SYSTEMS AND METHODS FOR BATCH CULTIVATION OF NON-TRANSGENIC HETEROGAMETES

Abstract

A system for batch production of the heterogametic sex of a biological species generally includes a first strain of a biological species genetically engineered to include a conditional Y-linked (or Z-linked) genetic lethal circuit and a second strain of the biological species genetically engineered to include a conditional X-linked (or W-linked) genetic lethal circuit.

Claims

1. A system comprising: a first strain of a biological species genetically engineered to comprise a conditional P-linked genetic lethal circuit; and a second strain of the biological species genetically engineered to comprise a conditional X-linked genetic lethal circuit.

2. The system of claim 1, wherein the X-linked genetic lethal circuit is the same as the Y-linked genetic lethal circuit.

3. The system of claim 1, wherein the X-linked genetic lethal circuit is different than the Y-linked genetic lethal circuit.

4. The system of claim 1, wherein the biological species is a pest species.

5. The system of claim 1, wherein the biological species is a species in which one sex has greater commercial value than the other sex.

6. The system of claim 1, wherein the conditional X-linked genetic lethal circuit is lethal only to females under conditions effective to express the conditional X-linked lethal circuit.

7. A method of selecting non-transgenic males of a biological species, the method comprising: providing a first strain of the biological species genetically engineered to comprise a conditional Y-linked genetic lethal circuit; providing a second strain of the biological species genetically engineered to comprise a conditional X-linked genetic lethal circuit; performing a first cross mating males of the first strain and females of the first strain under conditions effective to express the conditional Y-linked genetic lethal circuit, thereby producing non-transgenic female progeny; and mating the non-transgenic progeny of the first cross with males of the second strain under conditions effective to express the conditional X-linked genetic lethal circuit, thereby producing non-transgenic males.

8. The method of claim 7, wherein the X-linked genetic lethal circuit is the same as the Y-linked genetic lethal circuit.

9. The method of claim 7, wherein the X-linked genetic lethal circuit is different than the Y-linked genetic lethal circuit.

10. The system of claim 7, wherein the biological species is a pest species.

11. The system of claim 7, wherein the biological species is a species in which one sex has greater commercial value than the other sex.

12. A method of producing a population of sterile non-transgenic males of a biological species, the method comprising: providing the non-transgenic males selected using the method of claim 7; and subjecting the non-transgenic males to a treatment effective to sterilize the males.

13. The method of claim 12, wherein the males are sterilized by subjecting the males to X-ray irradiation.

14. The method of claim 12, wherein the biological species is a pest species.

15. A system comprising: a first strain of a biological species genetically engineered to comprise a conditional W-linked genetic lethal circuit; and a second strain of the biological species genetically engineered to comprise a conditional Z-linked genetic lethal circuit.

16. The system of claim 15, wherein the Z-linked genetic lethal circuit is the same as the W-linked genetic lethal circuit.

17. The system of claim 15, wherein the Z-linked genetic lethal circuit is different than the W-linked genetic lethal circuit.

18. The system of claim 15, wherein the biological species is a pest species.

19. The system of claim 15, wherein the biological species is a species in which one sex has greater commercial value than the other sex.

20. A method of selecting non-transgenic females of a biological species, the method comprising: providing a first strain of the biological species genetically engineered to comprise a conditional W-linked genetic lethal circuit; providing a second strain of the biological species genetically engineered to comprise a conditional Z-linked genetic lethal circuit; performing a first cross mating males of the first strain and females of the first strain under conditions effective to express the conditional W-linked genetic lethal circuit, thereby producing non-transgenic male progeny; and mating the non-transgenic progeny of the first cross with females of the second strain under conditions effective to express the conditional Z-linked genetic lethal circuit, thereby producing non-transgenic females.

21. The method of claim 20, wherein the Z-linked genetic lethal circuit is the same as the W-linked genetic lethal circuit.

22. The method of claim 20, wherein the Z-linked genetic lethal circuit is different than the W-linked genetic lethal circuit.

23. The system of claim 20, wherein the biological species is a pest species.

24. The system of claim 20, wherein the biological species is a species in which one sex has greater commercial value than the other sex.

25. A method of selecting non-transgenic females of a biological species, the method comprising: providing females of the biological species genetically engineered to comprise a conditional Z-linked genetic lethal circuit; and mating wild-type males with females of the biological species under conditions effective to express the conditional Z-linked genetic lethal circuit, thereby producing non-transgenic females.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] FIG. 1. Overview of STSS. Minimal requirements for each strain to be used in STSS, including true-breeding population with conditional Y-linked lethality or conditional X-linked lethality. (A) Schematic illustration of Y-linked lethality. (B) Schematic illustration of X-linked lethality. (C) Mating scheme in absence of lethal gene repressor. Combining non-transgenic females produced from the Y.sup.L strain with adult flies from the X.sup.L strain results in death of all offspring except for non-transgenic males. This includes offspring from mating events of X.sup.L males and females. Tet, tetracycline.

[0027] FIG. 2. Application of STSS in sex selection of chicken. (A) In chicken, females determine the sex of the offspring. A conditional Z-linked lethal strain would allow maintenance of the strain in presence of the permissible medium. (B) Mating scheme in absence of lethal gene repressor. Mating between wildtype males and Z-linked lethal strain females results in death of all offspring except for non-transgenic males.

[0028] FIG. 3. Sex-chromosome linked tet-repressible lethal circuits are effective. (A) Construct-level schematic of tet-repressible lethal circuit used in this study. (B) Proportion of male and female offspring generated from self-mating of DmX.sup.L-tTA or with non-transgenic (w1118) flies in presence or absence of tetracycline. Genotypes of parental files are indicated below x-axis. Numbers above bars indicate total number of progeny produced from six biological replicates. (C) Proportion of male and females generated from mating DmY.sup.L-tTA with non-transgenic (w1118) flies in presence or absence of tetracycline. Numbers above bars show total number of progeny produced from three biological replicates. * indicates statistically significant difference from expected 50:50 male:female sex ratio (chi-squared test, p<0.05). ** indicates a statistically significant difference between the +tet and -tet groups (chi-squared test, p<0.001).

[0029] FIG. 4. Batch production of adult males via STSS. (A) Average number of adult males obtained from mating between different proportions of non-transgenic female flies obtained from DmY.sup.L-tTA in the absence of tetracycline (‘XX*’) when combined with adult DmX.sup.L-tTA flies in absence of tetracycline. Data represent mean numbers from two or three biological replicates with error bars showing standard deviation. Average numbers of females produced are indicated numerically above bars. Numbers below x-axis indicate ratio of genotypes in parental generation. Total numbers from all replicates are (from left to right): N=822, N=1151, N=1292, N=1301, N=1086, N=1078. (B) Bright-field (left) and fluorescent (right) images of parental (i) 10 DmY.sup.L-tTA females produced in the absence of tetracycline (‘XX*’), parental (ii) 10 DmX.sup.L-tTA males, and (iii) approximately 400 offspring from the batch production of non-transgenic males. Files in (i) and (ii) are only included for visual comparison to the STSS males; they were not present in the final batch of STSS flies. (C) PCR amplification of transgene cassette from genomic DNA isolated from 10 DmX.sup.L-tTA flies (+), 1180 batch-produced STSS males (male symbol), or STSS gDNA spiked with DNA from DmX.sup.L-tTA flies at five-fold dilutions from 1:5 (right) to 1:3125 (left). L denotes 1 kb plus DNA ladder (ThermoFisher Scientific, Inc., Waltham, Mass.).

[0030] FIG. 5. An exemplary approach of STSS. (A) Reproductive behavior of X-linked Female Lethal construct used in female-lethal (FL-) STSS. (B) Mating scheme for producing non-transgenic males via FL-STSS. Combining non-transgenic females produced from the YL strain with adult male flies produced from the X.sup.FL strain results in death of all offspring except for non-transgenic males. (C) Genetic design of FL construct.

[0031] FIG. 6. An exemplary approach of STSS. (A) Chromosomal location of FL constructs in two copy ‘FL12a-c’ flies. FL1 and FL2 have only one copy of the X-linked FL construct on their X-chromosome. (B) Proportion of male and female offspring generated from self-mating or outcrosses to wild-type (w1118) for DmX.sup.FL1, DmX.sup.FL2, and three independently generated DmX.sup.FL12 genotypes. Parental genotypes are indicated below the x-axis. Results are shown in the presence or absence of tetracycline. Numbers above bars indicate total number of progeny produced from at least three biological replicates. (C) Average number of adult males obtained from mating between different proportions of non-transgenic female flies obtained from DmY.sup.L-tTA in absence of tetracycline (′XX*′) when combined with adult male DmX.sup.FL12c flies in absence of tetracycline. Data represent mean numbers from 2 biological replicates with error bars showing standard deviation. Average numbers of females produced are indicated numerically above bars. Total numbers from all replicates are (from left to right): N=460, N=475, N=692, N=617. Numbers below x-axis indicate number of parental flies of each genotype. * indicates statistically significant difference from expected 50:50 male:female sex ratio (chi-squared test, p<0.05). ** indicates a statistically significant difference between the +tet and -tet groups (chi-squared test, p<0.001).

[0032] FIG. 7. Characterization of unexpected flies. Obtained female (♀*) from the final mating and male (♂*) from DmY.sup.L-tTA mating in absence of tetracycline were assayed for the presence of X and Y-chromosome by amplifying an X-chromosome specific gene, upd1 and Y-chromosome specific gene, kl-5. L denotes 1 kb plus DNA ladder (Thermo Fisher Scientific, Waltham, Mass.).

[0033] FIG. 8. An example of an inducible lethal system. A temperature sensitive promoter (pHsp70Bb) induces overexpression of a gene (hedgehog; Hh) that results in lethality in response to elevated temperature.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0034] This disclosure describes and demonstrates systems and methods for batch production of the heterogametic sex of a species that are suitable for, for example, sterile pest control. In many species, the heterogametic sex is male (XY, FIG. 1). In other species, the heterogametic sex is female (WZ, FIG. 2). The systems and methods described herein may be applied to generate the heterogametic sex (XY or WZ) in any organism with chromosomal sex determination. At least 99% sex-selection was observed with batch cultures as large as 6800 individuals. Transgenes were not detected in the surviving progeny.

[0035] Insect pests impose a major burden to food production and human health worldwide. The most successful population control method in use today is the sterile insect technique (SIT). SIT relies on mass rearing of pest insects followed by a sterilization treatment (e.g., X-ray irradiation). Sterilized insects are released into the wild where sterile males compete with wild males to seek out and mate with wild females. The female of many pest insects will typically only mate once in her lifetime. Mating with a sterile male therefore prevents successful reproduction. Sufficiently large releases of sterile insects can be used to eliminate wild populations or prevent their introduction in an area at threat of their introduction. Also, SIT is considered safe to humans and the environment, as there are fewer off-target effects compared to the application of chemical pesticides.

[0036] Existing SIT programs are used to control several major agricultural pests including the New World Screwworm (Cochliomyia hominivorax), Mediterranean Fruit Fly (Ceratitis capitata), and Queensland Fruit Fly (Bactrocera tryoni). All together, these programs produce and release billions of sterile insects on a weekly basis. SIT for many insects, including C. hominivorax and B. tryoni, currently involves releasing both sterilized males and females. However, the effectiveness of SIT can be substantially increased if only males are released since they will then seek out wild females instead of mating with co-released sterile females. In some insect pests, such as the Yellow Fever Mosquito (Aedes aegypti), SIT programs only release sterile males since sterile females can vector disease.

[0037] A variety of sex-sorting techniques have been developed. Mechanical separation of Aedes aegypti pupae based on size differences can be effective and flow cytometric separation of transgene-expressing female Anopholes gambiae has been demonstrated. These approaches can be labor intensive and/or require sophisticated equipment, however. Combining irradiation with temperature-sensitive lethal (tsl) mutant strains of C. capitata is presently in use in SIT programs. Repressible transgenic female-elimination constructs act as genetic biocontrol systems on their own and have been developed for Ae. aegypti, C. hominivorax, Sheep Blow Fly (Lucilia cuprina), Diamondback moth (Plutella xylostella), Pink Bollworm (Pectinophora gossypiella), and Silkworm (Bombyx mori). Despite their effectiveness, specificity, reduction of insecticide use, and safety, public resistance and regulatory hurdles have limited the broad use of released transgenic insects for pest control.

[0038] The sex selection methods described herein may have applications beyond controlling pest species. For example, selection of female-only progeny is desirable in egg-layer poultry production since there is little economical need for the male chicks in the egg-layer industry. Current practices of female-only chicken selection use physical characteristics in day-old chicks. In some cases, the separated male chicks are culled and discarded, often subjected to industrial grade mechanical grinders. This practice is banned in several countries as being inhumane. It is also laborious and error-prone. Other methods for in-egg sex selection require individual screening of eggs with sophisticated and time-consuming instrumentation. The methods described herein provide a humane, labor-free, and accurate strategy for sex selection in egg-laying poultry.

[0039] This disclosure describes a genetic approach to produce non-transgenic males in Drosophila melanogaster, referred to herein as Subtractive Transgene Sex Sorting (STSS). STSS relies on two transgenic strains, each of which has bi-sex lethal genetic circuit that can be induced or repressed. One of the strains has the lethal circuit on the Y-chromosome (Y.sup.L strain, FIG. 1A) and the other has the lethal circuit on the X-chromosome (X.sup.L strain, FIG. 1B). Non-transgenic males are produced (FIG. 1C) by first switching the Y.sup.L strain to media that activates the lethal circuit, resulting in non-transgenic females. These non-transgenic females are combined with the X.sup.L strain in media that activates the lethal circuit. Mating between the X.sup.L males and non-transgenic females results in non-transgenic males. All other offspring die. An alternative approach is to create a strain containing X-linked female lethal circuit (FL-STSS, FIG. 5A), which when active is selectively lethal to females containing the circuit. Males produced from this strain in the selection media when crossed with females obtained from Y.sup.L strain would result in non-transgenic males (FIG. 5B). This technique is transferable to any organism that relies on genetic, as opposed to environmental, sex determination (Smanski MJ & Zarkower, D. 2019. EMBO Reports 20:e48577).

Design and Construction of a Repressible Lethal and Female-Specific Lethal Transgenic Construct

[0040] A repressible lethal genetic construct can be designed with a conditionally-expressed promoter driving a toxic gene product. Two exemplary constructs are illustrated in FIG. 3A and FIG. 5C. A tetracycline-repressible hsp70 minimal promoter (pHsp70) was selected due to its well-characterized behavior in model and applied insect species. To drive lethality, the tet-transactivator (tTA) was expressed, the VP64 transactivation domain of which is toxic to cells when expressed strongly. Plasmids were constructed with a positive feedback loop where the hsp70 minimal promoter drives basal expression of the tet-transactivator (tTA) similar to what has been previously described (Shockett et al., 1995, Proc. Natl. Acad. Sci. USA, 92:6522-6526). In the absence of tetracycline, tTA binds to operators upstream of the hsp70 minimal promoter and establishes a positive feedback loop that generates lethal amounts of tTA. 5′ UTR translational enhancer features were incorporated to further boost tTA expression. Tuning gene expression in lethal transgenic constructs is a balance between (i) incurring fitness effects from leaky expression in the repressed ‘off’-state and (ii) incomplete penetrance due to weak expression in the de-repressed ‘on’-state. To ensure complete penetrance of the lethal phenotype in the derepressed state, the construct was designed to favor strong expression.

[0041] PhiC31-mediated transgenesis was used to integrate a single copy of the tTA circuit into AttP landing sites on the X and Y chromosomes of two separate strains (referred to as DmX.sup.L-tTA and DmY.sup.L-tTA from here on). Both of these strains were maintained in the presence of 200 μg/ml tetracycline. The genotype of transgenic flies was confirmed by PCR amplification and Sanger sequencing of engineered loci. The DmX.sup.L-tTA were mated with a X-chromosome balancer strain and then selfed to screen for females homozygous for the modified X-chromosome, which was confirmed by PCR. From this point on, DmX.sup.L-tTA and DmY.sup.L-tTA were maintained as true-breeding lines in the presence of tetracycline.

[0042] Similar methods were used to create X.sup.FL strain with the exception of there being insertions into two separate locations in the X-chromosome (FIG. 6A), referred to as DmX.sup.FL1 and DmX.sup.FL2. Both DmX.sup.FL1 and DmX.sup.FL2 were maintained as homozygous true breeding lines in the presence of 10 μg/ml tetracycline. In order to create a line containing inserts in both the locations of the X-chromosome, DmX.sup.FL1 and DmX.sup.FL2 were crossed with each other and several recombinants were isolated and identified by screening with PCR. They were balanced and selfed to create homozygous true breeding line and maintained in presence of 10 μg/ml tetracycline.

Performance of Repressible Lethal and Female Lethal Genetic Constructs

[0043] To test the efficiency of toxic gene expression, virgin females and males were mated on media lacking tetracycline. In each of three replicate crosses, no DmX.sup.L-tTA adults survived to adulthood (FIG. 3B). This suggests that the repressible lethal transgenic construct is sufficiently strong to cause lethality in two copies (females) or one copy (males). In an analogous experiment with DmY.sup.L-tTA flies ten replicate crosses produced 777 females and only two males (99.7% females, FIG. 3C). These males did not reproduce when subsequently mated with non-transgenic females and lacked a Y chromosome (XO, FIG. 7), likely a result of nondisjunction. Thus, both the DmX.sup.L-tTA and DmY.sup.L-tTA produced a sufficiently lethal phenotype in the absence of tetracycline to remove the transgene from the accessible gene pool (FIG. 3B, 3C). Both DmX.sup.FL1 and DmX.sup.FL2 strains were equally efficient at producing 100% males in absence of tetracycline only as true breeding line (FIG. 6B). To use a heterozygote DmX.sup.FL for biocontrol, the two DmX.sup.FL1 and DmX.sup.FL2 lines were combined. Three independent lines containing transgene in the two locations (DmX.sup.FL12a, DmX.sup.FL12b, DmX.sup.FL12c) produced almost 100% males in absence of tetracycline as a heterozygote (FIG. 6B). The females produced in absence of tetracycline were very sick and never produced any progeny.

Sub-Stoichiometric Ratio of Mixed-Sex DmXL.sup.tTA to Female DmYL.sup.tTA Sufficient for Non-Transgenic Male Production

[0044] Non-transgenic males can be generated by crossing non-transgenic females produced by the DmY.sup.L-tTA strain and males from a mixed-sex true-breeding population of DmX.sup.L-tTA flies (FIG. 1C). The number of non-transgenic males produced is directly related to the number of non-transgenic mothers, but is unaffected by decreasing numbers of DmX.sup.L-tTA fathers. This would be important for economically scaling-up the production of non-transgenic males for SIT programs. Experimental crosses were performed between non-transgenic females and DmX.sup.L-tTA mixed-sex populations to determine the minimum sufficient ratio of parental genotypes. A monotonically increasing number of total offspring were produced as the ratio of DmX.sup.L-tTA males to DmY.sup.L-tTA females increased from 1:20 to 3:10 (FIG. 4A). The offspring number appeared to plateau or even decline after further increasing the number of DmX.sup.L-tTA males. This suggests that a ˜1:3 ratio is sufficient to ensure that the number of DmX.sup.L-tTA males are not limiting the total number of offspring produced.

[0045] At or below the optimal ratios of DmX.sup.L-tTA males to DmY.sup.L-tTA females, 100% male offspring (N.sub.combined=5388 male offspring, 0 female offspring) were observed (FIG. 4A). A total of four female offspring across all replicates when the ratio of DmX.sup.L-tTA males to DmY.sup.L-tTA females was 10:10 or 20:10 (FIG. 4A; N.sub.combined=2142 male offspring, 4 female offspring). It is unclear how these females were able to survive, but they lacked a GFP phenotype, did not appear to be transgenic, and did not carry a Y chromosome (FIG. 7), indicating that they were not XXY females.

[0046] An alternative approach of creating STSS (FIG. 5A, 5B), where two copies of the female lethal construct illustrated in FIG. 5C are inserted into two different locations of X-chromosome (FIG. 6A) results in similar number of males and similar percentage of males from different ratios of GE males and non-GE females (FIG. 6B, 6C). Females observed in this method were very sick and failed to produce viable progeny when crossed with healthy wildtype males.

Large-Scale Cultivation of Non-Transgenic Males Suitable for Egg Release

[0047] Next, the effectiveness of producing only non-transgenic males by the mating scheme in FIG. 1C followed by batch cultivation was tested. A true-breeding culture of DmY.sup.L-tTA was transferred to media lacking tetracycline and cleared all adults after 24 hours. The resulting offspring from the tetracycline-free medium were mixed at a 2:1 ratio with adults from a true-breeding population of DmX.sup.L-tTA flies. Adults from this cross were cleared after three days. This mating yielded 2932 males (N=3, 977±144) and one female. None of the more than 1000 males screened contained the GFP transgene marker. To ensure the lack of GFP detection (FIG. 4B) was not due to transgene silencing, genomic DNA from was isolated more than 1000 male STSS flies and screened for presence of the transgene by PCR (expected fragment size of 821 bp). A clear band of the expected size from a positive control (gDNA isolated from 10 DmXL.sup.tTA flies) but not in gDNA isolated from the putative non-transgenic males (FIG. 4C). Spiking trace amounts of positive control gDNA confirmed that limit of detection via this assay at less than 1:3000 transgenic:non-transgenic gDNA. This confirmed that the assay was sufficiently powerful to detect any transgenic flies that would have been present in the screened population.

[0048] This disclosure therefore describes a method of Subtractive Transgene Sex Sorting (STSS) using D. melanogaster as a model system. While described in detail in the context of an exemplary embodiment in which species is D. melanogaster, the methods described herein can involve any organism that relies on genetic, as opposed to environmental, sex determination. Exemplary other species in which the method may be employed include, for example, an insect (e.g., mosquito, tstetse fly, spotted-wing drosophila, diamond back moth, fall army worm, soybean gall midge, white fly, Mediterranean fruit fly, olive fly, gypsy moth, codling moth, deer tick, etc.), a fish (e.g., salmon, carp, sea lamprey, etc.), a bird (e.g., poultry), a mammal (e.g., swine, a mouse, a rat, etc.), an amphibian (e.g., a cane toad, a bullfrog, etc.), a reptile (e.g., brown tree snake, etc.), or a crustacean (e.g., rusty crayfish, etc.).

[0049] The method involves the use of two genetically-engineered strains of a pest species and a mating protocol that produces only non-transgenic males. The males may be subsequently sterilized using, for example, SIT sterilization techniques to produce sterile non-transgenic males. The sterile non-transgenic males may be released to control the population of the pest species.

[0050] Each strain is engineered to possess a lethal genetic circuit that can be induced or repressed. While described herein in the context of an exemplary embodiment in which both genetically engineered strains possess a tet-transactivator (tTA) genetic circuit that is lethal in the absence of tetracycline, one or both strains may be constructed to include an alternate lethal genetic circuit (e.g. temperature inducible activation of a gene causing lethality, FIG. 8), lactose repressor, methionine repressor). The lethal genetic circuit used in one strain may be independent of—i.e., the same or different than—the lethal genetic circuit used in the other strain.

[0051] The basic genetic architecture has been demonstrated in numerous pest insects and STSS can be readily adapted to improve SIT programs by enabling efficient sex-sorting for male only release. Although described in the context of using PhiC31-mediated integration, CRISPR systems also may be used to target integration to many genomic loci in insects, including the repeat rich Y chromosome. This approach allows one to generate transgene-free males in species where males are heterogametic. For species where the female is heterogametic (i.e., lepidoptera), generation of transgene-free males is simplified and would only require a W′ construct. This approach can be applied to species with homomorphic sex chromosomes, assuming the sex-determining region of the sex chromosome is accessible to transgene integration technology.

[0052] Using the STSS system, no transgenic flies survives in the absence of tetracycline. The scale of the experiments described herein support a conclusion that the transgenic fly escape is less than 0.1%. SIT programs generate millions of flies for release on a weekly basis and some small number of transgenic flies may be produced and released using the STSS system, although they are likely to suffer fitness defects.

[0053] Production of males incapable of reproduction with wild females does not necessarily require radiation treatment. The incompatible insect technique relies on males infected with certain strains of Wolbachia bacteria. Mating between infected males and uninfected females results in embryonic lethality since the female-produced egg does not contain an antidote to a deubiquitylating enzyme toxin delivered by the sperm. Transgenic approaches have been developed or proposed to generate males that cannot reproduce with wild females. So far only Release of Insects with Dominant Lethal (RIDL) has made it to small scale commercial use for the control of Ae. aegypti. RIDL utilizes a bi-sex lethal genetic circuit where larvae, but not adults, require tetracycline to survive to adulthood. Mechanically sorted male pupae are subsequently used for release and their offspring die in the absence of tetracycline in the wild.

[0054] Combining STSS with cytoplasmic incompatibility can eliminate the need for any additional sterilization treatments and could enable the release of eggs/larvae to further reduce costs. Shipping of eggs, particularly of species such as Ae. aegypti that can be stored dry for extended periods, would allow for rearing facilities to be located far from control sites and reduce local infrastructure costs. However, this would require using non-antibiotic control of a lethal circuit such as temperature inducible activation of a lethal gene.

[0055] In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0056] In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0057] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0058] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Plasmid Construction—Repressible Lethal

[0059] The tetracycline repressible lethal circuit was made by adapting a previously described female-lethal piggybac vector, pB[FL3] (Yan et al., 2017, Sci Rep 7:2538). The female specific intron and 5′UTR were replaced with a myosin heavy chain intron and syn21 translational enhancers (Pfeiffer et al., 2012, Proc Natl Acad Sci USA 109: 6626-6631). The final plasmid, pMM7-10-1 (SEQ ID NO:1), was made by transferring the lethal circuit to pUB-EGFP (Schetelig et al., 2009, Proc Natl Acad Sci USA 106: 18171-18176), which contains an attB site for PhiC31 mediated integration and ubiquitin promoter driven EGFP expression.

Plasmid Construction—Female Lethal

[0060] The tetracycline female lethal circuit was made by adapting a previously described female-lethal piggybac vector, pB[FL3] (Li et. al., 2014. Insect Biochem & Mol Biol, 51:80-88). The final plasmid, pMM7-8-1 (SEQ ID NO:2), was made by transferring the lethal circuit to pUB-EGFP (Schetelig et al., 2009, Proc Natl Acad Sci USA 106:18171-18176), which contains an attB site for PhiC31 mediated integration and ubiquitin promoter driven EGFP expression.

Generating and Maintaining Transgenic Drosophila Strains

[0061] D. melanogaster strains were maintained at 25° C. and 12 hours light in cornmeal agar (FLYSTUFF, Genesee Scientific Corp., San Diego, Calif.) supplemented with 10-200 μg/ml tetracycline, as necessary. Transgenic D. melanogaster strains where generated by microinjection (BestGene Inc, Chino Hills, Calif.) and PhiC31 mediated integration of pMM7-10-1 into the X-chromosome attP site of y[1] w[*] P{y[+t7.7]=CaryIP}su(Hw)attP8 (BDSC #3233; Pfeiffer et al., 2010, Genetics 186: 735-755) to make DmXL.sup.tTA and the Y-chromosome attP of y1 w*/Dp(2;Y)G, P{CaryP}attPY (Szabad et al., 2012, Genes/Genomes/Genetics 2: 1095-1102) to make DmYL.sup.tTA.

Fly Viability Assays

[0062] Desired number of male and virgin female flies were moved to new tubes containing media either with or without tetracycline and allowed to lay eggs for five days at 25° C. and 12 hours light protocol. After five days, adults were removed from the tubes and offspring were allowed to develop in the incubator. Adult flies were counted as they emerged from the pupae for a total of 15 days from the start of experiment.

PCR Verification

[0063] Fly genomic DNA was isolated in a pool by grinding in 25 μl of “Squish Buffer” (10 mM Tris, 1 mM EDTA, 25 mM NaCl, 8 U/ml ProK (New England Biolabs, Inc., Ipswich, Mass.) per adult. ProK was heat inactivated at 98° C. for four minutes. For transgene PCR screen, 1181 STSS males were pooled together as one sample and compared to five male and five female DMX.sup.L-tTA flies in a separate pool of genomic DNA as positive control. The positive control samples were diluted in with STSS gDNA in the following ratios: 1:5, 1:25, 1:125, 1:625, and 1:3125. For each reaction, 1 μl, of template gDNA was used in a 20 μL PCR reaction with primers that anneal within the transgene. The following primers were used for amplification of the transgene,

TABLE-US-00001 (SEQ ID NO: 3) fwd: 5′-GCCGCAGAATTCTCTCTATC-3′, (SEQ ID NO: 4) rev: 5′-CTTAGCTTTCGCTTAGCGACG-3′; (SEQ ID NO: 5) upd1, fwd: 5′-TGCAGGTGACCTGGGAATAG-3′, (SEQ ID NO: 6) upd1, rev: 5′-GTGAGACCACTTGACCACAG-3′, (SEQ ID NO: 7) k1-5, fwd: 5′-CGCGACGATAGACAGCGG-3′, and (SEQ ID NO: 8) k1-5, rev: 5′-GAGAGCAATGCGCTCGTTGC-3′.

Data Analysis

[0064] All the experiments were performed with at least two and as high as 10 replicates. Raw offspring numbers from each experiment were converted into percent male/female and averaged across the replicates. Chi-squared test was performed to test difference between observed and expected sex ratio in different mating. Number of flies from each experiment across the different replicates was summed together then converted into percent male/female and used in the Chi-squared test. To test the effect of different parental male-female ratio on adult offspring numbers, One-way ANOVA was performed followed by Bonferroni's post-hoc test. P-value <0.05 was considered significant.

[0065] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0066] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0067] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0068] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence Listing Free Text

[0069]

TABLE-US-00002 GGCCCGGTAC GTACCCAATT CGCCCTATAG TGAGTCGTAT TACAATTCAC TGGCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG ACCAAGTTTA CTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCG AGCGCAGCGA GTCAGTGAGC GAGGAAGCGG AAGAGCGCCC AATACGCAAA CCGCCTCTCC CCGCGCGTTG GCCGATTCAT TAATGCAGCT GGCACGACAG GTTTCCCGAC TGGAAAGCGG GCAGTGAGCG CAACGCAATT AATGTGAGTT AGCTCACTCA TTAGGCACCC CAGGCTTTAC ACTTTATGCT TCCGGCTCGT ATGTTGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG GAAACAGCTA TGACCATGAT TACGCCAAGC TCGAAATTAA CCCTCACTAA AGGGAACAAA AGCTGGCTAG AACTAGTGTC GACATGCCCG CCGTGACCGT CGAGAACCCG CTGACGCTGC CCCGCGTATC CGCACCCGCC GACGCCGTCG CACGTCCCGT GCTCACCGTG ACCACCGCGC CCAGCGGTTT CGAGGGCGAG GGCTTCCCGG TGCGCCGCGC GTTCGCCGGG ATCAACTACC GCCACCTCGA CCCGTTCATC ATGATGGACC AGATGGGTGA GGTGGAGTAC GCGCCCGGGG AGCCCAAGGG CACGCCCTGG CACCCGCACC GCGGCTTCGA GACCGTGACC TACATCGTCG ACACTAGTGG ATCCAGCGGC CGCACCTGCA GGCCGGCCGT TAACACGCGT CCGCGGTCTA GACTCGAGGA TTCCAGATCT GGTACCGGGC CGCTGTATGG ATATTTGCAG GGCCGCAGAA TTCTCTCTAT CACTGATAGG GAGGTCTCTA TCACTGATAG GGAGTTCTCT ATCACTGATA GGGATGTCTC TATCACTGAT AGGGATTTCT CTATCACTGA TAGGGAAGTC TCTATCACTG ATAGGGACCT CTCTATCACT GATAGGGAAA TCTCTATCAC TGATAGGGAT CTCTCTATCA CTGATAGGGA CTTCTCTATC ACTGATAGGG ACGTCTCTAT CACTGATAGG GAACTCTCTA TCACTGATAG GGACATCTCT ATCACTGATA GGGACTTCTC TATCACTGAT AGGGAAGTAT GTTCTCTCTC TTCTCTTCTC TCTCTCTTTC TCGAATGTTC TCTCTCTTCT CTTCTCTCTC TCTTTCTCGA TGGCCGGGGC GCGCCAGGTT TCGACTTTCA CTTTTCTCTA TCACTGATAG GGAGTGGTAA ACTCGACTTT CACTTTTCTC TATCACTGAT AGGGAGTGGT AAACTCGACT TTCACTTTTC TCTATCACTG ATAGGGAGTG GTAAACTCGA CTTTCACTTT TCTCTATCAC TGATAGGGAG ATCCGAGCTC GTAAACTCGA CTTTCACTTT TCTCTATCAC TGATAGGGAG TGGTAAACTC GACTTTCACT TTTCTCTATC ACTGATAGGG AGTGGTAAAC TCGACTTTCA CTTTTCTCTA TCACTGATAG GGAGTGGTAA ACTCGAAgcg cCGGATCCGT CGAGGGAAAA GAGCGCCGGA GTATAAATAG AGGCGCTTCG TCTACGGAGC GACAATTCAA TTCAAACAAG CAAAGTGAAC ACGTCGCTAA GCGAAAGCTA AGCAAATAAA CAAGCGCAGC TGAACAAGCT AAACAATCTG CAGCCgatct aaaaggtagg ttcaaccact gatgcctagg cacaccgaaa cgactaaccc taattcttat cctttacttc aggcggccgg gctcgagggt accaacttaa aaaaaaaaat caaaATGGTC AGCCGTTTGG ATAAATCCAA AGTTATTAAT TCCGCTTTGG AATTGTTGAA TGAAGTTGGT ATTGAAGGTT TGACAACACG TAAATTGGCT CAAAAATTGG GTGTTGAACA ACCAACATTG TATTGGCATG TTAAAAATAA ACGTGCTTTG TTGGATGCTT TGGCTATTGA AATGTTGGAC CGTCATCATA CACATTTTTG CCCATTGGAA GGCGAATCCT GGCAAGATTT CTTGCGTAAT AATGCCAAAT CCTTCCGTTG TGCTTTGTTG TCCCATCGTG ATGGTGCCAA GGTTCATTTG GGCACACGTC CAACAGAAAA ACAATATGAA ACATTGGAAA ATCAATTGGC TTTCTTGTGT CAACAAGGCT TCAGCTTGGA AAATGCTTTG TATGCTTTGA GCGCCGTTGG TCATTTTACA TTGGGCTGTG TGTTGGAAGA TCAAGAACAT CAAGTCGCTA AAGAAGAACG TGAAACACCA ACAACAGATT CGATGCCCCC ATTGTTGCGT CAAGCAATTG AATTGTTCGA TCATCAAGGA GCCGAACCAG CATTCTTGTT CGGCTTGGAA TTGATTATTT GTGGATTGGA AAAACAATTG AAATGTGAAT CGGGCTCGGG CCCCGCGTAC AGCCGCGCGC GTACGAAAAA CAATTACGGG TCTACCATCG AGGGCCTGCT CGATCTCCCG GACGACGACG CCCCCGAAGA GGCGGGGCTG GCGGCTCCGC GCCTGTCCTT TCTCCCCGCG GGACACACGC GCAGACTGTC GACGGCCCCC CCGACCGATG TCAGCCTGGG GGACGAGCTC CACTTAGACG GCGAGGACGT GGCGATGGCG CATGCCGACG CGCTAGACGA TTTCGATCTG GACATGTTGG GGGACGGGGA TTCCCCGGGT CCGGGATTTA CCCCCCACGA CTCCGCCCCC TACGGCGCTC TGGATATGGC CGACTTCGAG TTTGAGCAGA TGTTTACCGA TGCCCTTGGA ATTGACGAGT ACGGTGGGTA GTAAGCTTGG ATCTTTGTGA AGGAACCTTA CTTCTGTGGT GTGACATAAT TGGACAAACT ACCTACAGAG ATTTAAAGCT CTAAGGTAAA TATAAAATTT TTAAGTGTAT AATGTGTTAA ACTACTGATT CTAATTGTTT GTGTATTTTA GATTCCAACC TATGGAACTG ATGAATGGGA GCAGTGGTGG AATGCCTTTA ATGAGGAAAA CCTGTTTTGC TCAGAAGAAA TGCCATCTAG TGATGATGAG GCTACTGCTG ACTCTCAACA TTCTACTCCT CCAAAAAAGA AGAGAAAGGT AGAAGACCCC AAGGACTTTC CTTCAGAATT GCTAAGTTTT TTGAGTCATG CTGTGTTTAG TAATAGAACT CTTGCTTGCT TTGCTATTTA CACCACAAAG GAAAAAGCTG CACTGCTATA CAAGAAAATT ATGGAAAAAT ATTCTGTAAC CTTTATAAGT AGGCATAACA GTTATAATCA TAACATACTG TTTTTTCTTA CTCCACACAG GCATAGAGTG TCTGCTATTA ATAACTATGC TCAAAAATTG TGTACCTTTA GCTTTTTAAT TTGTAAAGGG GTTAATAAGG AATATTTGAT GTATAGTGCC TTGACTAGAG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA TGTCTCGAGC ATGCGCAAAT TTAAAGCGCT GATATCGATC GCGCGCAGAT CTGTCATGAT GATCATTGCA ATTCTGCAGT CGACGGTACC CGATCTTGTC GCCGGAACGC AGCGACAGAG ATTCCAATGT GTCCGTATCT TTCAGGCTTT TGCCCTTCAG TTCCAGACGA AGCGACTGGC GATTCGCGTG TGGGGTCTGC TTCAGGGTCT TGTGAATTAG GGCGCGCAGA TCGCCGATGG GCGTGGCGCC GGAGGGCACC TTCACCTTGC CGTACGGCTT GCTGTTCTTC GCGTTCAAAA TCTCCAGCTC CATTTTGCTT TCGGTGCGCT TGCAATCAGT ACTGTCCAAA ATCGAAAATC GCCGAACCGT AGTGTGACCG TGCGGGGCTC TGCGAAAATA AACTTTTTTA GGTATATGGC CACACACGGG GAAAGCACAG TGGATTATAT GTTTTAATAT TATAATATGC AGGTTTTCAT TACTTATCCA GATGTAAGCC CACTTAAAGC GATTTAACAA TTATTTGCCG AAAGAGTAAA AACAAATTTC ACTTAAAAAT GGATTAAGAA AAGCTTGTGT AAGATTATGC GCAGCGTTGC CAGATAGCTC CATTTAAAAC ACTTCAAAAA CAATAAGTTT TGAAAATATA TACATAAATA GCAGTCGTTG CCGCAACGCT CAACACATCA CACTTTTAAA ACACCCTTTA CCTACACAGA ATTACTTTTT AAATTTCCAG TCAAGCTGCG AGTTTCAAAA TTATAGCCGG TAGAGAAGAC AGTGCTATTT CAAAAGCAAA CTAAATAAAC ACCAATCCTA ACAAGCCTTG GACTTTTGTA AGTTTAGATC AAAGGTGGCA TTGCATTCAA TGTCATGGTA AGAAGTAGGT CGTCTAGGTA GAAATCCTCA TTCAGCCGGT CAAGTCAGTA CGAGAAAGGT CTCAATTTGA AATTGTCTTA AAAATATTTT ATTGTTTTGT ACTGTGGTGA GTTTAAACGA AAAACACAAA AAAAAAGTGA TACACAGAAA TCATAAAAAA TTTTAATACA AGGTATTCGT ACGTATCAAA AACATTTCGG CACAATTTTT TTTCTCTGTA CTAAAGTGTT ACGAACACTA CGGTATTTTT TAGTGATTTT CAACGGACAC CGAAGGTATA TAAACAGCGT TCGCGAACGG TCGCCTTCAA AACCAATTGA CATTTGCAGC AGCAAGTACA AGCAGAAAGT AAAGCGCAAT CAGCGAAAAA TTTATACTTA ATTGTTGGTG ATTAAAGTAC AATTAAAAGA ACATTCTCGA AAGTCACAAG AAACGTAAGT TTTTAACTCG CTGTTACCAA TTAGTAATAA GAGCAACAAG ACGTTGAGTA ATTTCAAGAA AAACTGCATT TCAAGGTCTT TGTTCGGCCA TTTTTTTTTT ATTCAACGCT CTACGTAATT ACAAAATAAG AAATTGGCAG CCACGCATCT TGTTTTCCCA ATCAATTGGC ATCAAAACGC AAACAAATCT ATAAATAAAA CTTGCGTGTT GATTTTCGCC AAGATTTATT GGCAAATTGT GAAATTCGCA GTGACGCATT TGAAAATTCG AGAAATCACG AACGCACTCG AGCATTTGTG TGCATGTTAT TAGTTAGTTA GTTCTTTGCT TAATTGAAGT ATTTTACCAA CGAAATCCAC TTATTTTTAG CTGAAATAGA GTAGGTTGCT TGAAACGAAA GCCACGTCTG GAAAATTTCT TATTGCTTAG TAGTTGTGAC GTCACCATAT ACACACAAAA TAATGTGTAT GCATGCGTTT CAGCTGTGTA TATATACATG CACACACTCG CATTATGAAA ACGATGACGA GCAACGGAAC AGGTTTCTCA ACTACCTTTG TTCCTGTTTC TTCGCTTTCC TTTGTTCCAA TATTCGTAGA GGGTTAATAG GGGTTTCTCA ACAAAGTTGG CGTCGATAAA TAAGTTTCCC ATTTTTATTC CCCAGCCAGG AAGTTAGTTT CAATAGTTTT GTAATTTCAA CGAAACTCAT TTGATTTCGT ACTAATTTTC CACATCTCTA TTTTGACCCG CAGAATAATC CAAAATGCAG ATCGGGGATC CCACCCCACC CAAGAAGAAG CGCAAGGTGG AGGACGATCC CGTCGTTTTA CAACGTCGTG ACTGGGAAAA CCCTGGCGTT ACCCAACTTA ATCGCCTTGC AGCACATCCC CCTTTCGCCA GCTGGCGTAA TAGCGAAGAG GCCCGCACCG ATCGCCCTTC CCAACAGTTG CGGTCGACTC TAGAGGATCC CCGGGATCCA CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA GGGCGAGGGC GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG CCCTGGCCCA CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAG CCGCTACCCC GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT GAAGTTCGAG GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT CATGGCCGAC AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGACGGCAGC GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG CCCGACAACC ACTACCTGAG CACCCAGTCC GCCCTGAGCA AAGACCCCAA CGAGAAGCGC GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG CATGGACGAG CTGTACAAGT AAAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTAAAGC TTATCGATAC GCGTACGGCA CTAGAGCGGC CGCCACCGCG GTGGAGCTCC AGCTTTTGTT CCCTTTAGTG AGGGTTAATT AGATCGGCCG GCCTTGGCGC GCCTAGATCT TAATACGACT CACTATAGGG CGAATTGGGT ACCG

TABLE-US-00003 pMM7-8-1-PUbEGFP, 12394 bp ds-DNA SEQ ID NO: 2 1 GGCCCGGTAC GTACCCAATT CGCCCTATAG TGAGTCGTAT TACAATTCAC TGGCCGTCGT 61 TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC TTGCAGCACA 121 TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA 181 GTTGCGCAGC CTGAATGGCG AATGGAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG 241 TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT 301 TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT 361 CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT 421 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA 481 CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC 541 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA 601 GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG 661 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA 721 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA 781 AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA 841 TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT 901 CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG 961 AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC 1021 GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT 1081 CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA 1141 GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT 1201 CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT 1261 GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT 1321 GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA 1381 CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA 1441 CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT 1501 GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC 1561 GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT 1621 GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG ACCAAGTTTA CTCATATATA 1681 CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT 1741 GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC 1801 GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG 1861 CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT 1921 CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG 1981 TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG 2041 CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC 2101 TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA 2161 CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA 2221 GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC 2281 GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT 2341 GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG 2401 AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT 2461 TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC 2521 TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCG AGCGCAGCGA GTCAGTGAGC 2581 GAGGAAGCGG AAGAGCGCCC AATACGCAAA CCGCCTCTCC CCGCGCGTTG GCCGATTCAT 2641 TAATGCAGCT GGCACGACAG GTTTCCCGAC TGGAAAGCGG GCAGTGAGCG CAACGCAATT 2701 AATGTGAGTT AGCTCACTCA TTAGGCACCC CAGGCTTTAC ACTTTATGCT TCCGGCTCGT 2761 ATGTTGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG GAAACAGCTA TGACCATGAT 2821 TACGCCAAGC TCGAAATTAA CCCTCACTAA AGGGAACAAA AGCTGGCTAG AACTAGTGTC 2881 GACATGCCCG CCGTGACCGT CGAGAACCCG CTGACGCTGC CCCGCGTATC CGCACCCGCC 2941 GACGCCGTCG CACGTCCCGT GCTCACCGTG ACCACCGCGC CCAGCGGTTT CGAGGGCGAG 3001 GGCTTCCCGG TGCGCCGCGC GTTCGCCGGG ATCAACTACC GCCACCTCGA CCCGTTCATC 3061 ATGATGGACC AGATGGGTGA GGTGGAGTAC GCGCCCGGGG AGCCCAAGGG CACGCCCTGG 3121 CACCCGCACC GCGGCTTCGA GACCGTGACC TACATCGTCG ACACTAGTGG ATCCAGCGGC 3181 CGCACCTGCA GGCCGGCCGT TAACACGCGT CCGCGGTCTA GACTCGAGGA TTCCAGATCT 3241 GGTACCGGGC CGCTGTATGG ATATTTGCAG GGCCGCAGAA TTCTCTCTAT CACTGATAGG 3301 GAGGTCTCTA TCACTGATAG GGAGTTCTCT ATCACTGATA GGGATGTCTC TATCACTGAT 3361 AGGGATTTCT CTATCACTGA TAGGGAAGTC TCTATCACTG ATAGGGACCT CTCTATCACT 3421 GATAGGGAAA TCTCTATCAC TGATAGGGAT CTCTCTATCA CTGATAGGGA CTTCTCTATC 3481 ACTGATAGGG ACGTCTCTAT CACTGATAGG GAACTCTCTA TCACTGATAG GGACATCTCT 3541 ATCACTGATA GGGACTTCTC TATCACTGAT AGGGAAGTAT GTTCTCTCTC TTCTCTTCTC 3601 TCTCTCTTTC TCGAATGTTC TCTCTCTTCT CTTCTCTCTC TCTTTCTCGA TGGCCGGGGC 3661 GCGCCAGGTT TCGACTTTCA CTTTTCTCTA TCACTGATAG GGAGTGGTAA ACTCGACTTT 3721 CACTTTTCTC TATCACTGAT AGGGAGTGGT AAACTCGACT TTCACTTTTC TCTATCACTG 3781 ATAGGGAGTG GTAAACTCGA CTTTCACTTT TCTCTATCAC TGATAGGGAG ATCCGAGCTC 3841 GTAAACTCGA CTTTCACTTT TCTCTATCAC TGATAGGGAG TGGTAAACTC GACTTTCACT 3901 TTTCTCTATC ACTGATAGGG AGTGGTAAAC TCGACTTTCA CTTTTCTCTA TCACTGATAG 3961 GGAGTGGTAA ACTCGAACGG ATCCGTCGAG GGAAAAGAGC GCCGGAGTAT AAATAGAGGC 4021 GCTTCGTCTA CGGAGCGACA ATTCAATTCA AACAAGCAAA GTGAACACGT CGCTAAGCGA 4081 AAGCTAAGCA AATAAACAAG CGCAGCTGAA CAAGCTAAAC AATCTGCAGC CATGGTAATT 4141 TTCTTTACGT ATATCAAGTG TTACGGCTCC ATTTTTCTTT AGATATTTCC AGTATAGTTT 4201 TTTATTACCA ACATTTAAAA ACAAATTTTA GAAAGCATAC TGTTGGGATT TAAATGATTT 4261 TTTTATTAAA AAGTGAGACA AAATTTTCAA TACAGTTTTA ATAATGGCAA AAGAAAATAT 4321 ACTGAAAACG TTGCATTTTC CAAGAGGAAC AAACTACAAT CAACATACTA TGTCTTGGTT 4381 TGAAGAAGAA GTTGTGACAT ATTGGGCAGT TAAAACAAGA CTATAACAGT GAGTATTATA 4441 AAAAAATTGT TAAAATAACA TATTCCTATA TATATTTATA GCATTTTAAA TAAATATTAA 4501 ACATTTATTT ATCATTAAGT TATAAGACAT ATATTCAAAT ATTGTTGTAA CAGCTGTAAA 4561 AACAAGTTAG TTAATTGTTA TTATTCAGGT TCTGGTTAAA CTCCAGGTCA TGAAATTGTG 4621 TCTCTTCTAT AAGATAAGTC CCTGGATCTT CTGGAGTAAG AGTGGTAGGA TTGGCTTATC 4681 AGTTAAACAA ATGTTCTGTA TCATGTGGTT TGACCACATA CTGGACAAAT ATTAGGGACG 4741 GCACGGTCAA TACGAGACTA GTATGCATCG AGACTATTGA TCCACCCCAA GCGTAGTTGT 4801 TCCAGAGCTT CTTTTGTAGA CCTCGGTAGA TTCTGTTCTA TAAAAACCAA GTATCATGCT 4861 TCTTACTGAT GAGAATTAAC GAGAACGTAA AGCGCCTACA CATGTTTATA ATGTATTCCC 4921 GCAAGCATAT GTATACATTG TAATCTCTGT GTGACCATTA TATTTATAAT CTTTGATATA 4981 AACCTTATTC CAGGGTACTA AAGATATATT TTAATTTTAT TTCTTTTGAA TGTCTGTTGC 5041 AAAATATGTA AATATATAAA AACTTTTATG AAAATATTAT TTAAGTAAAG ATATAAAATA 5101 GTTGAAAAAA TCTTTGAATT GTAGAAAAAT AGTCCGCTCC CCTACTTGAT GCCCATATTC 5161 GCATTCGGTG TAGTCATCTT GTTCCATGCT TTAAGCCGCT TCTCACAAGT ATTCCAGTAA 5221 GAGGTCACCC GTGTTAATTA GGCATAGCTA TCAGTTTATT TTAGCACTAT TTGATCATCT 5281 AAATCCCTGC TCCTGCTAAC TACCACCTTG TACTTAAAGT ACATAAAATT TGGTCTTCAG 5341 CATCGTTCTT CGAGGGGCGG TCATAATATT TTTTATATTT TAAGGAGTGA GATCGAACGT 5401 TTTTAAAGTG CTGTAATTTT GCTCGAATAG GTAGTACATC TCGTTTTAAA ATCTAACACT 5461 TGGAAACCTA TTTTGTGCCC TTCAATTAAT AATTATCATG AGCATAATAT CTCTCTACTG 5521 GCCTTAGCTC CGGGCTTTTT GGAGAAAAAA AGTCGGCACA TAATGAAGTC TTATAAATGA 5581 AAACAGTCTT TCTTGTAAAC GTTCCTTGAT TTATATTTAT AGAGCCTGTT GTAACAAATA 5641 ATTAGCTTCT TAAAAAGAAC TTGACTGATT TTGGGTCCTA AATTTTTCTT CGACATTCTC 5701 TTGAGCATCA GCAACATAAA ATTTTTTATT AGGTAGTTGC AAAAAAACTG CCATGATTAG 5761 TCATCCATTA TGCAACAAGA ATACTTTTTC AAGAAAACTT GATTTTAATT TCCGTACTCT 5821 GTTTTAGGCC TCTAATTTTT TGAGCAACAT TCCTGTCAAA AACTTTTTGA GCCTTGTACG 5881 TTTTTAAACC TGCATCAGCT TTAACTTTTC GTACCAAATA GTCCGACCAA TGAGCTAACC 5941 GGGCTTTTCT ACCTAATATG TTGGCAGCTC TTTTGAAAAT ATTTTGAGAC ATCATGTGAA 6001 CCATTGCTTC CACATGAACC AGTTTTCTCT GGGTACTGTT TGAAAAACAT AGGAAACAGC 6061 TTGGCGGCAA ACCTTTGAAT GCTTGGGCAA CTTTTGTGGG ACAAAGTTTT GGTTTGTTGA 6121 AAATATTTAA TAATTTATTA GGCACTTTTT TCTGGTCCCT CATTTAAATC GGATTACCAA 6181 TTTCATTTGT TTTTAATTAA ACATTATAAA TTATATGTTT TACATGTTTC ACTAAACGTG 6241 TGTTACTAAT TTTTCGATCT CACTCCTTTT ATACCGTATT ATATTAATTC TATACTGTAA 6301 TTAAAGTTAT TTTCAAATTG TTGCAATATT TATTTAGCAA AATGTTTTCA TAACGTGAAA 6361 TTGTATGTTA TTTTTAGAAA AAATGTATAT TAAGTTCTCA AATTCATATT GTTTATTCAT 6421 TTAGATTCCC TTGAACAAAA GGGGTTTGGT ATTTTGTAAA AAATTACTAT ATTATTTAAT 6481 AAGTAGTTAA GATTAATGAA TTTCAGTGAA TAATAAAAGC TCAACAATCA ACATACTAAC 6541 ATTTTGAAGA TCAGCAATAT TAATCTATCA ACACTAATTA TAATTAACGA CAATCAACAT 6601 ACCATAGAAA AAAGGATAAT GGATAATGAA TACAAAACTA CAATCAACAT TTTCCTCAGG 6661 GCAACACACA TCTAGGTTTT GCAAGGATCA ACAAATCAAG AGTGCATTAA AAATAACAAC 6721 AATCAACATA CCATAATTGA AGATGTTGCA AATATTGAAA ATTTTTATTA AAAACATTTA 6781 AAATTTACTT AAATTTTTCC TTTAAACGCA AATAAAAAGA AAACTTAAAT TATTCTATTG 6841 CAAACAGAAA AAATCCCAAA TTAAAATTTA TTTAAAAATT ATTTTTGTTA TAAAACAAAT 6901 CTAAAATCTA TTTAATTTTA AAAATAATTA AAAAAAAACA TAAACGTGTT AAAACAATTT 6961 CACAGCTTAA AAATATCGAT AAAAAATATA TAATTTTTAA TAATTTATTT TAATTAATCA 7021 TCTTTATCAA CATACAAAAT GATAGATAGA TTTTAAAAGG ATCGAGGTTG CATGTATGAT 7081 AAATTTATTA TTCTTTTCTA TGTTTAGGTC AGCCGTTTGG ATAAATCCAA AGTTATTAAT 7141 TCCGCTTTGG AATTGTTGAA TGAAGTTGGT ATTGAAGGTT TGACAACACG TAAATTGGCT 7201 CAAAAATTGG GTGTTGAACA ACCAACATTG TATTGGCATG TTAAAAATAA ACGTGCTTTG 7261 TTGGATGCTT TGGCTATTGA AATGTTGGAC CGTCATCATA CACATTTTTG CCCATTGGAA 7321 GGCGAATCCT GGCAAGATTT CTTGCGTAAT AATGCCAAAT CCTTCCGTTG TGCTTTGTTG 7381 TCCCATCGTG ATGGTGCCAA GGTTCATTTG GGCACACGTC CAACAGAAAA ACAATATGAA 7441 ACATTGGAAA ATCAATTGGC TTTCTTGTGT CAACAAGGCT TCAGCTTGGA AAATGCTTTG 7501 TATGCTTTGA GCGCCGTTGG TCATTTTACA TTGGGCTGTG TGTTGGAAGA TCAAGAACAT 7561 CAAGTCGCTA AAGAAGAACG TGAAACACCA ACAACAGATT CGATGCCCCC ATTGTTGCGT 7621 CAAGCAATTG AATTGTTCGA TCATCAAGGA GCCGAACCAG CATTCTTGTT CGGCTTGGAA 7681 TTGATTATTT GTGGATTGGA AAAACAATTG AAATGTGAAT CGGGCTCGGG CCCCGCGTAC 7741 AGCCGCGCGC GTACGAAAAA CAATTACGGG TCTACCATCG AGGGCCTGCT CGATCTCCCG 7801 GACGACGACG CCCCCGAAGA GGCGGGGCTG GCGGCTCCGC GCCTGTCCTT TCTCCCCGCG 7861 GGACACACGC GCAGACTGTC GACGGCCCCC CCGACCGATG TCAGCCTGGG GGACGAGCTC 7921 CACTTAGACG GCGAGGACGT GGCGATGGCG CATGCCGACG CGCTAGACGA TTTCGATCTG 7981 GACATGTTGG GGGACGGGGA TTCCCCGGGT CCGGGATTTA CCCCCCACGA CTCCGCCCCC 8041 TACGGCGCTC TGGATATGGC CGACTTCGAG TTTGAGCAGA TGTTTACCGA TGCCCTTGGA 8101 ATTGACGAGT ACGGTGGGTA GTAAGCTTGG ATCTTTGTGA AGGAACCTTA CTTCTGTGGT 8161 GTGACATAAT TGGACAAACT ACCTACAGAG ATTTAAAGCT CTAAGGTAAA TATAAAATTT 8221 TTAAGTGTAT AATGTGTTAA ACTACTGATT CTAATTGTTT GTGTATTTTA GATTCCAACC 8281 TATGGAACTG ATGAATGGGA GCAGTGGTGG AATGCCTTTA ATGAGGAAAA CCTGTTTTGC 8341 TCAGAAGAAA TGCCATCTAG TGATGATGAG GCTACTGCTG ACTCTCAACA TTCTACTCCT 8401 CCAAAAAAGA AGAGAAAGGT AGAAGACCCC AAGGACTTTC CTTCAGAATT GCTAAGTTTT 8461 TTGAGTCATG CTGTGTTTAG TAATAGAACT CTTGCTTGCT TTGCTATTTA CACCACAAAG 8521 GAAAAAGCTG CACTGCTATA CAAGAAAATT ATGGAAAAAT ATTCTGTAAC CTTTATAAGT 8581 AGGCATAACA GTTATAATCA TAACATACTG TTTTTTCTTA CTCCACACAG GCATAGAGTG 8641 TCTGCTATTA ATAACTATGC TCAAAAATTG TGTACCTTTA GCTTTTTAAT TTGTAAAGGG 8701 GTTAATAAGG AATATTTGAT GTATAGTGCC TTGACTAGAG ATCATAATCA GCCATACCAC 8761 ATTTGTAGAG GTTTTACTTG CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA 8821 TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA 8881 AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG 8941 TTTGTCCAAA CTCATCAATG TATCTTATCA TGTCTCGAGC ATGCGCAAAT TTAAAGCGCT 9001 GATATCGATC GCGCGCAGAT CTGTCATGAT GATCATTGCA ATTCTGCAGT CGACGGTACC 9061 CGATCTTGTC GCCGGAACGC AGCGACAGAG ATTCCAATGT GTCCGTATCT TTCAGGCTTT 9121 TGCCCTTCAG TTCCAGACGA AGCGACTGGC GATTCGCGTG TGGGGTCTGC TTCAGGGTCT 9181 TGTGAATTAG GGCGCGCAGA TCGCCGATGG GCGTGGCGCC GGAGGGCACC TTCACCTTGC 9241 CGTACGGCTT GCTGTTCTTC GCGTTCAAAA TCTCCAGCTC CATTTTGCTT TCGGTGCGCT 9301 TGCAATCAGT ACTGTCCAAA ATCGAAAATC GCCGAACCGT AGTGTGACCG TGCGGGGCTC 9361 TGCGAAAATA AACTTTTTTA GGTATATGGC CACACACGGG GAAAGCACAG TGGATTATAT 9421 GTTTTAATAT TATAATATGC AGGTTTTCAT TACTTATCCA GATGTAAGCC CACTTAAAGC 9481 GATTTAACAA TTATTTGCCG AAAGAGTAAA AACAAATTTC ACTTAAAAAT GGATTAAGAA 9541 AAGCTTGTGT AAGATTATGC GCAGCGTTGC CAGATAGCTC CATTTAAAAC ACTTCAAAAA 9601 CAATAAGTTT TGAAAATATA TACATAAATA GCAGTCGTTG CCGCAACGCT CAACACATCA 9661 CACTTTTAAA ACACCCTTTA CCTACACAGA ATTACTTTTT AAATTTCCAG TCAAGCTGCG 9721 AGTTTCAAAA TTATAGCCGG TAGAGAAGAC AGTGCTATTT CAAAAGCAAA CTAAATAAAC 9781 ACCAATCCTA ACAAGCCTTG GACTTTTGTA AGTTTAGATC AAAGGTGGCA TTGCATTCAA 9841 TGTCATGGTA AGAAGTAGGT CGTCTAGGTA GAAATCCTCA TTCAGCCGGT CAAGTCAGTA 9901 CGAGAAAGGT CTCAATTTGA AATTGTCTTA AAAATATTTT ATTGTTTTGT ACTGTGGTGA 9961 GTTTAAACGA AAAACACAAA AAAAAAGTGA TACACAGAAA TCATAAAAAA TTTTAATACA 10021 AGGTATTCGT ACGTATCAAA AACATTTCGG CACAATTTTT TTTCTCTGTA CTAAAGTGTT 10081 ACGAACACTA CGGTATTTTT TAGTGATTTT CAACGGACAC CGAAGGTATA TAAACAGCGT 10141 TCGCGAACGG TCGCCTTCAA AACCAATTGA CATTTGCAGC AGCAAGTACA AGCAGAAAGT 10201 AAAGCGCAAT CAGCGAAAAA TTTATACTTA ATTGTTGGTG ATTAAAGTAC AATTAAAAGA 10261 ACATTCTCGA AAGTCACAAG AAACGTAAGT TTTTAACTCG CTGTTACCAA TTAGTAATAA 10321 GAGCAACAAG ACGTTGAGTA ATTTCAAGAA AAACTGCATT TCAAGGTCTT TGTTCGGCCA 10381 TTTTTTTTTT ATTCAACGCT CTACGTAATT ACAAAATAAG AAATTGGCAG CCACGCATCT 10441 TGTTTTCCCA ATCAATTGGC ATCAAAACGC AAACAAATCT ATAAATAAAA CTTGCGTGTT 10501 GATTTTCGCC AAGATTTATT GGCAAATTGT GAAATTCGCA GTGACGCATT TGAAAATTCG 10561 AGAAATCACG AACGCACTCG AGCATTTGTG TGCATGTTAT TAGTTAGTTA GTTCTTTGCT 10621 TAATTGAAGT ATTTTACCAA CGAAATCCAC TTATTTTTAG CTGAAATAGA GTAGGTTGCT 10681 TGAAACGAAA GCCACGTCTG GAAAATTTCT TATTGCTTAG TAGTTGTGAC GTCACCATAT 10741 ACACACAAAA TAATGTGTAT GCATGCGTTT CAGCTGTGTA TATATACATG CACACACTCG 10801 CATTATGAAA ACGATGACGA GCAACGGAAC AGGTTTCTCA ACTACCTTTG TTCCTGTTTC 10861 TTCGCTTTCC TTTGTTCCAA TATTCGTAGA GGGTTAATAG GGGTTTCTCA ACAAAGTTGG 10921 CGTCGATAAA TAAGTTTCCC ATTTTTATTC CCCAGCCAGG AAGTTAGTTT CAATAGTTTT 10981 GTAATTTCAA CGAAACTCAT TTGATTTCGT ACTAATTTTC CACATCTCTA TTTTGACCCG 11041 CAGAATAATC CAAAATGCAG ATCGGGGATC CCACCCCACC CAAGAAGAAG CGCAAGGTGG 11101 AGGACGATCC CGTCGTTTTA CAACGTCGTG ACTGGGAAAA CCCTGGCGTT ACCCAACTTA 11161 ATCGCCTTGC AGCACATCCC CCTTTCGCCA GCTGGCGTAA TAGCGAAGAG GCCCGCACCG 11221 ATCGCCCTTC CCAACAGTTG CGGTCGACTC TAGAGGATCC CCGGGATCCA CCGGTCGCCA 11281 CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG GTCGAGCTGG 11341 ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA GGGCGAGGGC GATGCCACCT 11401 ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG CCCTGGCCCA 11461 CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAG CCGCTACCCC GACCACATGA 11521 AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG CGCACCATCT 11581 TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT GAAGTTCGAG GGCGACACCC 11641 TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC ATCCTGGGGC 11701 ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT CATGGCCGAC AAGCAGAAGA 11761 ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGACGGCAGC GTGCAGCTCG 11821 CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG CCCGACAACC 11881 ACTACCTGAG CACCCAGTCC GCCCTGAGCA AAGACCCCAA CGAGAAGCGC GATCACATGG 11941 TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG CATGGACGAG CTGTACAAGT 12001 AAAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG TTTTACTTGC 12061 TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG CAATTGTTGT 12121 TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT 12181 CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT 12241 ATCTTAAAGC TTATCGATAC GCGTACGGCA CTAGAGCGGC CGCCACCGCG GTGGAGCTCC 12301 AGCTTTTGTT CCCTTTAGTG AGGGTTAATT AGATCGGCCG GCCTTGGCGC GCCTAGATCT 12361 TAATACGACT CACTATAGGG CGAATTGGGT ACCG

TABLE-US-00004 primer SEQ ID NO: 3 GCCGCAGAAT TCTCTCTATC primer SEQ ID NO: 4 CTTAGCTTTC GCTTAGCGAC G primer SEQ ID NO: 5 TGCAGGTGAC CTGGGAATAG primer SEQ ID NO: 6 GTGAGACCAC TTGACCACAG primer SEQ ID NO: 7 CGCGACGATA GACAGCGG primer SEQ ID NO: 8 GAGAGCAATG CGCTCGTTGC