cag tta gat acc gca atg tgg gac acc gct cag gcg aaa 672 Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln Ala Lys 210 215 220 gat aag atg gac gcc tgg ctg tct ggc ccg aac gcc aac aaa atc gaa 720 Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys Ile Glu 225 230 235 240 gtg gtt atc gcc aac aac gat gcg atg gca atg ggc gcg gtt gaa gcg 768 Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val Glu Ala 245 250 255 ctg aaa gca cac aac aag tcc agc att ccg gtg ttt ggc gtc gat gcg 816 Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val Asp Ala 260 265 270 ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt gca ctg gcg ggc acc 864 Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala Gly Thr 275 280 285 gta ctg aac gat gct aac aac cag gcg aaa gcg acc ttt gat ctg gcg 912 Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp Leu Ala 290 295 300 aaa aac ctg gcc gat tcg gct ggt atg gtg agc aag ggc gag gag ctg 960 Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu 305 310 315 320 ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac 1008 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 325 330 335 ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac 1056 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 340 345 350 ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg 1104 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 355 360 365 ccc tgg ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc 1152 Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe 370 375 380 agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc 1200 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 385 390 395 400 atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac 1248 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 405 410 415 ggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg 1296 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 420 425 430 gtg aac cgc atc gag ctg aag ggc atc gac ttc aag gag gac ggc aac 1344 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 435 440 445 atc ctg ggg cac aag ctg gag tac aac tac atc agc cac aac gtc tat 1392 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr 450 455 460 atc acc gcc gac aag cag aag aac ggc atc aag gcc aac ttc aag atc 1440 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 465 470 475 480 cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag 1488 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 485 490 495 cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac 1536 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 500 505 510 tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc 1584 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 515 520 525 gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc 1632 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 530 535 540 ggc atg gac gag ctg tac ggt agc cga ggt gcg gct gat ggc acc aac 1680 Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 tgg aaa atc gac aac aaa gtg gtc cgc gta cct tat gtt ggc gta gat 1728 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 aaa gac aac ctg gct gaa ttc agc aag aaa ggc gcc ggt acc ggt gga 1776 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 1824 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 1872 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 1920 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 1968 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 ttc ggc tac ggc ctg cag tgc ttc gcc cgc tac ccc gac cac atg aag 2016 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 2064 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 2112 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 2160 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 2208 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac 2256 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 2304 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 2352 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 ccc gtg ctg ctg ccc gac aac cac tac ctg agc tac cag tcc gcc ctg 2400 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 2448 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag taa 2496 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 18 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-mglBF16A-G275S-CFP-K276R-YFP Vector <400> SEQUENCE: 18 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Ala Asp Thr Arg Ile Gly Val Thr Ile Tyr Lys Tyr Asp 35 40 45 Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile Glu Gln Asp Ala Lys 50 55 60 Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp Ser Gln Asn Asp Gln 65 70 75 80 Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu Ala Lys Gly Val Lys 85 90 95 Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala Ala Gly Thr Val Ile 100 105 110 Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val Phe Phe Asn Lys Glu 115 120 125 Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys Ala Tyr Tyr Val Gly 130 135 140 Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly Asp Leu Ile Ala Lys 145 150 155 160 His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn Lys Asp Gly Gln Ile 165 170 175 Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His Pro Asp Ala Glu Ala 180 185 190 Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp Lys Gly Ile Lys Thr 195 200 205 Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp Thr Ala Gln Ala Lys 210 215 220 Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn Ala Asn Lys Ile Glu 225 230 235 240 Val Val Ile Ala Asn Asn Asp Ala Met Ala Met Gly Ala Val Glu Ala 245 250 255 Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val Phe Gly Val Asp Ala 260 265 270 Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly Ala Leu Ala Gly Thr 275 280 285 Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala Thr Phe Asp Leu Ala 290 295 300 Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu 305 310 315 320 Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn 325 330 335 Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr 340 345 350 Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val 355 360 365

Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe 370 375 380 Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala 385 390 395 400 Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp 405 410 415 Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu 420 425 430 Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn 435 440 445 Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr 450 455 460 Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile 465 470 475 480 Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln 485 490 495 Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His 500 505 510 Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg 515 520 525 Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu 530 535 540 Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala Ala Asp Gly Thr Asn 545 550 555 560 Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro Tyr Val Gly Val Asp 565 570 575 Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys Gly Ala Gly Thr Gly Gly 580 585 590 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 595 600 605 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 610 615 620 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 625 630 635 640 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 645 650 655 Phe Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 660 665 670 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 675 680 685 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 690 695 700 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 705 710 715 720 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 725 730 735 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 740 745 750 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 755 760 765 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 770 775 780 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu 785 790 795 800 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 805 810 815 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 820 825 830 <210> SEQ ID NO 19 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-Y12S-CFP-D13R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 19 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tcg gct ggt atg gtg agc aag ggc gag gag ctg ttc acc 912 Ile Tyr Lys Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr 290 295 300 ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac 960 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His 305 310 315 320 aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag 1008 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys 325 330 335 ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg 1056 Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp 340 345 350 ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc 1104 Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg 355 360 365 tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc 1152 Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro 370 375 380 gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac 1200 Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 385 390 395 400 tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac 1248 Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 405 410 415 cgc atc gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg 1296 Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu 420 425 430 ggg cac aag ctg gag tac aac tac atc agc cac aac gtc tat atc acc 1344 Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr 435 440 445 gcc gac aag cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac 1392 Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His 450 455 460 aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac 1440 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 465 470 475 480 acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg 1488 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 485 490 495 agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac 1536 Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 500 505 510 atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg 1584 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 515 520 525 gac gag ctg tac ggt agc cga gat aac gcg atg tct gta gtg cgc aag 1632 Asp Glu Leu Tyr Gly Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys 530 535 540 gct att gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg 1680 Ala Ile Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met 545 550 555 560 aat gat tct cag aat gac cag tcc aag cag aac gat cag atc gac gta 1728 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 ttg ctg gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg 1776 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 gca gct gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg 1824 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605

gtg gtt ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac 1872 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 gac aaa gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att 1920 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 caa ggc gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat 1968 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 ctg aac aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg 2016 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 ggc cat ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg 2064 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 aac gat aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg 2112 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 tgg gac acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc 2160 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 ccg aac gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg 2208 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 gca atg ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att 2256 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 ccg gtg ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa 2304 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 tcc ggt gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg 2352 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 aaa gcg acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg 2400 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 20 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-Y12S-CFP-D13R Vector <400> SEQUENCE: 20 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr 290 295 300 Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His 305 310 315 320 Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys 325 330 335 Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp 340 345 350 Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg 355 360 365 Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro 370 375 380 Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn 385 390 395 400 Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn 405 410 415 Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu 420 425 430 Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr 435 440 445 Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His 450 455 460 Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn 465 470 475 480 Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu 485 490 495 Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His 500 505 510 Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met 515 520 525 Asp Glu Leu Tyr Gly Ser Arg Asp Asn Ala Met Ser Val Val Arg Lys 530 535 540 Ala Ile Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met 545 550 555 560 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 21 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-P32S-CFP-D33R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 21 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60

gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg tcg gct ggt atg gtg agc aag ggc gag 960 Glu Gln Asp Ala Lys Ala Ala Ser Ala Gly Met Val Ser Lys Gly Glu 305 310 315 320 gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac ggc gac 1008 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 325 330 335 gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gat gcc 1056 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 340 345 350 acc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg 1104 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 355 360 365 ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tgg ggc gtg cag 1152 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln 370 375 380 tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag 1200 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 385 390 395 400 tcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag 1248 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 405 410 415 gac gac ggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac 1296 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 420 425 430 acc ctg gtg aac cgc atc gag ctg aag ggc atc gac ttc aag gag gac 1344 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 435 440 445 ggc aac atc ctg ggg cac aag ctg gag tac aac tac atc agc cac aac 1392 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn 450 455 460 gtc tat atc acc gcc gac aag cag aag aac ggc atc aag gcc aac ttc 1440 Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe 465 470 475 480 aag atc cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac 1488 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 485 490 495 tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac 1536 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 500 505 510 aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag 1584 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 515 520 525 aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc 1632 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 530 535 540 act ctc ggc atg gac gag ctg tac ggt agc cga gtt cag ctg ctg atg 1680 Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Val Gln Leu Leu Met 545 550 555 560 aat gat tct cag aat gac cag tcc aag cag aac gat cag atc gac gta 1728 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 ttg ctg gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg 1776 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 gca gct gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg 1824 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 gtg gtt ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac 1872 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 gac aaa gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att 1920 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 caa ggc gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat 1968 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 ctg aac aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg 2016 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 ggc cat ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg 2064 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 aac gat aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg 2112 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 tgg gac acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc 2160 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 ccg aac gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg 2208 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 gca atg ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att 2256 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 ccg gtg ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa 2304 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 tcc ggt gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg 2352 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 aaa gcg acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg 2400 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 22 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-P32S-CFP-D33R Vector <400> SEQUENCE: 22 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255

Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Ser Ala Gly Met Val Ser Lys Gly Glu 305 310 315 320 Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp 325 330 335 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala 340 345 350 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 355 360 365 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln 370 375 380 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys 385 390 395 400 Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys 405 410 415 Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp 420 425 430 Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 435 440 445 Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn 450 455 460 Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe 465 470 475 480 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 485 490 495 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp 500 505 510 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 515 520 525 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 530 535 540 Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Val Gln Leu Leu Met 545 550 555 560 Asn Asp Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val 565 570 575 Leu Leu Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro 580 585 590 Ala Ala Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro 595 600 605 Val Val Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr 610 615 620 Asp Lys Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile 625 630 635 640 Gln Gly Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp 645 650 655 Leu Asn Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro 660 665 670 Gly His Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu 675 680 685 Asn Asp Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met 690 695 700 Trp Asp Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly 705 710 715 720 Pro Asn Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met 725 730 735 Ala Met Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile 740 745 750 Pro Val Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys 755 760 765 Ser Gly Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala 770 775 780 Lys Ala Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 23 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-G275S-CFP-K276R Vector <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 23 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat 960 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 tct cag aat gac cag tcc aag cag aac gat cag atc gac gta ttg ctg 1008 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct 1056 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt 1104 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa 1152 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc 1200 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac 1248 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat 1296 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat 1344 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg tgg gac 1392 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac 1440 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg 1488 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg 1536 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt 1584 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly

515 520 525 gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg 1632 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 acc ttt gat ctg gcg aaa aac ctg gcc gat tcg gct ggt atg gtg agc 1680 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser 545 550 555 560 aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg 1728 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 565 570 575 gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag 1776 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 580 585 590 ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc 1824 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 595 600 605 ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tgg 1872 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp 610 615 620 ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac 1920 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp 625 630 635 640 ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atc 1968 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 645 650 655 ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag gtg aag ttc 2016 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 660 665 670 gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc atc gac ttc 2064 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 675 680 685 aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac aac tac atc 2112 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile 690 695 700 agc cac aac gtc tat atc acc gcc gac aag cag aag aac ggc atc aag 2160 Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys 705 710 715 720 gcc aac ttc aag atc cgc cac aac atc gag gac ggc agc gtg cag ctc 2208 Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 725 730 735 gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg 2256 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 740 745 750 ctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac 2304 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp 755 760 765 ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc 2352 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 770 775 780 gcc ggg atc act ctc ggc atg gac gag ctg tac ggt agc cga ggt gcg 2400 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala 785 790 795 800 gct gat ggc acc aac tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 24 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-G275S-CFP-K276R Vector <400> SEQUENCE: 24 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Ser Ala Gly Met Val Ser 545 550 555 560 Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu 565 570 575 Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu 580 585 590 Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr 595 600 605 Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp 610 615 620 Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp 625 630 635 640 Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile 645 650 655 Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe 660 665 670 Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe 675 680 685 Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile 690 695 700 Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys 705 710 715 720 Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu 725 730 735 Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu 740 745 750 Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp 755 760 765 Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala 770 775 780 Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Gly Ser Arg Gly Ala 785 790 795 800 Ala Asp Gly Thr Asn Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 25 <211> LENGTH: 2496 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-T282S-CFP-N283R Vector

<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2496) <400> SEQUENCE: 25 atg cgg ggt tct cat cat cat cat cat cat ggt atg gct agc atg act 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 ggt gga cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat 96 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 ccg ggc cgc atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg 144 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc 192 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg 240 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc 288 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc gac 336 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac 384 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc 432 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140 cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag 480 Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag 528 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac aag 576 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc gag 624 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc 672 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag 720 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg 768 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg 816 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 tac aag ggt ggt acc gga ggc gcc gct gat act cgc att ggt gta aca 864 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 atc tat aag tac gac gat aac gcg atg tct gta gtg cgc aag gct att 912 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 gag caa gat gcg aaa gcc gcg cca gat gtt cag ctg ctg atg aat gat 960 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 tct cag aat gac cag tcc aag cag aac gat cag atc gac gta ttg ctg 1008 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 gcg aaa ggg gtg aag gca ctg gca atc aac ctg gtt gac ccg gca gct 1056 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 gcg ggt acg gtg att gag aaa gcg cgt ggg caa aac gtg ccg gtg gtt 1104 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 ttc ttc aac aaa gaa ccg tct cgt aag gcg ctg gat agc tac gac aaa 1152 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 gcc tac tac gtt ggc act gac tcc aaa gag tcc ggc att att caa ggc 1200 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 gat ttg att gct aaa cac tgg gcg gcg aat cag ggt tgg gat ctg aac 1248 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 aaa gac ggt cag att cag ttc gta ctg ctg aaa ggt gaa ccg ggc cat 1296 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 ccg gat gca gaa gca cgt acc act tac gtg att aaa gaa ttg aac gat 1344 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 aaa ggc atc aaa act gaa cag tta cag tta gat acc gca atg tgg gac 1392 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 acc gct cag gcg aaa gat aag atg gac gcc tgg ctg tct ggc ccg aac 1440 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 gcc aac aaa atc gaa gtg gtt atc gcc aac aac gat gcg atg gca atg 1488 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 ggc gcg gtt gaa gcg ctg aaa gca cac aac aag tcc agc att ccg gtg 1536 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 ttt ggc gtc gat gcg ctg cca gaa gcg ctg gcg ctg gtg aaa tcc ggt 1584 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 gca ctg gcg ggc acc gta ctg aac gat gct aac aac cag gcg aaa gcg 1632 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 acc ttt gat ctg gcg aaa aac ctg gcc gat ggt aaa ggt gcg gct gat 1680 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp 545 550 555 560 ggc tcg gct ggt atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg 1728 Gly Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 565 570 575 gtg ccc atc ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc 1776 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 580 585 590 agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc 1824 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 595 600 605 ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc 1872 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 610 615 620 ctc gtg acc acc ctg acc tgg ggc gtg cag tgc ttc agc cgc tac ccc 1920 Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro 625 630 635 640 gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc 1968 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 645 650 655 tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag 2016 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 660 665 670 acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc 2064 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 675 680 685 gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac 2112 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 690 695 700 aag ctg gag tac aac tac atc agc cac aac gtc tat atc acc gcc gac 2160 Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp 705 710 715 720 aag cag aag aac ggc atc aag gcc aac ttc aag atc cgc cac aac atc 2208 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile 725 730 735 gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc 2256 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 740 745 750 atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc 2304 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 755 760 765 cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc 2352 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 770 775 780 ctg ctg gag ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag 2400 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 785 790 795 800 ctg tac ggt agc cga tgg aaa atc gac aac aaa gtg gtc cgc gta cct 2448 Leu Tyr Gly Ser Arg Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 tat gtt ggc gta gat aaa gac aac ctg gct gaa ttc agc aag aaa taa 2496 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 26 <211> LENGTH: 831 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: FLIP-YFP-mglBF16A-T282S-CFP-N283R Vector <400> SEQUENCE: 26 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Gly Arg Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val 35 40 45 Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser 50 55 60 Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu 65 70 75 80 Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu 85 90 95 Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp 100 105 110 His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr 115 120 125 Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr 130 135 140

Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu 145 150 155 160 Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys 165 170 175 Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys 180 185 190 Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu 195 200 205 Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile 210 215 220 Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln 225 230 235 240 Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu 245 250 255 Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu 260 265 270 Tyr Lys Gly Gly Thr Gly Gly Ala Ala Asp Thr Arg Ile Gly Val Thr 275 280 285 Ile Tyr Lys Tyr Asp Asp Asn Ala Met Ser Val Val Arg Lys Ala Ile 290 295 300 Glu Gln Asp Ala Lys Ala Ala Pro Asp Val Gln Leu Leu Met Asn Asp 305 310 315 320 Ser Gln Asn Asp Gln Ser Lys Gln Asn Asp Gln Ile Asp Val Leu Leu 325 330 335 Ala Lys Gly Val Lys Ala Leu Ala Ile Asn Leu Val Asp Pro Ala Ala 340 345 350 Ala Gly Thr Val Ile Glu Lys Ala Arg Gly Gln Asn Val Pro Val Val 355 360 365 Phe Phe Asn Lys Glu Pro Ser Arg Lys Ala Leu Asp Ser Tyr Asp Lys 370 375 380 Ala Tyr Tyr Val Gly Thr Asp Ser Lys Glu Ser Gly Ile Ile Gln Gly 385 390 395 400 Asp Leu Ile Ala Lys His Trp Ala Ala Asn Gln Gly Trp Asp Leu Asn 405 410 415 Lys Asp Gly Gln Ile Gln Phe Val Leu Leu Lys Gly Glu Pro Gly His 420 425 430 Pro Asp Ala Glu Ala Arg Thr Thr Tyr Val Ile Lys Glu Leu Asn Asp 435 440 445 Lys Gly Ile Lys Thr Glu Gln Leu Gln Leu Asp Thr Ala Met Trp Asp 450 455 460 Thr Ala Gln Ala Lys Asp Lys Met Asp Ala Trp Leu Ser Gly Pro Asn 465 470 475 480 Ala Asn Lys Ile Glu Val Val Ile Ala Asn Asn Asp Ala Met Ala Met 485 490 495 Gly Ala Val Glu Ala Leu Lys Ala His Asn Lys Ser Ser Ile Pro Val 500 505 510 Phe Gly Val Asp Ala Leu Pro Glu Ala Leu Ala Leu Val Lys Ser Gly 515 520 525 Ala Leu Ala Gly Thr Val Leu Asn Asp Ala Asn Asn Gln Ala Lys Ala 530 535 540 Thr Phe Asp Leu Ala Lys Asn Leu Ala Asp Gly Lys Gly Ala Ala Asp 545 550 555 560 Gly Ser Ala Gly Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 565 570 575 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 580 585 590 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 595 600 605 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 610 615 620 Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro 625 630 635 640 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 645 650 655 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 660 665 670 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 675 680 685 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 690 695 700 Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp 705 710 715 720 Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile 725 730 735 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 740 745 750 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 755 760 765 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 770 775 780 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 785 790 795 800 Leu Tyr Gly Ser Arg Trp Lys Ile Asp Asn Lys Val Val Arg Val Pro 805 810 815 Tyr Val Gly Val Asp Lys Asp Asn Leu Ala Glu Phe Ser Lys Lys 820 825 830 <210> SEQ ID NO 27 <211> LENGTH: 909 <212> TYPE: DNA <213> ORGANISM: Escherichia coli K12 <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(909) <400> SEQUENCE: 27 atg caa tta cgt aaa cct gcc aca gca atc ctc gcc ctg gcg ctt tcc 48 Met Gln Leu Arg Lys Pro Ala Thr Ala Ile Leu Ala Leu Ala Leu Ser 1 5 10 15 gca gga ctg gca cag gca gat gac gcc gcc ccg gca gcg ggc agt act 96 Ala Gly Leu Ala Gln Ala Asp Asp Ala Ala Pro Ala Ala Gly Ser Thr 20 25 30 ctg gac aaa atc gcc aaa aac ggt gtg att gtc gtc ggt cac cgt gaa 144 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 35 40 45 tct tca gtg cct ttc tct tat tac gac aat cag caa aaa gtg gtg ggt 192 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 50 55 60 tac tcg cag gat tac tcc aac gcc att gtt gaa gca gtg aaa aag aaa 240 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 65 70 75 80 ctc aac aaa ccg gac ttg cag gta aaa ctg att ccg att acc tca caa 288 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 85 90 95 aac cgt att cca ctg ctg caa aac ggc act ttc gat ttt gaa tgt ggt 336 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 100 105 110 tct acc acc aac aac gtc gaa cgc caa aaa cag gcg gct ttc tct gac 384 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 115 120 125 act att ttc gtg gtc ggt acg cgc ctg ttg acc aaa aag ggt ggc gat 432 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 130 135 140 atc aaa gat ttt gcc aac ctg aaa gac aaa gcc gta gtc gtc act tcc 480 Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 145 150 155 160 ggc act acc tct gaa gtt ttg ctc aac aaa ctg aat gaa gag caa aaa 528 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 165 170 175 atg aat atg cgc atc atc agc gcc aaa gat cac ggt gac tct ttc cgc 576 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 180 185 190 acc ctg gaa agc ggt cgt gcc gtt gcc ttt atg atg gat gac gct ctg 624 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 195 200 205 ctg gcc ggt gaa cgt gcg aaa gcg aag aaa cca gac aac tgg gaa atc 672 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 210 215 220 gtc ggc aag ccg cag tct cag gag gcc tac ggt tgt atg ttg cgt aaa 720 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 225 230 235 240 gat gat ccg cag ttc aaa aag ctg atg gat gac acc atc gct cag gtg 768 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 245 250 255 cag acc tcc ggt gaa gcg gaa aaa tgg ttt gat aag tgg ttc aaa aat 816 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 260 265 270 cca att ccg ccg aaa aac ctg aac atg aat ttc gaa ctg tca gac gaa 864 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 275 280 285 atg aaa gca ctg ttc aaa gaa ccg aat gac aag gca ctg aac taa 909 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 290 295 300 <210> SEQ ID NO 28 <211> LENGTH: 302 <212> TYPE: PRT <213> ORGANISM: Escherichia coli K12 <400> SEQUENCE: 28 Met Gln Leu Arg Lys Pro Ala Thr Ala Ile Leu Ala Leu Ala Leu Ser 1 5 10 15 Ala Gly Leu Ala Gln Ala Asp Asp Ala Ala Pro Ala Ala Gly Ser Thr 20 25 30 Leu Asp Lys Ile Ala Lys Asn Gly Val Ile Val Val Gly His Arg Glu 35 40 45 Ser Ser Val Pro Phe Ser Tyr Tyr Asp Asn Gln Gln Lys Val Val Gly 50 55 60 Tyr Ser Gln Asp Tyr Ser Asn Ala Ile Val Glu Ala Val Lys Lys Lys 65 70 75 80 Leu Asn Lys Pro Asp Leu Gln Val Lys Leu Ile Pro Ile Thr Ser Gln 85 90 95 Asn Arg Ile Pro Leu Leu Gln Asn Gly Thr Phe Asp Phe Glu Cys Gly 100 105 110 Ser Thr Thr Asn Asn Val Glu Arg Gln Lys Gln Ala Ala Phe Ser Asp 115 120 125 Thr Ile Phe Val Val Gly Thr Arg Leu Leu Thr Lys Lys Gly Gly Asp 130 135 140

Ile Lys Asp Phe Ala Asn Leu Lys Asp Lys Ala Val Val Val Thr Ser 145 150 155 160 Gly Thr Thr Ser Glu Val Leu Leu Asn Lys Leu Asn Glu Glu Gln Lys 165 170 175 Met Asn Met Arg Ile Ile Ser Ala Lys Asp His Gly Asp Ser Phe Arg 180 185 190 Thr Leu Glu Ser Gly Arg Ala Val Ala Phe Met Met Asp Asp Ala Leu 195 200 205 Leu Ala Gly Glu Arg Ala Lys Ala Lys Lys Pro Asp Asn Trp Glu Ile 210 215 220 Val Gly Lys Pro Gln Ser Gln Glu Ala Tyr Gly Cys Met Leu Arg Lys 225 230 235 240 Asp Asp Pro Gln Phe Lys Lys Leu Met Asp Asp Thr Ile Ala Gln Val 245 250 255 Gln Thr Ser Gly Glu Ala Glu Lys Trp Phe Asp Lys Trp Phe Lys Asn 260 265 270 Pro Ile Pro Pro Lys Asn Leu Asn Met Asn Phe Glu Leu Ser Asp Glu 275 280 285 Met Lys Ala Leu Phe Lys Glu Pro Asn Asp Lys Ala Leu Asn 290 295 300 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 30 Gly Asn Asn Ser Ala Gly 1 5 <210> SEQ ID NO 31 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 31 Gly Ser Ala Asp Asp Gly 1 5 <210> SEQ ID NO 32 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 32 Gly Gly Thr Gly Gly Ala 1 5 <210> SEQ ID NO 33 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: linker sequence <400> SEQUENCE: 33 ggtggtaccg gaggcgcc 18 <210> SEQ ID NO 34 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 34 agcgctggta tggtg 15 <210> SEQ ID NO 35 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 35 actctcggta gcgct 15 <210> SEQ ID NO 36 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 36 Ala Gly Met Val 1 <210> SEQ ID NO 37 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB N58V-ECFP232-Q59N <400> SEQUENCE: 37 Thr Leu Gly Ser 1 <210> SEQ ID NO 38 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 38 aaagcgggca acaacagcgc tggtgacggc 30 <210> SEQ ID NO 39 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 39 atcgagggta gcgctgacga cggaaagaaa 30 <210> SEQ ID NO 40 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 40 Lys Ala Gly Asn Asn Ser Ala Gly Asn Gly 1 5 10 <210> SEQ ID NO 41 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from pRSETB A216-cpVenus-K217 <400> SEQUENCE: 41 Ile Glu Gly Ser Ala Asp Asp Gly Lys Lys 1 5 10 <210> SEQ ID NO 42 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII275 Pglu plasmid <400> SEQUENCE: 42 actctcggca tggacgagct gtacaagggt ggtaccggag gcgcc 45 <210> SEQ ID NO 43 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 43 actctcggca tggacgagct gtacggtagc cga 33 <210> SEQ ID NO 44 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 44 gaattcagca agaaaggcgc cggtaccggt ggaatggtga gcaagggcga g 51 <210> SEQ ID NO 45 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 45 gaattcagca agaaaggtac catggtgagc aagggcgag 39 <210> SEQ ID NO 46 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid

<400> SEQUENCE: 46 gaattcagca agaaaatggt gagcaagggc gag 33 <210> SEQ ID NO 47 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 47 ttcagcaaga aaggcgccgg taccggtgga atggtgagca agggcgag 48 <210> SEQ ID NO 48 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 48 agcaagaaag gcgccggtac cggtggaatg gtgagcaagg gcgag 45 <210> SEQ ID NO 49 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 49 aagaaaggcg ccggtaccgg tggaatggtg agcaagggcg ag 42 <210> SEQ ID NO 50 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 50 aaaggcgccg gtaccggtgg aatggtgagc aagggcgag 39 <210> SEQ ID NO 51 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 51 ggcgccggta ccggtggaat ggtgagcaag ggcgag 36 <210> SEQ ID NO 52 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 52 gccggtaccg gtggaatggt gagcaagggc gag 33 <210> SEQ ID NO 53 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 53 ggtaccggtg gaatggtgag caagggcgag 30 <210> SEQ ID NO 54 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 54 accggtggaa tggtgagcaa gggcgag 27 <210> SEQ ID NO 55 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 55 ggtggaatgg tgagcaaggg cgag 24 <210> SEQ ID NO 56 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 56 ggaatggtga gcaagggcga g 21 <210> SEQ ID NO 57 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 57 atggtgagca agggcgag 18 <210> SEQ ID NO 58 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 58 gtgagcaagg gcgag 15 <210> SEQ ID NO 59 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: partial sequence from FLII2 Pglu plasmid <400> SEQUENCE: 59 agcaagggcg ag 12

Claims

1.-98. (canceled)

99. An isolated nucleic acid comprising a polynucleotide sequence encoding a periplasmic binding protein (PBP) that specifically binds a ligand of interest, and at least one of a donor fluorophore protein fused to the PBP or an acceptor fluorophore protein fused to the PBP, wherein the coding region of the donor fluorophore protein or the coding region of the acceptor fluorophore protein is inserted at an internal site of the coding region of the PBP.

100. The isolated nucleic acid of claim 99, wherein the coding region of the donor fluorophore protein is fused to an internal site of the PBP.

101. The isolated nucleic acid of claim 99, wherein the coding region of the acceptor fluorophore protein is fused to an internal site of the PBP.

102. The isolated nucleic acid of claim 99, wherein the coding region of the donor fluorescent protein and the coding region of the acceptor fluorescent protein are fused to the same lobe of the PBP.

103. The isolated nucleic acid of claim 99, wherein the PBP specifically binds an amino acid.

104. The isolated nucleic acid of claim 99, wherein the PBP specifically binds a sugar.

105. The isolated nucleic acid of claim 99, wherein the coding region of the acceptor fluorophore protein is inserted at the C-terminus of the PBP.

106. The isolated nucleic acid of claim 99, wherein the donor fluorophore protein is selected from the group consisting of a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy) and monomeric CoralHue Kusabira-Orange (mKO).

107. The isolated nucleic acid of claim 106, wherein the donor fluorophore protein is eCFP.

108. The isolated nucleic acid of claim 106, wherein the donor fluorophore protein is YFP VENUS.

109. The isolated nucleic acid of claim 99, wherein the acceptor fluorophore protein is selected from the group consisting of a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy) and monomeric CoralHue Kusabira-Orange (mKO).

110. The isolated nucleic acid of claim 100, further comprising at least one peptide linker that links the internal site of the PBP to the at least one donor fluorophore protein.

111. The isolated nucleic acid of claim 101, further comprising at least one peptide linker that links the internal site of the PBP to the at least one acceptor fluorophore protein.

112. The isolated nucleic acid of claim 99, wherein the PBP comprises a glucose-galactose binding protein (GBP) or a glutamate-aspartate receptor (YbeJ).

113. The isolated nucleic acid of claim 99, wherein the polynucleotide sequence encoding the periplasmic binding protein (PBP) comprises one or more substitution mutations that modify the affinity of the PBP to its ligand.

114. An expression vector comprising the nucleic acid of claim 99.

115. A host cell comprising the vector of claim 114.

116. A ligand binding fluorescent indicator encoded by the nucleic acid of claim 99.

117. The isolated nucleic acid of claim 100, wherein the nucleic acid further comprises a coding region of an acceptor fluorophore protein that is fused to the coding region of the PBP.

118. The isolated nucleic acid of claim 101, wherein the nucleic acid further encodes a coding region of a donor fluorophore protein that is fused to the coding region of the PBP.

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