Controlled transcription of polynucleotides

Abstract

Compositions and methods are provided for the transcription of a polynucleotide sequence encoding a polypeptide in a cell. The cells and methods provided are useful for controlling the expression of polypeptides for a wide variety of purposes.

Description

[Technical field]

[0001] This application claims priority to U.S. Application No. 63 / 256,831, filed October 18, 2021, which is incorporated by reference in its entirety. The invention described herein provides, inter alia, cells, cell cultures, polynucleotide and polypeptide constructs, systems and methods for controlling the transcription of one or more polynucleotide sequences of interest. The invention described herein further provides stable cell lines in which the transcription of at least one polynucleotide (particularly polydeoxyribonucleotide) sequence of interest can be tightly controlled to control the expression of a polypeptide. If RNA is not transcribed from DNA, a polypeptide cannot be translated from RNA, allowing the control of protein expression by controlling transcription.

[0002] Reference to Electronic Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated by reference herein in its entirety. The .XML copy, created on October 5, 2022, is named "135975--66502.xml" and is 98,737 bytes in size. The Sequence Listing contained in this .XML file is a part of this specification and is incorporated by reference herein in its entirety. [Background technology]

[0003] Various methods for the controlled transcription of a polynucleotide sequence of interest in cells are known in the art and are described in U.S. Patent No. 9,469,856.For example, No et al., Proc. Natl. Acad. Sci. USA 93:3346-3351 (1996) report a controllable gene expression system that utilizes a chimeric transactivator consisting of the ecdysone nuclear receptor fused to the VP16 transactivation domain from herpes simplex virus.Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992) report a single system for controlling the transcription of a polynucleotide sequence of interest based on a chimeric protein consisting of the tetracycline repressor protein fused to the VP16 transactivation domain. Gossen et al., Science 268:1766-69 (1995) report a fusion of the VP16 activation domain with a mutated "reverse" Tet repressor that requires tetracycline for induction. Tetracycline-inducible gene expression is discussed in Ortiz and Johnson, Molec. Biochem. Parasitology 128:43-40 (2003). Other reported single-regulatory systems are cited in U.S. Patent No. 9,469,856 and include, for example, Sadowski et al., Nature 335:563-564 (1988), Brent et al., Cell 40:729-736 (1985), and Labow et al., Mol. Cell. Biol. 10:3342-3356 (1990).

[0004] Problems due to transcriptional leakage associated with sole reliance on minimal promoters have led to systems that use fusions of the steroid-binding domains of glucocorticoid or estrogen nuclear receptors. See, e.g., Mattioni et al., Methods Cell Biol. 43:335-352 (1994); Louvion et al. Gene 131:129-134 (1993); Iida et al. J. Virol. 70:6054-6059 (1996).

[0005] Further improvements in regulated expression systems are described and claimed in U.S. Patent No. 9,469,856. However, ever tighter control of polynucleotide transcription and polypeptide expression is desired. Summary of the Invention

[0006] The present invention advantageously includes and utilizes regulatory fusion proteins (RFPs) (which can act as activators or repressors) and repressor proteins (which act as repressors), such as antibiotic repressors, and operators arranged in tandem to control the transcription of at least one polynucleotide of interest. Transcription of a single polynucleotide of interest can be controlled according to the present invention, or multiple polynucleotides in an operon-like arrangement can be controlled according to the present invention.

[0007] As used herein, a first RFP can be bound to a first operator, which can be located 5' of the promoter and the polynucleotide of interest to be transcribed. A second RFP or repressor protein can be bound to a second operator. The second operator can be located 3' of the promoter but 5' of the polynucleotide to be transcribed. In some embodiments, a second operator can be located 3' of the promoter but 5' of the polynucleotide to be transcribed, and a separate second operator can optionally be operably linked to the polynucleotide encoding the first RFP or repressor protein.

[0008] The description of aspects and embodiments of the present invention provides a method for controlling transcription of a polynucleotide of interest in a cell, the method comprising: (I) maintaining the cell in a medium that does not have an effective amount of a ligand of both an activator and a repressor, the cell comprising: (A) a promoter, the promoter being operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, the transcription of the polynucleotide of interest being inhibited in the absence of the ligand of both the activator and the repressor; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of a ligand of both the activator and the repressor. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The activator can bind to the first operator in the presence of the ligand to enable transcription of the polynucleotide of interest. The repressor can be a repressor protein, such as an antibiotic repressor, where transcription of the polynucleotide of interest is inhibited in the absence of a ligand and transcription is permitted in the presence of a ligand. The repressor protein can bind to a second operator in the absence of a ligand. The activator can be a regulatory fusion protein (RFP). The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator. The repressor protein can be an antibiotic repressor, such as a tetracycline repressor.

[0009] Further provided is a method for controlling transcription of a polynucleotide of interest in a cell, the method comprising: (I) maintaining the cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of a ligand of a repressor (repressor ligand), the cell comprising: (A) a promoter, the promoter being operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter, (B) a polynucleotide encoding an activator, (C) a second operator, and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of the activator ligand and in the presence of the repressor ligand, and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The activator can bind to the first operator in the presence of an activator ligand to allow transcription of the polynucleotide of interest. The activator can be a regulatory fusion protein (RFP). The repressor can be a regulatory fusion protein (RFP), where transcription of the polynucleotide of interest is inhibited in the presence of the repressor ligand and transcription is allowed in the absence of the repressor ligand. The activator RFP can be a reverse tetracycline transactivator. The activator ligand can be selected from the group consisting of tetracycline and doxycycline. The repressor RFP can be ArcEr, where the repressor RFP ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, and 4-hydroxytamoxifen (OHT).

[0010] Also provided is a method for controlling transcription of a polynucleotide of interest in a cell culture, the method comprising: I. maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, the cell comprising: (A) a promoter, the promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding the first RFP, the first RFP comprising: (1) a transcriptional activation domain fused to a first DNA binding domain, and (2) a ligand binding domain, the first ligand capable of binding to the ligand binding domain of the first RFP, the DNA binding domain of the first RFP being activated in response to the presence of the first ligand. (C) a second operator; and (D) a polynucleotide encoding a second RFP different from the first RFP, the second RFP comprising (1) a second DNA binding domain and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, and the second RFP being capable of binding to the second operator in the presence of the second ligand, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand; and (II) controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the first ligand and not having an effective amount of the second ligand. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide sequence encoding the protein of interest. A separate second operator can optionally be operably linked to the polynucleotide sequence encoding the first RFP. The first RFP as an activator can be a reverse tetracycline transactivator (rtTA).The second RFP as a repressor can comprise an Arc repressor binding domain fused to an estrogen receptor ligand binding domain (ArcEr). The first operator can be a Tet response element (TRE). The second operator can be an Arc operator (AO). The cell can further comprise a repressor that is altered by the first ligand. The repressor can be a tet repressor protein (TetR). Additionally, the polynucleotide encoding the first RFP can be operably linked to a promoter and optionally a second Arc operator. The promoter can be a CMV promoter, such as CMVmin. ArcEr can control the transcription of the polynucleotide encoding rtTA.

[0011] Also provided is a method for controlling transcription of a polynucleotide of interest in a cell culture, the method comprising: (I) maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and an effective amount of a second ligand of a second RFP, wherein the cell is adapted to express a polynucleotide of interest comprising: (A) a promoter, the promoter being controlled by a Tet response element (TRE) operably linked to the polynucleotide of interest and operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding the first RFP, the first RFP comprising: (1) a transcriptional activation domain fused to a DNA binding domain, and (2) a ligand binding domain, wherein the first ligand is capable of binding to the ligand binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to a 5'-positioned operator when in the presence of the first ligand. (C) an Arc operator operably linked and positioned 3' to the promoter and 5' to the polynucleotide encoding the protein of interest; and (D) a polynucleotide encoding a second RFP, the second RFP comprising (1) an Arc repressor DNA binding domain and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the Arc operator in the presence of the second ligand, wherein transcription of the polynucleotide encoding the protein of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand; and (II) controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the first ligand and not having an effective amount of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline, and derivatives thereof. The second ligand may be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof.The ligand binding domain of the second RFP can be a ligand binding domain of a steroid receptor. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be an ArcER with an Arc repressor binding domain fused to an estrogen receptor ligand binding domain (Arc is a repressor from phage P22). The promoter operably linked to the polynucleotide sequence encoding the polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. The CMV promoter and the Arc operator can optionally be operably linked to the polynucleotide encoding the first RFP. The SV40E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP.

[0012] Further provided is a method for controlling transcription of a polynucleotide of interest in a cell, the method comprising: (I) maintaining at least one cell in a medium that does not have an effective amount of a regulatory fusion protein (RFP) and a ligand of a repressor protein, the cell comprising: (A) a promoter, the promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding the RFP, the RFP comprising (1) a transcriptional activation domain fused to a DNA binding domain, and (2) a ligand binding domain, the ligand binding to the RFP. (C) a second operator; and (D) a polynucleotide encoding a repressor protein, the repressor protein being capable of binding to the second operator only in the absence of the ligand, wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the ligand; and (II) controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the ligand. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The repressor protein can bind to the second operator in the absence of the ligand to inhibit transcription of the polynucleotide of interest. The RFP can bind to the first operator in the presence of the ligand to allow transcription of the polynucleotide of interest. The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator (rtTA). The repressor protein can be a tetracycline repressor (TetR). The first operator can be a Tet response element (TRE). The second operator can be a Tet operator.

[0013] Additionally provided is a method for controlling transcription of a polynucleotide of interest in a cell culture, the method comprising: (I) maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, the cell being adapted to culture a medium containing: (A) a promoter, the promoter being operably linked to a polynucleotide of interest and controlled by a Tet response element (TRE) operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding the first RFP, the first RFP comprising: (1) a transcriptional activation domain fused to a DNA binding domain, and (2) a ligand binding domain, the first ligand being capable of binding to the ligand binding domain of the first RFP, the DNA binding domain of the first RFP being positioned 5' when in the presence of the first ligand. (C) a Tet operator operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest, and (D) a polynucleotide encoding a second RFP, the second RFP comprising (1) an Arc repressor DNA binding domain, and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the Arc operator in the presence of the second ligand, wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand, and (II) controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the first ligand and not having an effective amount of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline, and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof. The ligand-binding domain of the second RFP can be the ligand-binding domain of a steroid receptor.The first regulatory fusion protein (RFP) as an activator can be reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding the polypeptide of interest can be a CMV promoter, such as CMVmin. The CMV promoter and Arc operator can optionally be operably linked to the polynucleotide encoding the first RFP. The SV40E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP. The cell can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR.

[0014] Also provided is a method for controlling transcription of a polynucleotide of interest in a cell culture, the method comprising maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and an effective amount of a second ligand of a second RFP, the cell comprising (A) a promoter, (B) an Arc operator, and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B), and (C) are operably linked, the transcription of the rtTA polynucleotide being controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr), the rtTA being capable of controlling the transcription of the polynucleotide of interest. The promoter can be a CMV promoter, such as CMVmin. The first ligand can be selected from the group consisting of tetracycline and doxycycline, and derivatives thereof. The second ligand may be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof.

[0015] The present invention also provides a cell that allows for controlled transcription of at least one polynucleotide of interest, the cell comprising: (A) a promoter, the promoter being operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of a ligand of both the activator and the repressor and permitted in the presence of a ligand of both the activator and the repressor. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The activator can bind to the first operator in the presence of the ligand to permit transcription of the polynucleotide of interest. The repressor can be a repressor protein, wherein transcription of the polynucleotide of interest is inhibited in the absence of the ligand and transcription is permitted in the presence of the ligand. The repressor protein can bind to the second operator in the absence of the ligand. The activator can be a regulatory fusion protein (RFP). The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator. The repressor protein can be a tetracycline repressor.

[0016] Additionally, a cell is provided that allows for controlled transcription of at least one polynucleotide of interest, the cell comprising: (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding a first regulatory fusion protein (RFP), the first RFP comprising (1) a transcription activation domain fused to a first DNA binding domain, and (2) a ligand binding domain, wherein the first ligand is capable of binding to the ligand binding domain of the first RFP, and the DNA binding domain of the first RFP is capable of activating a transcription factor in the presence of the first ligand. (C) a second operator; and (D) a polynucleotide encoding a second RFP different from the first RFP, the second RFP comprising (1) a second DNA binding domain and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the second operator in the presence of the second ligand, and wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand and is permitted in the presence of the first ligand and in the absence of the second ligand. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide sequence encoding the protein of interest. The second operator can optionally be operably linked to the polynucleotide sequence encoding the first RFP. The cell can include a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR. The polynucleotide (B) encoding the first RFP can be operably linked to a promoter and a second Arc operator. The promoter can be a CMV promoter, such as CMVmin.The first RFP as an activator can be a reverse tetracycline transactivator fusion protein (rtTA), and the second RFP as a repressor can be a fusion protein containing an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr). ArcEr can control the transcription of a polynucleotide encoding rtTA.

[0017] Further provided is a cell that allows for controlled transcription of a polynucleotide of interest, the cell comprising: (A) a promoter, the promoter being operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of an activator ligand and in the presence of an effective amount of a repressor ligand, and is allowed in the presence of an effective amount of an activator ligand and in the absence of an effective amount of a repressor ligand. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The activator can bind to the first operator in the presence of the activator ligand to allow transcription of the polynucleotide of interest. The activator can be a regulatory fusion protein (RFP). The repressor can be a regulatory fusion protein (RFP), where transcription of the polynucleotide of interest is inhibited in the presence of the repressor ligand and transcription is permitted in the absence of the repressor ligand. The activator RFP can be a reverse tetracycline transactivator. The activator ligand can be selected from the group consisting of tetracycline and doxycycline. The repressor RFP can be ArcEr. The repressor ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, and 4-hydroxytamoxifen (OHT).

[0018] Also provided is a cell that allows for controlled transcription of at least one polynucleotide of interest, the cell comprising: (A) a promoter that is operably linked to a polynucleotide of interest and that is controlled by a Tet response element (TRE) operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), the first RFP comprising (1) a transcriptional activation domain fused to a DNA binding domain, and (2) a ligand binding domain, wherein the first ligand is capable of binding to the ligand binding domain of the first RFP, and the DNA binding domain of the first RFP is capable of binding to a 5'-located operator when in the presence of the first ligand; and (C) an Arc operator operably linked and positioned 3' to the promoter and 5' to the polynucleotide encoding a protein of interest. (D) a polynucleotide encoding a second RFP, the second RFP comprising (1) an Arc repressor DNA binding domain and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the Arc operator in the presence of the second ligand, and wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand, and is permitted in the presence of the first ligand and in the absence of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline, and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof. The ligand binding domain of the second RFP can be the ligand binding domain of a steroid receptor. The first regulatory fusion protein (RFP) as an activator can be reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding the polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. The CMV promoter and Arc operator can optionally be operably linked to the polynucleotide encoding the first RFP. The SV40E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP.

[0019] Additionally, a cell is provided that allows for controlled transcription of a polynucleotide of interest, the cell comprising: (A) a promoter, the promoter being operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding a regulatory fusion protein (RFP), the RFP comprising (1) a transcription activation domain fused to a DNA binding domain, and (2) a ligand binding domain, the ligand being capable of binding to the ligand binding domain of the RFP, the DNA binding domain of the RFP being capable of binding to the first operator when in the presence of the ligand; (C) a second operator; and (D) a polynucleotide encoding a repressor protein, the polynucleotide being capable of binding to the second operator only in the absence of the ligand, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of a ligand for both the activator and the repressor, and is permitted in the presence of an effective amount of a ligand for both the activator and the repressor. The second operator can be operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest. The repressor protein can bind to the second operator in the absence of a ligand to inhibit transcription of the polynucleotide of interest. The RFP can bind to the first operator in the presence of a ligand to allow transcription of the polynucleotide of interest. The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator (rtTA). The repressor protein can be a tetracycline repressor (TetR). The first operator can be a Tet response element (TRE). The second operator can be a Tet operator.

[0020] Additionally, a cell is provided that allows for controlled transcription of at least one polynucleotide of interest, the cell comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a Tet response element (TRE) operably linked and positioned 5' to the promoter; and (B) a polynucleotide encoding a first regulatory fusion protein (first RFP), the first RFP comprising (1) a transcription activation domain fused to a DNA binding domain, and (2) a ligand binding domain, wherein the first ligand is capable of binding to the ligand binding domain of the first RFP, and the DNA binding domain of the first RFP binds to the 5'-positioned TRE when in the presence of the first ligand. (C) a Tet operator operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest; and (D) a polynucleotide encoding a second RFP, the second RFP comprising (1) an Arc repressor DNA binding domain and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the Arc operator in the presence of the second ligand, wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand and permitted in the presence of the first ligand and in the absence of the second ligand. The first ligand may be selected from the group consisting of tetracycline, doxycycline, and derivatives thereof. The second ligand may be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof. The first regulatory fusion protein (RFP) as an activator can be reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding the polypeptide of interest can be a CMV promoter, such as CMVmin.The CMV promoter and Arc operator can optionally be operably linked to a polynucleotide encoding a first RFP. The SV40E / L promoter, or other constitutive promoter, can be operably linked to a polynucleotide encoding a second RFP. The cell can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR.

[0021] Additionally, a cell is provided that allows for the regulated transcription of at least one polynucleotide of interest when present, the cell comprising: (A) a polynucleotide sequence encoding a first regulatory fusion protein (first RFP), the first RFP comprising: (1) a transcriptional activation domain fused to a DNA binding domain; and (2) a ligand binding domain, wherein the first ligand is capable of binding to the ligand binding domain of the first RFP, and the DNA binding domain of the first RFP is capable of binding to a 5'-located operator when in the presence of the first ligand. The polynucleotide comprises: (A) a polynucleotide encoding a second regulatory fusion protein (second RFP), the second RFP comprising: (1) a DNA binding domain comprising an Arc repressor DNA binding domain; and (2) a ligand binding domain, the second ligand being capable of binding to the ligand binding domain of the second RFP, the second RFP being capable of binding to the Arc operator in the presence of the second ligand; and (C) one or more insertion sites for a polynucleotide of interest operably linked to a promoter and at least one operator. The first ligand can be selected from the group consisting of tetracycline, doxycycline, and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof. The first regulatory fusion protein (RFP) as an activator can be reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding the polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. The CMV promoter and Arc operator can optionally be operably linked to the polynucleotide encoding the first RFP. The SV40E / L promoter or other constitutive promoter can be operably linked to the polynucleotide encoding the second RFP.The cell can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR.

[0022] Also provided is a cell capable of controlling the transcription of a polynucleotide of interest, the cell comprising: (A) a promoter; (B) an Arc operator; and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B), and (C) are operably linked, and the transcription of the rtTA polynucleotide is controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr), and the rtTA is capable of controlling the transcription of the polynucleotide of interest. The promoter can be a CMV promoter, such as CMVmin. The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and derivatives thereof.

[0023] Also provided are master cell banks, working cell banks, developmental cell banks, cell cultures, seed cultures, and production cultures comprising cells according to the invention, as well as bioreactors and fermenters comprising cell cultures comprising cells according to the invention as described herein.

[0024] Aspects of the embodiment include maintaining the cells in the absence of the first ligand and in the presence of the second ligand, or alternatively in the presence of the first ligand as well. Under these maintenance conditions, the percentage of cells containing copies of the DNA polynucleotide sequence encoding the polypeptide of interest is reduced by less than about 5%. Under the same maintenance conditions, expression of the polypeptide of interest can be at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or at least 95% less than expression of the polypeptide in the cells in the presence of the first ligand and in the absence of the second ligand after a period of time sufficient to cause disappearance of the previously present second ligand (usually about 4-14 days depending on the second ligand and culture conditions). Under the same maintenance conditions, the number of RNA copies encoding the polypeptide of interest can be at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, or at least 95% less than the number of RNA copies encoding the polypeptide in the cells in the presence of the first ligand and after a period of time sufficient to allow the second ligand to disappear (usually about 4-14 days, depending on the second ligand and culture conditions), in the absence of the previously present second ligand.

[0025] In certain embodiments, transcription of a polynucleotide sequence encoding a first regulatory fusion protein (first RFP) is inhibited in the presence of a second ligand, such as OHT, and a second RFP, such as ArcER.

[0026] In certain embodiments, the promoter operably linked to the polynucleotide sequence encoding the protein of interest is a CMV promoter. The promoter may be a CMV min promoter.

[0027] In certain embodiments, the polynucleotide sequence of interest encodes a polypeptide and / or product of interest. The polynucleotide sequence of interest can encode a polypeptide of interest. The polypeptide of interest can be a protein that is toxic or inhibitory to a cell, such as a viral protein.

[0028] In certain embodiments, the cells retain the ability to transcribe a polynucleotide of interest in the presence of a first ligand and in the absence of a second ligand after being frozen and thawed at least once, at least twice, at least three times, or at least four times.

[0029] In certain embodiments, the cell culture has a cell density of at least 400,000 to 1 million viable cells per ml while in an inhibited state in the presence of the second ligand. The cell culture can have a cell density of at least 600,000 to 2 million viable cells per ml while in an induced state in the presence of the first ligand and in the absence of the second ligand. In further embodiments, the cells are grown in a research or production bioreactor having a volume of, for example, at least 2 liters, at least 5 liters, at least 10 liters, 50 liters, at least 75 liters, at least 100 liters, at least 150 liters, at least 200 liters, at least 500 liters, at least 1,000 liters, at least 2,000 liters, at least 5,000 liters, at least 10,000 liters, at least 15,000 liters, at least 20,000 liters, or more.

[0030] In certain embodiments, the cell is a mammalian cell, such as a primate cell, a canine cell, or a rodent cell. In more specific embodiments, the cell is a CHO cell, such as a CHO-K1 cell, a BHK cell, a human amniotic cell, or a HEK293 cell.

[0031] In certain embodiments, in the absence of the first ligand and in the presence of the second ligand, the transcription of the polynucleotide sequence encoding the polypeptide of interest is substantially reduced. For example, at least a 10-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand. In certain embodiments, at least a 20-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand. In certain embodiments, at least a 50-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand. In certain embodiments, at least a 100-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand. In certain embodiments, at least a 500-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand.

[0032] In certain embodiments, the degree of transcription of the polynucleotide sequence of interest achieved in the presence of the first ligand and in the absence of the second ligand may be at least 10 times higher than in the absence of the first ligand and in the presence of the second ligand. In certain embodiments, the degree of transcription of the polynucleotide sequence of interest achieved in the presence of the first ligand and in the absence of the second ligand may be at least 20 times higher than in the absence of the first ligand and in the presence of the second ligand. In certain embodiments, the degree of transcription of the polynucleotide sequence of interest achieved in the presence of the first ligand and in the absence of the second ligand may be at least 50 times higher than in the absence of the first ligand and in the presence of the second ligand. In certain embodiments, the degree of transcription of the polynucleotide sequence of interest achieved in the presence of the first ligand and in the absence of the second ligand may be at least 100 times higher than in the absence of the first ligand and in the presence of the second ligand. In certain embodiments, the degree of transcription of a polynucleotide sequence of interest achieved in the presence of a first ligand and in the absence of a second ligand may be at least 500-fold higher than in the absence of the first ligand and in the presence of the second ligand.

[0033] Other embodiments, aspects, objects, and advantages will become apparent from review of the following detailed description. [Brief description of the drawings]

[0034] [Figure 1] Induction of transcription of a gene of interest (in this example, a polynucleotide encoding a crimson fluorescent protein) in the presence of dox and in the absence of OHT is shown. The left side shows the repressed state in the absence of dox and the presence of OHT. The right side shows the induced state in the presence of dox and the absence of OHT. The diagram shows an example of a tandem arrangement of a Tet response element (TRE) and an Arc operator (AO).

[0035] [Diagram 2]The results of transcription of a gene of interest (GOI) (in this example, a polynucleotide encoding a crimson fluorescent protein) in the presence or absence of a ligand are shown. See FIG. 1. A polynucleotide encoding a crimson fluorescent protein was transcribed under the control of rtTA bound to ArcER (TRE-AO) or under the control of CMV-TO. In the presence of E2 and in the absence of Dox, a very low level of transcription of the GOI (crimson) is observed compared to the control (negative, unmodified cells) and CMV-TO (TRE-TO suppression (+E2 / -Dox). In the presence of Dox and in the absence of E2, a high level of transcription of the GOI (crimson) is observed (TRE-AO induction).

[0036] [Diagram 3] Optional embodiments are shown in which expression of a regulatory fusion protein, such as rtTA, or a repressor protein, can be regulated by a second RFP and associated elements such as ArcER, AO, and OHT.

[0037] [Figure 4] This shows an embodiment in which the polynucleotide encoding the GOI, crimson fluorescent protein, was under the control of rtTA and TetR. The Tet response element (TRE) and the Tet operator (TetO) are in tandem arrangement. Induction of transcription of the gene of interest (in this example, the polynucleotide encoding the crimson fluorescent protein) occurs in the presence of dox and in the absence of a ligand such as E2 or OHT, thereby allowing the rtTA fusion protein to be expressed. See FIG. 3 for optional regulated expression of a regulatory fusion protein such as rtTA.

[0038] [Diagram 5]The results of transcription of a gene of interest (GOI) (in this example, a polynucleotide encoding crimson fluorescent protein) in the presence or absence of a ligand are shown. The transcription of the polynucleotide encoding crimson fluorescent protein was under the control of rtTA and TetR (TRE-TO). See FIG. 4. In the presence of E2 and in the absence of dox, a very low level of transcription of the GOI (crimson) is observed (TRE-TO suppression (+E2 / -Dox)), almost the same as that of the control (negative, unmodified cells). In the presence of dox and in the absence of E2, a high level of transcription of the GOI (crimson) is observed (TRE-TO induction).

[0039] [Figure 6] The control of transcription of a gene of interest (in this figure, a polynucleotide encoding a cytotoxic gene) in the absence of dox and in the presence of OHT is shown to provide a repressed state.

[0040] [Figure 7] The control of transcription of a gene of interest (in this figure, a polynucleotide encoding a cytotoxic gene) in the presence of dox and in the absence of OHT is shown to provide an induced state.

[0041] [Figure 8] Western blot of proteins produced by HEK293 cells transformed with Rep78 and Rep52 genes under strict control by TRE-AO. When HEK293 cells were in a repressed state (-) (no Dox and E2), Rep78 and Rep52 were not produced. In an induced state (+) (Dox and E2), both Rep78 and Rep52 are produced. The left column of the Western blot has size markers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] definition The term "about" in the context of numerical values ​​and ranges refers to a value or range that is approximately or very close to the recited value or range so that the invention can be performed to have the desired rate, amount, degree, increase, decrease, degree of transcription, or degree of expression, concentration, or time, as is clear from the teachings contained herein. Thus, the term encompasses values ​​beyond those that simply result from systematic error. For example, "about" can indicate either a value above or below the recited value by a range of approximately + / - 10% or more or less, depending on the ability to perform.

[0044] An "effective amount" of a compound refers to the amount of the compound necessary to bring about an intended result, and is typically defined in terms of the molar or weight concentration of the compound when present in culture. A ligand is an example of a compound.

[0045] "Capable of binding" refers to the ability of a molecule, such as a regulatory fusion protein or portion thereof, to bind to another molecule or portion thereof, such as a ligand binding domain, a nucleic acid binding domain, an operator, a response element, etc. Typically, binding can enable an action or function or block an action or function.

[0046] A "nucleic acid portion" includes any arrangement of single- or double-stranded nucleotide sequences. A nucleic acid portion can include, but is not limited to, polynucleotides, promoters, enhancers, operators, repressors, transcription termination signals, ribosome entry sites, and polyadenylation signals.

[0047] A "DNA cassette" or "cassette" is a type of nucleic acid segment that contains at least a promoter, at least one open reading frame, and optionally, a polyadenylation signal. Also, one or more operators are optional. Thus, a DNA cassette is a polynucleotide that contains two or more shorter polynucleotides. A cassette can contain one or more genes, as well as promoters, enhancers, operators, repressors, transcription termination signals, ribosome entry sites, introns, and polyadenylation signals.

[0048] "Operably linked" refers to one or more nucleotide sequences in a functional relationship with one or more other nucleotide sequences. Such a functional relationship refers to being able to directly or indirectly control, induce, generate, regulate, enhance, promote, permit, affect, attenuate, stop, prevent, inhibit, and / or block one or more actions or activities according to a selected design for a selected purpose. Typical include single-stranded or double-stranded nucleic acid portions and can include two or more nucleotide sequences arranged within a given portion such that the sequence(s) can exert at least one functional effect on the other(s). For example, a promoter operably linked to a coding region of a DNA polynucleotide sequence can promote transcription of the coding region. Other elements such as enhancers, operators, repressors, transcription termination signals, ribosome entry sites, and polyadenylation signals can also be operably linked to a polynucleotide of interest to control its transcription. The location and spacing to achieve operably linked can be confirmed by approaches available to those skilled in the art, such as screening using Western blots and RT-PCR.

[0049] "Operator" refers to a DNA sequence that is introduced into or near a polynucleotide sequence such that the polynucleotide sequence can be regulated by the interaction of a molecule that can bind to the operator, resulting in preventing or allowing transcription of the polynucleotide sequence, as the case may be. Those skilled in the art will recognize that an operator must be placed close enough to the promoter to be able to control transcription by the promoter, and can be considered a type of operable linkage. The operator can be placed either downstream or upstream of the promoter. These include, but are not limited to, the operator region of the LexA gene of E. coli that binds to the LexA peptide and lactose, and the 45 tryptophan operator that binds to the repressor protein encoded by the Lad and trpR genes of E. coli. Bacteriophage operators from lambda Pi and phage P22 Mnt, as well as Arc. In an alternative embodiment, when the transcription blocking domain of the RFP is a restriction enzyme, the operator is the recognition sequence for that enzyme. Preferred operators are the Tet operator and the Arc operator. The operator can have a native sequence or a mutant sequence (e.g., synthetic or semi-synthetic). For example, mutant sequences of the Tet operator are disclosed in Wissmann et al., Nucleic Acids Res. 14:4253-66 (1986). A TRE can also function as an operator and can include a native operator sequence, a mutant operator sequence, or a combination of native and mutant operator sequences.

[0050] The phrases "percent identity" or "% identical" in their various grammatical forms, when describing a sequence, are meant to include homologous sequences that exhibit the recited identities along the contiguous regions of homology, but the presence of gaps, deletions, or insertions in the compared sequences that do not have a homolog are not taken into account when calculating the percent identity. As used herein, the determination of "percent identity" or "% identical" between homologs does not include the comparison of sequences where the homolog does not have a homologous sequence to compare in an alignment. Thus, "percent identity" and "% identical" do not include penalties for gaps, deletions, and insertions.

[0051] "Homologous sequence" in the context of nucleic acid sequences refers to a sequence that is substantially homologous to a reference nucleic acid sequence. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is a complete (i.e., entire) sequence.

[0052] A "polynucleotide" includes a sequence of covalently linked nucleotides, including RNA and DNA. An oligonucleotide is considered a shorter polynucleotide. A gene is a DNA polynucleotide (polydeoxyribonucleic acid) that ultimately codes for a polypeptide, typically transcribed from DNA and translated from RNA (polyribonucleic acid). A DNA polynucleotide can also code for an RNA polynucleotide that is not translated, but rather functions as an RNA "product". The type of polynucleotide (i.e., DNA or RNA) is clear from the context of the use of the term. A polynucleotide referred to or specified by the polypeptide it encodes refers to and encompasses all suitable sequences according to codon degeneracy. Polynucleotides, including those disclosed herein, include percent identity and homologous sequences, where indicated.

[0053] A "polypeptide" comprises a sequence of covalently linked amino acids. Polypeptides include natural, semi-synthetic, and synthetic proteins, as well as protein fragments. "Polypeptide" and "protein" can be used interchangeably. An oligopeptide is considered a shorter polypeptide.

[0054] "Promoter" refers to a DNA sequence that causes transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to allow transcription of a nucleotide sequence of interest when appropriate signals are present and / or repressors are absent. Transcription of a polynucleotide of interest can be under the control of any promoter or enhancer element known in the art. Eukaryotic promoters can be operably linked to a TATA box, and most eukaryotic promoters have a TATA box. The TATA box is typically located upstream of the transcription start site.

[0055] Useful promoters that may be used include, but are not limited to, the SV40 early promoter region, the SV40E / L (early late) promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequences of the metallothionein gene, the mouse or human cytomegalovirus major immediate early (CMV-MIE) promoter, and other CMV promoters, including the CMVmin promoter, plant expression vectors including the nopaline synthase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase; promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase I, insulin, immunoglobulins, mouse mammary tumor virus, albumin, C-fetoprotein, C.1-antitrypsin, 3-globin, and myosin light chain-2. Various forms of the CMV promoter are preferred, with the CMVmin promoter exemplified herein.

[0056] Minimal promoters, such as the CMVmin promoter, tend to be truncated or core promoters and can be used in controlled expression systems. Minimal promoters are more suitable for control. Minimal promoters and development approaches are widely known and are disclosed, for example, in Saxena et al., Methods Molec.Biol.1651:263-73(2017); Ede et al., ACS Synth Biol.5:395-404(2016); Brown et al., Biotech Bioeng.111:1638-47(2014); Morita et al., Biotechniques 0:1-5(2012); Lagrange et al., Genes Dev.12:34-44(1998). There are many CMVmin promoters reported in the field. It is also possible to use a TATA box sequence to perform the role of the promoter.

[0057] "Recombinase recognition sites" (RRS), also known as "heterospecific recombination sites", are used in recombinase-mediated cassette exchange (RMCE). For example, Cre / Lox, Dre / Rox, Vre / Vlox, SCre / Slox, and Flp / Frt are suitable systems. RRS suitable for use with the present invention include LoxP, Lox66, Lox71, Lox511, Lox2272, Lox2372, Lox5171, LoxM2, LoxM3, loxM7, and LoxM11. These sites may be generally referred to as the first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is clear from the context of use.

[0058] A "regulatory fusion protein" or "RFP" is a protein that contains a ligand-binding domain and a DNA-binding domain from different proteins. The steroid-binding domain of the glucocorticoid or estrogen nuclear receptor can be used as a ligand-binding domain. Also useful as a ligand-binding domain is the reverse Tet DNA-binding domain (rTet), which can similarly bind to DNA. An exemplary RFP for use in accordance with the invention described herein is a fusion protein that includes the reverse tetracycline transactivator (rtTA) and the Arc repressor binding domain (Arc) with the estrogen receptor ligand-binding domain (ArcER). Other components in RFP include the DNA binding domain of yeast activator GAL4 fused to HSV VP16, the KRAB domain of human Kox1 fused to the prokaryotic Tet repressor (TetR-KRAB), the carboxyl terminus of the tTA transactivator and the ligand binding domain of the estrogen receptor (ER) (TetR-VP16), and a catalytically inactive form of Cas9 fused to a repeat of the minimal activation domain of VP16 (dCas9-VP64). Other fusion proteins include LexA-VP16 and LacI-VP16. Polynucleotides encoding regulatory fusion proteins (e.g., rtTA and ArcEr) can be integrated into the cellular genome as described herein.

[0059] A "repressor protein", also referred to as a "repressor", is a protein capable of binding to DNA for repressor transcription. Repressors are of eukaryotic and prokaryotic origin. Prokaryotic repressors are preferred. Examples of repressor families include the TetR, LysR, LacI, ArsR, IcIR, MerR, AsnC, MarR, DeoR, GntR, and Crp families. Repressor proteins in the TetR family include ArcR, ActII, AmeR, AmrR, ArpR, BpeR, EnvR, EthR, HemR, HydR, IfeR, LanK, LfrR, LmrA, MtrR, Pip, PqrA, QacR, RifQ, RmrR, SimReg2, SmeT, SrpR, TcmR, TetR, TtgR, TrgW, UrdK, VarR YdeS, ArpA, BarA, Aur1B, CalR1, CprB, FarA, JadR*, JadR2, MphB, NonG, PhlF, TylQ, VanT, T arA, TylP, BM1P1, Bm3R1, ButR, CampR, CamR, DhaR, KstR, LexA-like, AcnR, PaaRR, PsbI, Th1R, Ui dR, YDH1, BetI, McbR, MphR, PhaD, Q9ZF45, TtK, Yhgd, YixD, CasR, IcaR, LitR, LuxR, LuxT, O Examples include paR, Orf2, SmcR, HapR, Ef0113, HlyIIR, BarB, ScbR, MmfR, AmtR, PsrA, and YjdC proteins. See Ramos et al., Microbiol. Mol. Biol. Rev., 69:326-56 (2005). Still other repressors include PurR, LacR, MetJ, and PadR, and the repressor proteins are encoded by genes referred to as "repressor genes" or "repressor protein genes."

[0060] A "reporter protein" as used herein refers to any protein capable of directly or indirectly generating a detectable signal. Reporter proteins typically fluoresce or catalyze a colorimetric or fluorescent reaction and are often referred to as "fluorescent proteins" or "color proteins". However, reporter proteins can also be non-enzymatic and non-fluorescent, so long as they can be detected by another protein or moiety, such as a cell surface protein detected with a fluorescent ligand. Reporter proteins can also be inactive proteins that become functional through interaction with another protein that is fluorescent or catalyzes a reaction. Thus, any suitable reporter protein can be used, as will be appreciated by those of skill in the art. In some aspects, the reporter protein can be selected from fluorescent proteins, luciferase, alkaline phosphatase, β-galactosidase, β-lactamase, dihydrofolate reductase, ubiquitin, and variants thereof. Fluorescent proteins are useful in some cases for the recognition of gene cassettes that may or may not have been successfully inserted and / or exchanged. Fluid cytometry and fluorescence activated cell sorting are suitable for detection. Examples of fluorescent proteins are known in the art and include Discosoma Reporter proteins include, but are not limited to, a reporter protein such as a reporter gene, a reporter protein encoding a reporter gene, a reporter protein encoding a reporter protein ...A reporter can be considered a type of marker. "Color" or "fluorescence" in their various grammatical forms can also be used to refer more specifically to a reporter protein or gene.

[0061] "Selectable" or "selectable" marker proteins include proteins that confer a particular trait, including but not limited to drug resistance or other selective advantage. A selectable marker can result in a cell that has received a selectable marker gene that is resistant to a particular toxin, drug, antibiotic, or other compound, allowing the cell to produce the protein and grow in the presence of the toxin, drug, antibiotic, or other compound, and is often referred to as a "positive selectable marker." Suitable examples of antibiotic resistance markers include, but are not limited to, proteins that confer resistance to various antibiotics, such as kanamycin, spectinomycin, neomycin, gentamicin (G418), ampicillin, tetracycline, chloramphenicol, puromycin, hygromycin, zeocin, and / or blasticidin. There are other selectable markers, often referred to as "negative selectable markers," that cause the cell to stop growing, stop producing protein, and / or are lethal to the cell in the presence of the negative selectable marker protein. Thymidine kinase and certain fusion proteins can function as negative selectable markers, including but not limited to GyrB-PKR. See White et al., Biotechniques, 50:303-309 (May 2011). Selectable marker proteins and corresponding genes can be generally referred to as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as will be clear from the context of use.

[0062] A "stable integration site" or "SIS" is a region for site-specific integration of a DNA polynucleotide, comprising a cassette containing a gene and / or other open reading frame, a promoter, and optionally other elements. Stable integration sites can be created according to the methods of the invention described and depicted herein. Constructs can be inserted into the SIS by a variety of approaches. Multiple stable integration sites can be created and located on different chromosomes, different regions of the same chromosome, or in different locations in the same region of a chromosome.

[0063] "Tetracycline response elements" or "TREs" contain seven copies of the 19 nucleotide TetO separated by spacers containing 17-18 nucleotides and are commercially available. The TetO sequence can vary and nucleotide substitutions are known. For example, altered sequences based on the Tet operator are disclosed in Wissmann et al., Nucleic Acids Res. 14:4253-66 (1986). The spacers are not sequence specific. The spacers can be similar, but need not all be identical. TREs are considered a type of operator as used herein.

[0064] All numerical limits and ranges set forth herein include all numbers or values ​​around or between the numerical values ​​of the range or limit. Ranges and limits set forth herein expressly represent and represent all integer, decimal, and fractional values ​​defined and encompassed by the range or limit. Ranges and limits set forth herein expressly represent and represent all integer, decimal, and fractional values ​​defined and encompassed by the range or limit. Thus, the recitation of a range of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within that range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually set forth herein.

[0065] Detailed Description The present invention generally relates to constructs that allow for tight control of the transcription of a polynucleotide sequence in a cell. The present invention is based on research and determination that it is possible to establish stable cells that transcribe a polynucleotide sequence such that the transcription of the polynucleotide sequence is controlled by a controllable expression system. Cells expressing a polynucleotide sequence such that the transcription of a polynucleotide sequence of interest is controlled by the controllable expression system described herein can be used in a wide variety of applications. The polynucleotide sequence of interest can encode a polypeptide of interest or a product of interest. The controllable expression system described herein is particularly useful for controlling the expression of a polypeptide of interest and / or a product of interest that is toxic or inhibitory to a host cell.

[0066] The cells described herein provide the particular advantage that the cells are stable. By "stable" it is meant that the cells can be used to establish cell lines with regions of interest that are functionally homogeneous in culture. Regions of interest include, for example, polynucleotides of interest and associated promoters, operators, internal ribosome entry sites (IRES), polyadenylation signals, and non-translated RNAs that can be monitored.

[0067] Additionally, the cells described herein provide the additional advantage that the transcription of the polynucleotide encoding the polypeptide of interest is tightly controlled, thereby allowing the cells to survive to a stage that allows large-scale expression of the polypeptide of interest. By "tightly controlled" is meant that in the absence of the first ligand (alternatively, in the presence of the first ligand as well) and in the presence of the second ligand, the transcription of the polynucleotide sequence encoding the polypeptide of interest is substantially reduced. The tightest control is achieved in the absence of the first ligand and in the presence of the second ligand. For example, in certain embodiments of the repressed state, at least a 10-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand in the induced state. In certain embodiments of the repressed state, at least a 20-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand in the induced state. In certain embodiments of the repressed state, at least a 50-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand in the induced state. In certain embodiments of the repressed state, at least a 100-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand in the induced state. In certain embodiments of the repressed state, at least a 500-fold reduction in transcription is achieved compared to the level of transcription observed in the presence of the first ligand and in the absence of the second ligand in the induced state.

[0068] As a result of the repressed state, the degree of induction of transcription of the polynucleotide sequence of interest observed in the presence of the first ligand and in the absence of the second ligand may be at least 10 times higher than the repressed state in certain embodiments. In certain embodiments, the degree of induction of transcription of the polynucleotide sequence of interest observed in the presence of the first ligand and in the absence of the second ligand may be at least 20 times higher than the repressed state in certain embodiments. In certain embodiments, the degree of induction of transcription of the polynucleotide sequence of interest observed in the presence of the first ligand and in the absence of the second ligand may be at least 50 times higher than the repressed state in certain embodiments. In certain embodiments, the degree of induction of transcription of the polynucleotide sequence of interest observed in the presence of the first ligand and in the absence of the second ligand may be at least 100 times higher than the repressed state in certain embodiments. In certain embodiments, the degree of induction of transcription of a polynucleotide sequence of interest observed in the presence of a first ligand and in the absence of a second ligand can, in certain embodiments, be at least 500-fold higher than in a repressed state.

[0069] The degree or amount of transcription of a polynucleotide sequence of interest can be determined by methods known to those skilled in the art.For example, the expression level of a polypeptide of interest in a host cell can be determined based on the amount of corresponding mRNA present in the cell.The messenger RNA transcribed from a polynucleotide sequence can be quantified by various methods known to those skilled in the art, including but not limited to Northern blot hybridization, RNase RNA protection, in situ hybridization to cellular RNA, or PCR.

[0070] As a further example, the expression level of a polypeptide of interest in a host cell can also be determined based on the amount of the polypeptide of interest encoded by a selected sequence. The polypeptide encoded by a polynucleotide sequence can be quantified by a variety of methods known to those of skill in the art, including, but not limited to, ELISA, Western blotting, radioimmunoassay, immunoprecipitation, assays for biological activity of the polypeptide, immunostaining of the polypeptide by FACS analysis, or homogeneous time-resolved fluorescence assay (HTRF).

[0071] Controllable transcription and expression systems The present invention relates to a controllable transcription and expression system that can be used to control the transcription of any polynucleotide sequence of interest. The controllable transcription and expression system described comprises at least two controllable operator systems. One of the operator systems can be located 5' to a promoter operably linked to the polynucleotide sequence of interest, and the second operator system can be located 3' to the promoter. The operator system can include an operator operably linked to a promoter that drives transcription of the polynucleotide sequence of interest. The polynucleotide of interest can encode a polypeptide and / or product (e.g., RNA) of interest.

[0072] The controllable transcription described herein allows for transcription of a polynucleotide of interest in the presence of a first ligand and in the absence of a second ligand. Briefly, the first ligand, if present, binds to a ligand binding site on a first regulatory fusion protein (RFP), which includes (1) a transcription activation domain fused to a DNA binding domain, and (2) a ligand binding domain. When the first ligand binds to the ligand binding domain of the first RFP, the DNA binding domain of the first RFP binds to the first operator and allows transcription from the promoter, but only if transcription is not inhibited by the second operator system. The second operator system is controlled by a second ligand. Briefly, the second ligand, if present, binds to a ligand binding site on a second regulatory fusion protein (RFP), which includes (1) a transcription blocking domain fused to a DNA binding domain, and (2) a ligand binding domain. When the second ligand binds to the ligand binding domain of the second RFP, the DNA binding domain of the second RFP binds to the second operator and blocks transcription from the promoter. Thus, in the presence of the second ligand and in the absence of the first ligand, transcription is repressed, whereas in the absence of the second ligand and in the presence of the first ligand, transcription is permitted. Figures 1, 4, 6, and 7 show examples of transcriptional control utilizing this system.

[0073] The first operator system may include at least one operator operably linked to a promoter that drives transcription of a polynucleotide sequence of interest. The operator of the first operator system may be located 5' to the promoter that is operably linked to the polynucleotide sequence of interest. Examples of such configurations are shown in Figures 1, 4, 6, and 7, in which the first operator system includes a TRE.

[0074] The first operator system may also include a regulatory fusion protein (RFP) that includes (1) a transcription activation domain fused to a DNA binding domain, and (2) a ligand binding domain. The first operator system may further include a ligand that binds to the ligand binding domain of the first RFP. When the ligand binds to the ligand binding domain of the first RFP, the DNA binding domain of the RFP binds to an operator, e.g., a TRE, thereby allowing transcription from the promoter, but only if transcription is not inhibited by the second operator system discussed herein. Other system components are known to those of skill in the art, such as the tetracycline-on system (Tet-On), advanced tetracycline (Tet-OnAdvanced), tetracycline-on 3G system (Tet-On 3G), cumate inducible system, lactose inducible system, and modifications thereof.

[0075] The second operator system can be located 3' to a promoter operably linked to a polynucleotide sequence encoding a polypeptide of interest. Examples of such configurations are shown in Figures 1, 4, 6, and 7. The second regulatory element comprises at least one operator operably linked to a promoter that drives transcription of the polynucleotide sequence of interest. Examples of such configurations are shown in Figures 1, 4, 6, and 7, where the second operator system comprises either a tetracycline operator (TetO) or an Arc operator (ArcO). In some embodiments, the second operator is TetO (Figure 4). In some embodiments, the second operator is ArcO (Figures 1, 6, and 7).

[0076] As a non-limiting example, a controllable operator component suitable for the second operator system is described in US Pat. No. 9,469,856.

[0077] Suitable promoters for use in the described system are known and can be determined by those skilled in the art in a selection combination. In some embodiments, the promoter operably linked to the polynucleotide sequence can be selected from, but is not limited to, the SV40 early promoter region, the SV40E / L promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequence of the metallothionein gene, the mouse or human cytomegalovirus major early (MIE) promoter; the CMVmin promoter, a plant expression vector containing the nopaline synthase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase; promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and animal transcriptional control regions that exhibit tissue specificity and are utilized in transgenic animals: elastase I, insulin, immunoglobulins, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1-antitrypsin, beta-globin, and myosin light chain-2. In some embodiments, the promoter is a human CMV-MIEmin or other CMVmin promoter. Approaches to develop minimal promoters are described in Saxena et al., Methods Molec. Biol. 1651: 263-73 (2017), Ede et al., ACS Synth Biol. 5: 395-404 (2016), Brown et al., Biotech Bioeng. 111: 1638-47 (2014), Morita et al., Biotechniques 0: 1-5 (2012), Lagrange et al. Genes Dev. 12: 34-44 (1998).

[0078] In some embodiments, the polynucleotide sequence of interest encodes a polypeptide of interest. The polynucleotide of interest can be a native gene, including variants thereof, or a synthetic, semi-synthetic, or optimized sequence. In other embodiments, the polynucleotide sequence of interest encodes a product of interest (e.g., RNA). More specifically, the product of interest can be a non-coding RNA.

[0079] A "protein of interest" or "polypeptide of interest" (POI) can have any amino acid sequence and includes any protein, polypeptide, or peptide, as well as derivatives, components, domains, chains, and fragments thereof. Included are, but are not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins, and animal (including human) proteins. Protein types can include, but are not limited to, antibodies, bispecific antibodies, multispecific antibodies, antibody chains (including heavy and light chains), antibody fragments, Fv fragments, Fc fragments, Fc-containing proteins, Fc fusion proteins, receptor Fc fusion proteins, receptors, receptor domains, trap and mini-trap proteins, enzymes, factors, inhibitors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters, and contractile proteins. Derivatives, components, chains, and fragments of the above are also included. The sequences can be natural, semi-synthetic, or synthetic. Proteins of interest and polypeptides of interest are encoded by "genes of interest," which may also be referred to as "polynucleotides of interest." Where multiple genes (the same or different) are integrated, they may be referred to as "first," "second," "third," "fourth," "fifth," "sixth," "seventh," "eighth," "ninth," "tenth," etc., as is clear from the context of usage.

[0080] The polypeptide of interest can also include cytotoxic proteins such as viral proteins. For example, adenovirus E1A, E1B, E2A, and E4 are used to perform functions for the production of adeno-associated virus (AAV), but have been reported to have toxic effects in certain cell types. AAV Rep has also been reported to have cytotoxic effects in certain cell types. Additionally, proteins used in genetic modification, such as Cre recombinase, Flp recombinase, zinc finger (ZFN) proteins and dimers, TALEN, bxb1 integrase, CRISPR-associated proteins (types I-VI, including Cas1, Cas2, Cas3, Cas4, Cas, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, and Cas13), and other nucleases and integrases, can be POIs and thereby controlled according to the present invention.

[0081] Cells capable of regulated transcription and expression In one embodiment, a cell is provided, comprising a promoter operably linked to a polynucleotide sequence of interest, the promoter being controlled by at least two operators operably linked to the promoter. The promoter operably linked to the polynucleotide sequence of interest, the operator operably linked to the promoter can be integrated into the cell genome. The transcription of the polynucleotide sequence of interest is controlled by the operator, allowing or preferably suppressing the transcription of the polynucleotide sequence of interest.

[0082] Cells suitable for use with these inventions can be readily selected by one of skill in the art. In some embodiments, the cell line is a eukaryotic cell line, such as a yeast cell line, an insect cell line (e.g., Sf9 and Sf21 cells), or a mammalian cell line. Preferred mammalian cells include primate cells (including human), canine cells, and rodent cells. The cells can be primary or immortalized cells. Suitable cells can be selected from cell lines transfected with viral genes, such as AD5 E1, including, but not limited to, Vero cells, COS cells, HEK293 cells, HeLa cells, CHO cells, BHK cells, Sp2 / 0 cells, MDCK cells, amniotic cells (including human), embryonic cells, immortalized human retinal cells transfected with adenoviral genes, such as PER.C6 cells, or NSO cells. In some embodiments, the cells are Chinese Hamster Ovary (CHO) cell lines. Some examples of CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants / variants thereof. In a further preferred embodiment, the CHO cell may be a CHO cell line designated K1. Examples of HEK293 cells include, but are not limited to, HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T, and mutants and variants thereof.

[0083] An example of a construct used in generating a cell expressing a polynucleotide sequence encoding a crimson fluorescent protein is shown in Figure 1. The construct includes an expression cassette that includes a polynucleotide sequence encoding a gene of interest (e.g., crimson), at least one promoter, and at least two operators. Briefly, as shown in Figure 1, transcription of the polynucleotide sequence encoding crimson from the CMVmin promoter is controlled by the tetracycline operator (TetO) and the Arc operator (AO or ArcO).

[0084] In some embodiments, the cells further comprise a polynucleotide sequence encoding one or more regulatory fusion proteins (RFPs). The RFP may comprise (a) a transcriptional activation domain fused to a DNA binding domain, and (b) a ligand binding domain. The ligand may bind to the ligand binding domain of the RFP.

[0085] In some embodiments, the cell further comprises a polynucleotide sequence encoding one or more regulatory fusion proteins (RFPs), regulatory proteins, or repressor proteins. The RFP may comprise (a) a transcription blocking domain fused to a DNA binding domain, and (b) a ligand binding domain. The transcription blocking domain may comprise an Arc repressor DNA binding domain. The regulatory protein may be TetR. Inhibition of transcription of a polynucleotide of interest by binding to a Tet or Arc operator. In some embodiments, the operator is Tet and the transcription blocking domain is a Tet repressor. In other embodiments, the operator is Arc and the transcription blocking domain is an Arc repressor DNA binding domain.

[0086] The cell may further comprise an element that regulates the transcription of the polynucleotide sequence(s) encoding one or more regulatory fusion proteins (RFPs). As a non-limiting example, the transcription of the polynucleotide sequence(s) encoding one or more regulatory fusion proteins (RFPs) may be controlled by the Tet-On system, such that the polynucleotide sequence(s) encoding one or more regulatory fusion proteins (RFPs) are only transcribed in the presence of Dox. In another embodiment, the transcription of rtTA (RFP) may be optionally under the control of ArcER and AO.

[0087] In some cases, the polynucleotide sequence of interest and operably linked promoter and operator may be introduced into the cell by transfection of a plasmid containing the polynucleotide sequence and elements. Thus, the invention includes the production of the described cells and cells containing the described plasmid constructs.

[0088] Suitable plasmid constructs can be made by those skilled in the art. Useful regulatory elements already reported or known in the art can also be included in the plasmid constructs used to transfect cells. Some non-limiting examples of useful regulatory elements include, but are not limited to, promoters, enhancers, sequences encoding suitable mRNA ribosome binding sites, and sequences that control the termination of transcription and translation. Suitable plasmid constructs can also include non-transcribed elements such as origins of replication, other 5' or 3' adjacent non-transcribed sequences, and 5' or 3' non-translated sequences such as splice donor and acceptor sites. One or more marker genes can also be incorporated. Selectable marker proteins and reporter proteins useful for use in the present invention are known and can be easily identified by those skilled in the art.

[0089] Plasmid constructs encoding the gene of interest can be delivered to cells using viral vectors or via non-viral transfer methods.

[0090] Non-viral methods of nucleic acid transfer include naked nucleic acid, liposomes, and protein / nucleic acid conjugates. The plasmid construct introduced into the cell can be linear or circular, single-stranded or double-stranded, and can be DNA, RNA, or any modification or combination thereof.

[0091] The plasmid construct can be introduced into cells by transfection. Those skilled in the art are aware of many different transfection protocols and can select the appropriate system for use in transfecting cells. In general, transfection methods include, but are not limited to, viral transduction, cationic transfection, liposomal transfection, dendrimer transfection, electroporation, heat shock, nucleofection transfection, magnetofection, nanoparticles, biolistic delivery (gene gun), and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene, or DreamFect.

[0092] Upon introduction into a cell, some polynucleotide sequences from the plasmid construct may be integrated into the cell genome, such as a chromosome. In some cases, integration of the polynucleotide sequence into the genome may be achieved by lox sites.

[0093] In embodiments, a promoter operably linked to a polynucleotide sequence of interest, a first operator operably linked to the promoter, and a second operator are integrated into the cell genome. Other polynucleotides, such as polynucleotides encoding regulatory fusion proteins (e.g., rtTA and ArcEr) and repressor proteins (e.g., TetR), can also be integrated into the cell genome as described herein.

[0094] In some embodiments, the genome integration is random. Methods for achieving random genome integration are known by those skilled in the art, and suitable means can be identified by those skilled in the art. For example, a linearized plasmid carrying a selectable marker can be used for genome integration at random positions.

[0095] In some embodiments, the genomic integration is site-specific. Site-specific integration refers to integration at a specific site within a chromosome. Methods for achieving site-specific integration are known by those skilled in the art, and suitable approaches can be identified by those skilled in the art. As a non-limiting example, one approach for site-specific integration in CHO cells is described in U.S. Pat. No. 7,771,997 ("Stable Site 1"), which is incorporated herein by reference, including sequence information. U.S. Pat. No. 7,771,997 describes an integration site located in an enhanced expression and stability region. Another suitable integration site is described in U.S. Pat. No. 9,816,110 ("Stable Site 2"), which is incorporated herein by reference, including sequence information. Regeneron offers a line of products and services called EESYR®. CHO cells with sequences integrated into Stable Site 1 and Stable Site 2 are disclosed in US2019 / 0233544A1, which is incorporated herein by reference, including sequence information. The sequences set forth in these patents and in Examples 6 and 7 can be used in accordance with the invention described and illustrated herein. Additionally, the AAVS1-like region and the COSMC locus in hamster cells can be used in accordance with the invention.

[0096] In human cells, such as HEK293 cells, integration can be achieved using the adeno-associated virus integration site 1 (AAVS1) via an appropriate plasmid. See Lou et al., Human Gene Therapy Methods, 28:124-38 (2017); Liu et al., BMC Research Note, 7:626 (2014). AAVS1 is reported to be located on chromosome 19. Additional sites include CCR5 and Rosa26.

[0097] Modification of the cell genome can be performed by known approaches such as Cre / Lox, Flp / Frt, transcription activator-like effector nucleases (TALEN), TAL effector domain fusion proteins, zinc finger nucleases (ZFN), ZFN dimers, or RNA-guided DNA endonuclease systems such as CRISPR / Cas9. See U.S. Pat. No. 9,816,110 at cols. 17-18, Sajgo et al., PLoS ONE 9:e91435 (2014), Suzuki et al., Nucl. Acids. Res. 39:e49 (2011). Modification can also be performed using Bxb1 integrase in human, mouse, and rat cells. As disclosed in Russell et al., Biotechniques 40:460-64 (2006).

[0098] To maximize stability and efficiency and to facilitate integration and control of the present invention, Stable Integration Sites (SIS) can be created using genomic safe harbors and the like in a wide variety of cell types and cell lines following the teachings of US63 / 256,675. The descriptions (including examples) and figures providing methods and cells resulting from the methods of US63 / 256,675 are incorporated herein by reference.

[0099] Thus, site-specific and random integration approaches can be used in cells. Finally, the polynucleotide can be present integrated in a non-chromosomal location known to those skilled in the art, such as an episome.

[0100] In some embodiments, the cells described herein may include a polynucleotide sequence encoding a marker. In embodiments, the polynucleotide sequence encoding the marker is linked to a polynucleotide sequence encoding a polynucleotide of interest. The polynucleotide sequence encoding the marker may be linked to a polynucleotide sequence of interest such that when the polynucleotide sequence of interest is integrated, the marker polynucleotide sequence is similarly integrated. Markers useful for use in the present invention are known and can be readily identified by those of skill in the art, and include, but are not limited to, selectable markers (such as drug resistance markers) and reporter proteins such as colorimetric / fluorescent markers.

[0101] Methods for controlling the expression of a polypeptide In another aspect, the invention provides a method of controlling the transcription of a gene of interest in a cell as described, which has utility in a wide variety of applications, including, but not limited to, the production of a protein / product of interest for therapeutic purposes.

[0102] The present invention provides methods of controlling the expression of a polypeptide of interest in a cell. In some embodiments, the polypeptide of interest is toxic or inhibitory to the cell, such as a viral gene.

[0103] Production of a protein or product of interest begins with a working cell bank (WCB), which is typically cryopreserved. The WCB contains cells in the absence or presence of a first ligand (e.g., dox) and in the presence of a second ligand (e.g., OHT), as determined by one of skill in the art. The WCB is thawed and used to create a seed culture (also known as a "seed train"). In the seed culture stage, cells can be grown in the absence or presence of a first ligand and in the absence of a second ligand, as determined by one of skill in the art. The effectiveness of inhibition of transcription by the second ligand typically decreases about 4-14 days after seeding (however, the time course can be modified, for example, by adding additional second ligand to the cell culture medium to delay loss of effectiveness). After the seed culture stage, production of the protein or product of interest can begin. Non-limiting examples of embodiments are described below to further illustrate aspects of the invention.

[0104] Methods and compositions are provided to facilitate control of transcription of a polynucleotide sequence of interest in cell culture at various scales (e.g., from benchtop to bioreactor). Also provided are methods and compositions for achieving delayed transcription of a polynucleotide of interest in cells in culture that is dependent on transitioning from the presence of a second ligand to the presence of a first ligand and the absence of a second ligand. Delayed transcription involves transcription of the polynucleotide of interest only after the cells have grown to a desired density, or at a time thereafter. In general, under some circumstances, it is desirable to allow the cells to reach a desired density, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (i.e., about 10-100%) of the maximum achievable density in culture, before a desired amount of transcription of the polynucleotide sequence of interest occurs. In some embodiments, a cell density of about 90 to about 100% is desirable before complete transcription of the polynucleotide sequence of interest.

[0105] In some embodiments, a cell density of at least 400,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 500,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 600,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 700,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 800,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 900,000 viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 1 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 2 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 3 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 4 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 5 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 6 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 7 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 8 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 9 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest.In some embodiments, a cell density of at least 10 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 12 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. In some embodiments, a cell density of at least 15 million viable cells per ml is desired prior to complete transcription of the polynucleotide sequence of interest. A typical range can be 400,000 to 3 million. Other acceptable ranges include 1 million to 15 million, 2 million to 12 million, 3 million to 10 million, 4 million to 9 million, 5 million to 8 million, and 6 million to 7 million, as well as any subranges within any of these ranges as would be apparent to one of skill in the art in light of these teachings.

[0106] Delayed induction of transcription can be achieved by any suitable method described herein. In various embodiments, delayed induction can be achieved by growing cells to a desired cell density in the presence of an effective amount of a second ligand. In light of the teachings contained herein, the time course of inhibition can be defined by the selection of a time course of maintaining an effective amount of the second ligand in the cell culture. Removal of the second ligand can be achieved, for example, by (i) separating the cells from the medium containing the second ligand, (ii) diluting the cell culture with medium that does not contain the second ligand, and / or (iii) splitting the mixture of cells and medium such that the second ligand is then present at a level below an effective amount (e.g., an amount of the second ligand that does not substantially inhibit or does not inhibit transcription of the polynucleotide sequence of interest), sometimes referred to as disappearance. In various embodiments, the cells are first grown and / or stored (e.g., in a working cell bank (WCB)) with an effective amount of the second ligand.

[0107] In various embodiments, the cell culture can be grown in medium that is absent an effective amount of a second ligand. More specifically, during growth, the cell culture has an effective amount of a first ligand and is absent an effective amount of a second ligand for a period of time sufficient to allow the second ligand to disappear, usually about 4-14 days, depending on the second ligand and culture conditions.

[0108] In other embodiments, once the desired culture size is reached and the second ligand no longer effectively inhibits the transcription of the cell culture, the polynucleotide of interest can be transcribed in the presence of an effective amount of the first ligand.The effective amount of the first ligand can be added to the cell culture at any suitable time.For example, the effective amount of the first ligand can be added during seeding.In another example, the effective amount of the first ligand can be added at a later time, such as when the second ligand is disappearing or has already disappeared.

[0109] Those skilled in the art will be able to determine suitable concentrations of the first and second ligands to achieve effective amounts throughout the process. Exemplary concentrations of the ligands may include about 100 nM to about 1,000 nM, about 100 to 900 nM, 100 to 800 nM, about 100 nM to 700 nM, 100 nM to 600 nM, 100 nM to 500 nM, 100 nM to 400 nM, 100 nM to 300 nM, in certain embodiments, about 200 nM to about 500 nM, and in another particular embodiment, about 400 nM. Other concentrations may be used as well.

[0110] All methods described herein can be carried out in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples described herein, or the use of exemplary terms (e.g., "etc.") are intended merely to better illustrate the invention and do not limit the scope of the claims unless otherwise stated. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Since various changes can be made in the above compositions and methods without departing from the scope of the invention, it is intended that all matters contained in the above description and the examples given below should be construed as illustrative and not in a limiting sense. EXAMPLES

[0111] The following examples are offered to provide one of ordinary skill in the art with how to make and use the cells, methods, and compositions described herein and are not intended to limit the scope of what the inventors regard as their invention.

[0112] Example 1 Two regulatory fusion proteins, rtTA and ArcER, allow for tight control. CHO-K1 cells were constructed that stably express the crimson fluorescent protein under the control of both TRE and ArcO. As shown in Figure 1, transcription of crimson is from the CMVmin promoter with a TATA box. The CMVmin promoter is flanked by a TRE at 5' and an ArcO (AO) at 3'. Reverse tetracycline transcription activator (rtTA) is an RFP composed of a reverse Tet DNA binding domain (rTet) and a VP16 transactivation domain (VP16 AD). The rTet portion can bind the ligands tetracycline, doxycycline (dox), and their derivatives. ArcER is an RFP in which the Arc repressor binding domain (Arc) is fused to the estrogen receptor ligand binding domain (ER). The ER portion can bind estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT), and other derivatives thereof.

[0113] Figure 1 shows a repressed and an induced gene of interest. In the repressed state, the first ligand, here dox, is not present, meaning that rtTA cannot bind to the TRE and allow transcription (trx) to proceed. Additionally, a second ligand, here OHT, is present and binds to the ArcER regulatory fusion protein (RFP), thereby resulting in the inhibition of transcription of Crimson.

[0114] In the induced state, the first ligand, here dox, binds to rtTA, allowing it to bind to the TRE and allowing transcription (trx). In the absence of the second ligand, here OHT, ArcER does not inhibit transcription of Crimson. It is believed that ArcER is not transported into the nucleus in the absence of estrogen receptor ligands such as OHT or E2. Thus, in the presence of the first ligand and the absence of the second ligand, transcription can proceed.

[0115] For example, Crimson-expressing CHO-K1 cells (TRE-AO) can be made by inserting DNA cassettes encoding a selectable marker and a reporter protein, as well as polynucleotides encoding rtTA and ArcER, into the genome of the cell at stable site 1 according to U.S. Patent No. 7,771,997. For example, a cassette containing a polynucleotide encoding Crimson with a CMVmin promoter can be inserted into the genome of the cell at site 2 according to the teachings of U.S. Patent No. 9,816,110. The reporter protein was included to confirm integration of the expression cassette into the genome of the cell.

[0116] Stable integration of the two expression cassettes was confirmed by utilizing the included selectable markers.

[0117] The ability to tightly control the transcription of Crimson in TRE-AO CHO-K1 cells was then tested and compared to the negative control (unmodified cells). As expected, when the ligand for ArcER, here E2, was present and the ligand for rtTA, here Dox, was absent, the expression of Crimson was highly suppressed. As shown in Figure 2, the level of Crimson expression was close to that of the negative control. Additionally, the level of Crimson expression in TRE-AO CHO-K1 cells was lower than that in control cells (standard CMV-TO) that were able to express Crimson in the presence of Dox. This demonstrates that the TRE-AO system provided tighter control of transcription compared to TetO alone. Furthermore, high levels of Crimson expression were observed with +dox and -E2 (Figure 2, TRE-AO induction).

[0118] Thus, the TRE-AO system provides a means to tightly control the transcription of a polynucleotide of interest.

[0119] Example 2 Controlling the production of regulatory fusion proteins or repressor proteins Optionally, the production of a regulatory fusion protein such as rtTA, or a repressor protein, can be controlled by another regulatory fusion protein and ligand, such as ArcER and AO, as shown in Figure 3. An exemplary construct has a CMV promoter and a TATA box, with the gene encoding rtTA following downstream of the AO. ArcER in the presence of the ligand OHT can bind to the AO and block transcription (trx). In the absence of a ligand such as OHT, ArcER is no longer available to bind to the AO, allowing transcription (trx) of the gene encoding rtTA.

[0120] The use of an RFP such as ArcER to control the expression level of another RFP, such as rtTA, is another optional approach to control the transcription of a polynucleotide of interest that is under the control of that RFP (in this example, rtTA) in accordance with the invention described herein.

[0121] Example 3 A regulatory fusion protein (rtTA) with a repressor protein (TetR) allows for tight control CHO-K1 cells were constructed that stably express the crimson fluorescent protein under the control of TRE and a separate TetO. As shown in Figure 4, transcription of crimson is from the CMVmin promoter with a TATA box. The CMVmin promoter is flanked by TRE at 5' and TetO (TO) at 3'. In the absence of the ligand dox, rtTA cannot bind to the TRE and prevents transcription. Additionally, the Tet repressor protein (TetR) binds to the tetracycline operator (TO) in the absence of the ligand dox, which also blocks transcription.

[0122] When the TetR ligand, here dox, is present, it binds to rtTA, thereby allowing transcription. Additionally, the dox ligand binds to TetR, reducing the affinity of the Tet repressor for TO, allowing transcription. Polynucleotides encoding repressor proteins, such as TetR, can be inserted randomly into the genome or site-specifically into the genome.

[0123] As shown in Figure 5, transcription of the polynucleotide encoding the crimson fluorescent protein was under the control of rtTA and TetR (TRE-TO). See Figure 4. In the presence of E2 and in the absence of dox, a very low level of transcription of the GOI (crimson) is observed (TRE-TO repression (+E2 / -Dox)), almost the same as that of the control (negative, unmodified cells). In the presence of dox and in the absence of E2, a high level of transcription of the GOI (crimson) is observed (TRE-TO induction).

[0124] Example 4 Two regulatory fusion proteins (rtTA and ArcER) allow for tight control of cytotoxic genes Figures 6 and 7 show rtTA bound to ArcER, providing tight control over the transcription of cytotoxic genes. Figure 6 shows the repressed state in the absence (-dox) of dox (first type of ligand) and the presence (+OHT) of OHT (second type of ligand). The absence of dox means that rtTA cannot bind to the TRE and does not allow transcription to begin. The presence of OHT allows ArcEr to bind to the AO, blocking transcription. The presence of OHT also prevents transcription of the rtTA polynucleotide, where the embodiment of Example 2 and Figure 3 is used.

[0125] Figure 7 shows the induced state: dox is present (+dox), allowing rtTA to bind to the TRE and initiate transcription (trx). OHT is absent (-OHT), so that ArcER can no longer bind to AO, meaning that ArcER does not block transcription of cytotoxic genes, and in certain embodiments allows transcription of rtTA, as described in Example 2 and shown in Figure 3.

[0126] Example 5 The virulent AAV Rep gene can be regulated and expressed under tight control A HEK293 cell line was constructed with TRE and AO according to the teachings contained herein to control genes for AAV Rep78 and Rep52. See Examples 1 and 4 and Figures 1, 6, and 7. The embodiment shown in Figures 6 and 7 replaces the color genes of Figure 1 with cytotoxin-encoding genes such as Rep genes. Both AAV Rep78 and the truncated form, Rep52, are known to be toxic to human cells. Schmidt et al, J. Virol. 9441-50 (2000).

[0127] In the repressed state, rtTA is without its ligand (e.g., dox) and ArcER is in the presence of its ligand (e.g., OHT). See Figure 6. In the induced state, rtTA is in the presence of its ligand and ArcEr is in the absence of its ligand, which allows transcription of the cytotoxic gene (trx) to proceed. See Figure 7.

[0128] In this example, HEK293 cells were transformed with Rep78 and Rep52, both genes under the control of the TRE-AO system, and the ligands used were dox and E2 (instead of OHT).

[0129] Figure 8 discloses the results: when HEK293 cells were in a repressed state (-), Rep78 and Rep52 were not produced. In an induced state (+), both Rep78 and Rep52 are produced. Example 6 Exemplary sequences are provided below, other sequences (including homologues and variants) are available to those of skill in the art. Nucleic acid and amino acid sequences rtTA nucleotide sequence (SEQ ID NO:1) rtTA amino acid sequence (SEQ ID NO:2) MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPP LLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG* ArcER nucleotide sequence (SEQ ID NO:3) ArcER amino acid sequence (SEQ ID NO:4) MKGMSKMPQFNLRWPREVLDLVRKVAEENGRSVNSEIYQRVMESFKEGRIGAGGGSGGGTGGGSGGGMKGMSKMPQFNLRWPREVLDLVRKVAEENGRSVNSEIYQRVME SFKKEGRIGAAYSGSRELIRLSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTL HDQVHLLECAWLEILMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITD TLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV* TetR nucleotide sequence (SEQ ID NO:5) ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGT ATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGAT AGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCGTACAGCGGATCCCGGGAATTCAGATCTTATTAA TetR amino acid sequence (SEQ ID NO:6) MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQ YETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESAYSGSREFRSY Tet Operator (SEQ ID NO:7) TCCCTATCAGTGATAGAGA Tet response element (SEQ ID NO:8) TCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGGGAACGTATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATAGAGA Arc Operator (SEQ ID NO:9) ATGATAGAAGCACTCTACTATTC hCMVmin promoter (SEQ ID NO:10) GCGTATAAGCTTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTC CHO and Mouse Stable Site 1 Sequences - U.S. Patent No. 7,771,997 211>6473 <212> DNA <213> Cricetulus griseus <400> 1 (SEQ ID NO:11) tctagaaaca aaaccaaaaa tattaagtca ggcttggctt caggtgctgg ggtggagtgc 60 tgacaaaaat acacaaattc ctggctttct aaggcttttt cggggattca ggtattgggt 120 gatggtagaa taaaaatctg aaacataggt gatgtatctg ccatactgca tgggtgtgta 180 tgtgtgtgta tgtgtgtctg tgtgtgtgcc cagacagaaa taccatgaag gaaaaaaaca 240 cttcaaagac aggagagaag agtgacctgg gaggactcc ccaatgagat gagaactgag 360. cacatgccag aggaggtgag gactgaacca ttcaacacaa gtggtgaata gtcctgcaga 420. cacagagagg gccagaagca ctcagaactc cagggggtca ggagtggttc tctggaggct tctgcccttg gaggttcctg aggaggaggc ttccatattg aaaatgtagt tagtggccgt 480 ttccattagt acagtgacta gagagagctg agggaccact ggactgaggc ctagatgctc agtcagatgg ccatgaaagc ctagacaagc acttccgggt ggaaaggaa cagcaggtgt gaggggtcag gggcaagtta gtgggagagg tcttccagat gaagtagcag gaacggagac 660 gcactggatg gccccacttg tcaaccagca aaagcttgga tcttgttcta agaggccagg 720 gacatgacaa gggtgatctc ggtttttaaa aggctttgtg ttacctaatc acttctatta 840. gtcagatact ttgtaacaca aatgagtact tggcctgtat tttagaaact tctgggatcc tgaaaaaaca caatgacatt ctggctgcaa cacctggaga ctcccagcca ggccctggac ccgggtccat tcatgcaaat actcagggac agattcttca ctaggtactg atgagctgtc ttggatgcaa atgtggcctc ttcattttac tacaagtcac catgagtcag gaggtgctgt 1020 ttgcacagtg tgactaagtg atggagtgtt gactgcagcc attcccggcc ccagcttgtg 1080 agagagatcc ttttaaattg aaagtaagct caaagttacc acgaagccac acatgtataa 1140 actgtgtgaa taatctgtgc acatacacaa accatgtgaa taatctgtgt acatgtataa 1200 actgtgtgaa taatctgtgt gcagcctttc cttacctact accttccagt gatcaggttt 1260 ggactgcctg tgtgctactg gaccctgaat gtccccaccg ctgtcccctg tcttttacga 1320 ttctgacatt tttaataaat tcagcggctt cccctctgct ctgtgcctag ctataccttg 1380 gtactctgca ttttggtttc tgtgacattt ctctgtgact ctgctacatt ctcagatgac 1440 atgtgacaca gaaggtgttc cctctggaga catgtgatgt ccctgtcatt agtggaatca 1500 gatgccccca aactgttgtc cagtgtttgg gaaagtgaca cgtgaaggag gatcaggaaa 1560 agaggggtgg aaatcaagat gtgtctgagt atctcatgtc cctgagtggt ccaggctgct 1620 gacttcactc ccccaagtga gggaggccat ggtgagtaca cacacctcac acatactata 1680 tccaacacac acacacacac acacacacac acgcacgcac gcacgcacgc acgcacacat 1740 gcacacacac gaactacatt tcacaaacca catacgcata ttacacccca aacgtatcac 1800 ctatacatac cacacataca cacccctcca cacatcacac acataccaca cccacacaca 1860 gcacacacat acataggcac acattcacac accacacata tacatttgtg tatgcataca 1920 tgcatacaca cacaggcaca cagacaccac acacatgcat tgtgtacgca cacatgcata 1980 cacacacata ggcacacatt gagcacacac atacatttgt gtacgcacac tacatagaca 2040 tatatgcatt tgtatatgca cacatgcatg cacacataca taggcacaca tagagcacac 2100 acatacattt gtgtatgcac acatgcacac accaatcaca tgggaagact caggttcttc 2160 actaaggttc acatgaactt agcagttcct ggttatctcg tgaaacttgg aagattgctg 2220 tggagaagag gaagcgttgg cttgagccct ggcagcaatt aaccccgccc agaagaagta 2280 ggtttaaaaa tgagagggtc tcaatgtgga acccgcaggg cgccagttca gagaagagac 2340 ctacccaagc caactgagag caaaggcaga gggatgaacc tgggatgtag tttgaacctc 2400 tgtaccagct gggcttcatg ctattttgtt atatctttat taaatattct tttagtttta 2460 tgtgcgtgaa taccttgctt gcataaatgt atgggcactg tatgtgttct tggtgccggt 2520 ggaggccagg agagggcatg gatcctccgg agctggcgtt tgagacagtt gtgacccaca 2580 gtgtggggtc tgggaactgg gtcttagtgt tccgcaagtg cagctggggc tcttaacctc 2640 tgagccatcc ctccagcttc aagaaactta ttttcttagg acatggggga agggatccag 2700 ggctttaggc ttgtttgttc agcaaatact cttttcgtgt attttgaatt ttattttatt 2760 ttactttttt gggatagaat cacattctgc agctcaggct gggcctgaac tcatcaaaat 2820 cctcctgtct cagtctacca ggtgataaga ttactgatgt gagcctggct ttgacaagca 2880 ctttagagtc cccagccctt ctggacactt gttccaagta taatatatat atatatatat 2940 atatatatat atatatatat atatattgtg tgtgtgtgtt tgtgtgtgta tgagacactt 3000 gctctaaggg tatcatatat atccttgatt tgcttttaat ttatttttta attaaaaatg 3060 attagctaca tgtcacctgt atgcgtctgt atcatctata tatccttcct tccttctctc 3120 tctttctctc ttcttcttct cacccccaag catctatttt caaatccttg tgccgaggag 3180 atgccaagag tctcgttggg ggagatggtg aggggcgat acaggggaag agcaggagga 3240 3300 gtccctggtg tcacctctta cagccaacac cattttgtgg cctggcagaa gagttgtcaa 3360 gctggtcgca ggtctgccac acaaccccaa tctggcccca agaaaaggca cctgtgtgtg 3420 actctggggt taaaggcgct gcctggtcgt ctccagctgg acttgaaact cccgtttaat 3480 aaagagttct gcaaataat acccgcagag tcacagtgcc aggttcccgt gctttcctga 3540 agcgccaggc acgggttccc taggaaatgg ggccttgctt gccaagctcc cacggcttgc 3600 cctgcaaacg gcctgaatga tctggcactc tgcgttgcca ctgggaatgaa atggaaaaaa 3660 gaaaagaag aagtgtctct ggaagcgggc gcgctcacac aaacccgcaa cgattgtgta 3720 aacactctcc attgagaatc tggagtgcgg ttgccctcta ctggggagct gaagacagct 3780 agtggggcg gggggaggac cgtgctagca tccttccacg gtgctcgctg gctgtggtgc 3840 atgccgggaa ccgaaacgcg gaactaaagt caagtcttgc tttggtggaa ctgacaatca 3900 acgaaatcac ttcgattgtt ttcctctttt tactggaatt cttggatttg atagatgggg 3960 gaggatcaga gggggagggg aggggcgggg agacggaggg aggaggggag gaggggagga 4020 ggggaggagg ggaggagggg aagggatgga ggaaaatact aacttttcta attcaacatg 4080 acaaagattc ggagaaagtg caccgctagt gaccgggagg aggaatgccc tattgggcat 4140 tatattccct gtcgtctaat ggaatcaaac tcttggttcc agcaccaagg attctgagcc 4200 tatcctattc aagacagtaa ctacagccca cacggaagag gctatacaac tgaagaaata 4260 aaatttcac tttatttcat ttctgtgact gcatgttcac atgtagagag ccacctgtgt 4320 ctaggggctg atgtgctggg cagtagagtt ctgagcccgt taactggaac aacccagaac 4380 tcccaccaca gttagagctt gctgagagag ggaggccctt ggtgagattt ctttgtgtat 4440 ttatttagag acagggtctc atactgtagt ccaagctagc ctccagctca cagaaattct 4500 cctgttccgg tttccaaagt actggagtta tgagtgtgtg ttaattgaac gctaagaatt 4560 tgctgattga agaaaacctc aagtggtttt ggctaatccc cacgacccca gaggctgagg 4620 caggaggaat gagagaatttc aaggttgcc agagccacag ggtgagctca atgtggagac 4680 tgtgaggtg agctcaatgt ggagactgtg agggtgagct caatgtggag actgtgaggg 4740 tgagctcaat gtggagactg tgaggtgag ctcaatgtgg agactgtgag ggtgagctca 4800 atgtggagac ctgtatcaag ataatatag tagtagtac atgcaggcg agggtgtggt 4860 tgagtgtag agcagttagt tgattgaca tgcttgaggt ctcccggtcc atctgtggcc 4920 ctgcaacagg agggaggga gggagggggg gagagagagagagagag agahagaagc 4980 taggatagggg atgagag gagggaa acgggaa attcagactc cttcctgagt 5040 tccgccaacg cctagtgaca tcctgtgcac accctaggt ggccttgtg tgcactgc 5100 ttgggtggtc gggaaaggca tttcagctt gttgcagaac tgccacagta gcatgctggg 5160 tccgtgaaag ttctcccg ttaacaagaa gtcttacta cttgtgacct caccagtgaa 5220 aatttcttta attgtctcct gttgttctgg gttttgcatt ttgtttcta aggatacatt 5280 cctgggtgat gtcatgaagt ccccaagac acagtggggc tgtgttgat tgggaagat 5340 gatttatctg gggtgtcaa aggaaagaa gggaacagg cacttgggaa atgtcctcc 5400 cgcccacccg aattttggct tggcaccgt ggtggaggag cagaacac gtggacgttt 5460 gaggaggcat ggggtcctag gaggacagga agcagaagga gagagctggg ctgacagcct 5520 gcaggcattg cacagttca gaggagatt acagcatgac tgagtttta gggatccaac 5580 agggacctgg gtagagattc tgtgggctct gaggcaactt gacctcagcc agatgtatt 5640 tgaataacct gctcttagag ggaaaacaga catagcaac agagccacgt ttagtgag 5700 aactctcact ttgcctgagt catgtgcggc catgcccagg gtcaggctg acacctcact 5760 caaaaacaag tgagaattg agacaatcc gtggtggcag ctactggaag ggccaccaca 5820 tccccagaaa gagtggagct gctaaaaagc cattgtgat aggcacagtt atcttgaatg 5880 catggagcag agattacgga aaatcgaga atgttaatga ggcaacattc gagttgagtc 5940 attcagtgtg ggaaacccag acgctccat ccctaaag gaacatctg ctctcagtca 6000 aaatggaaat aaaaattggg gcttgaattt ggcaaatgat tcagaactct gtgtaggtat 6060 tttcacacgc acagtggata attttcatgt tggagtttat ttgtgctaaa aggcagaaaa 6120 gggtaaaaag cacatcttaa gagttatgag gttctacgaa taaaaataat gttacttaca 6180 gctattcctt aattagtacc cccttccacc tgtggtaatt tcctgagata gtcagtgggg 6240 aaaagatctc tccttctctt ctttctcccc ctcccctcct ctccctccct ccctccctcc 6300 ctccctcctc tccctccctc cccctttcct tctttctttg ctccttctcc tctgcctcct 6360 tctccctttc ttcttcattt attctaagta gcttttaaca gcacaccaat tacctgtgta 6420 taacgggaaa acacaggctc aagcagctta gagaagattg atctgtgttc act 6473 <211>7045 <212>DNA <213>Cricetulus griseus <400>2 (SEQ ID NO: 12) actagcgtgc aattcagagg tgggtgaaga taaaaggcaa acatttgagg ccatttcctt 60 atttggcacg gcacttagga agtggaacat gcctaatcta ctggtttgta ccacctttcc 120 ctataatgga ctgtttggga agctcctggg caacgattc tggcatca tggtcagag 180 gcctgttaaa tggtactctt atttgcaaag aaggctgtaa cttgtagctt taaaagccctc 240 tcctcaagaa agaagggaga aaggatatgg ctagacatat ctaatagact taaccactgt 300 gaaaagcctt agtatgaatc agatagaacc tattttaac tcagttttga aaaaaataat 360 ctttatattt atttgtgtgt gtgtgtgt gtgtgtgt gtgtgtgt gtgtgtgtgt 420 gaaccacatg tagcaggtgc tggaggc cagagaggg caccagatct cctggactg 480 acaccacaca tggttatgag ctgcctgatg tggtgctgg gaactgaact ctcgtgttct 540 gcaagagcag caacgttct cttaacgat gagccatctc tccagccccc cccataattt 600 taattgttca ttttagtaaa ttttattcat atcaattat cacagtataa aacaatgatt 660 ttatatatat catatacata tcaggatga cagtgagggg gatatgtgtg tgtgtgtgtg 720 tgtgtgtgtg tgtgtgtgtg tgtgttattt gtgtgtgtgc ttttaagaa ggtgccatag 780 tcactgcatt tctctgaagg attcaagg aatgagacat gtctgtctgc caggaaccct atcttcctct ttgggaatct gacccaaatg aggtattctg aggaactgaa tgaagagctc 900 aagtagcagt gtcttaaacc caaatgtgct gtctagagaa agtcaacgtc atcagtgagc 960 tgaggagaga tttactgagc ggaagacaag cgctctttga tttaagtggc tcgaacagtc 1020 acggctgtgg agtggagcct gtgctcaggt ctgaggcagt ctttgctagc cagctgtgat 1080 gagcagtgaa gaaagggtgg agatggaggc agggtgggag cagggctatg gttcagacta 1140 ggtatcgtga gcacaccagc tggttgactt gtggtctgtg ggtcaggcgt tgtaaacgcc 1200 ctcagggtca ggcagtcaca ttgcttgaag ctgaatgggt gaggcaacac agagagtgca 1260 aagaaggcaa agtaccacct cttccccgac ccaggtcact tctggggtat agctgagact 1320 ccggacagca tgcaaccagc tggttagagc ttcagggaaa acttgatgtc tgcatgttgc 1380 tatgaaatgt gattcggtac atctggagaa aatttataat gctggctcag tcaagcactg 1440 aaaaggta ccttggcttt gggagctaca tgacattgac ttgtaggcag actttttttt 1500 ttctgcccgc caattcccag ataaccaata tggaggctca atattaatta taaatgctcg 1560 gctgatagct caggcttgtt actagctaac tcttccaact taaatgaacc catttctatt 1620 atctacattc tgccacgtga cttaccttg tacttcctgt ttcctctcct tgtctgactc 1680 tgcccttctg cttcccagag tccttagtct ggttctcctg cctaacctta tcctgcccag 1740 ctgctgacca agcatttata attaatatta agtctcccag tgagactctc atccagggag 1800 gacttgggtg ctcccccctc ctcattgcca tccgtgctt cctctccct cgcttccccc 1860 tccttctct gctcttcctc ctccacccct cctttcatag tattgatggc aagggtgttc 1920 tagaatggag gagtgcccat aggcatgcaa agaaaccagt taggatgctc tgtgaggggt 1980 tgtaatcata agcgatggac acaattcaag ccacagagtg aagacggaag gatgcactgt 2040 gctctagagc aacttctggg gcagaatcac agggtgagtt tctgacttga gggcgaagag 2100 gccacgagga agggagtgag tttgtctgag ctagaagcta cggcccacct cttggtagca 2160 gacctgccca caagcatgct ttgttaatca tgtgggatct gattttcctc taaatctatg 2220 ttcaactctt aagaaaatgt gaattctcac attaaaattt agatatacgt cttttggtgg 2280 ggggggtgta aaaaatcctc aagaatatgg atttctgggg gccggagaga tggctcagag 2340 gttaagagaa ctggttgctc ttctagacat tctgagttca attcccagca accacatggt 2400 ggctcacaac catctgtaat gcgacctggt gccatctttct gacatgcatg gatacatgca 2460 ggcagaaagc tgtatacata gtaaattgat aaatcttttt ttaaaaagag tatggattct 2520 gccgggtgtt ggtggcgcac gcctttaatc ccagcactct ggaggcagag gcaggtggat 2580 ctctgtgagt tcgagaccag cctggtctat aagagctagt tccaggacag cctccaaagc 2640 cacagagaaa ccctgtctcg aaaaaccaaa aaaaaaaaaa aaaaaaaaa aaaaaaaaga 2700 gtatggattc taagaaagcc gtaacagctg gagctgtgta cggagttcag cgtggtacta 2760 gaagaacaga cattcatgat gaaacacccc aggattttta cttagtatct agtttccatt 2820 gttgttttga gaccggctct tatgctctcc aggctggcct caaactgctg atcttcccgc 2880 ctctacctct caagtcctgg gactacttgg ctcataaaac agttttgtc gggctccctg 2940 aagttatggt tgtacaaacc gtgggggtca atatactcac ttgggcagag agagaaggtc 3000 tgaatcccag acaatgactg catctcagga cagttgggaa gaggacaatg gcagaaggac 3060 ttagaaaaga tagactggag ggtggaaaag cagcaggaac agagaaacaa aacaggaagc 3120 ttgctatcca gggccactct ggagtcctgt ggcaagatgg aagcgggcta ggggaataca 3180 tttgtgctac tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgat caatgcctat 3240 caatgttgaa ggggaaatat gtataccaca ttgattctgg gagcaattct cagtatctgg 3300 cctagagaaa ggaatggcccc ctgcagaata gacagagtga atggtgccct ttatcatttg 3360 ctaaagtgaa ggagaaataa acatccttcc atagagtttc aggtaaatga accccacagt 3420 tcatctgtgc cgtggtggag gcctggccaa cagttaaaaa gattagacac ggacaaagtc 3480 tgaaggaaac acctcgaata ggaagaggag agccacctca ttctgtaact ttcctcaagg 3540 ggaagatgtt ccaagagtgg gaataaatgg tcaaaggggg gatttttaat taggaaaacg 3600 atttcctgta tcacttgtga aactggaggt tgatttgggg cataggacaa tagatttgat 3660 gctttgcaaa aagctgtttc aaagcagaga aatggaatag agacaattat gtagcgagga 3720 gggagggtgg ggcgaagatg gagacagaga agtggaagct gactttaggg aagagaaca 3780 tagaccacag gggcggggcg gggggcaggg gcggggggcg gggctcaaag gaggcagtgg 3840 gaacgttgct agtgttcgca gcgtaagcgt gaatgtgcaa gcgtctttgt ggtgtgtgac 3900 caggagtagc gtggctggct tgtgtgctgc ttgtaatccc agtctttgag gtttccacac 3960 tgttccacag tgggtgtgat tttccctcgg agagcatgag ggctctgctt tccccacatc 4020 ctccccagcg ttcgttggta tttgtttcca agatgttagt gggtgagaca aagcctctct 4080 gttgatttgc ctttaacagg tgacaaaaaa agctcaacca ggagacattt ttgccttctt 4140 ggaaggtaat gctcccatgt agagcaatgg gacccatctc taaggtgagg ctactcttgc 4200 agtttgcacc cagctcttct gatgcaggaa ggaagttggt gggcaagcaa gactgtttgc 4260 ttcttgcgat ggacacattc tgcacaaa ggctcaggag gggagaaggc tgtttgatgt 4320 ttagcactca ggaaggcccc tgatgcatct gtgattagct gtctccatct gtggagcaga 4380 cacggactaa ctaaaaacca gtgtttttaa attgtcaagc ctttaaggtg aggaaattga 4440 cttattgtgc tgggccatac gtagagcaag tgctctgcat tgggccaacc cccggctctg 4500 gttctaggc accagaatgg cctagaacta actcacaatc ctcccattc aggtctcagg 4560 tgctagaatg aaccactata ccagcctgcc tgcctgccta cctgccttcc taaattttaa 4620 atcatgggga gtaggggaga atacacttat cttagttagg gtttctattg ctgtgaagag 4680 acaccatgag catggcaact cttataaagg aaacattta gttgggtggc agtttcagag 4740 gttttagtac attgtcatca tggctgggaa catgatggca tgcagacaga catggtgctg 4800 gagaaaggga tgagagtcct acatcttgca ggcaacagga cctcagctga gacactggct 4860 ggtaccctga gcataggaaa cctcacagcc caccctcaca gtgacatatt tccttcaaca 4920 aagccatacc tcctaatagt gccactccct atgagatgac agggccaatt acattcaaac 4980 tgctataaca ctttaaagta ttttattttt attattgtaa attatgtatg tagctgggtg 5040 gtggcagccg aggtgcacgc cttaatccc agcacttggg aggcagaggc agatggatct 5100 ctgtgagttc aagaccagcc tggtctataa gagctagttg caaggaagga tatacaaaga 5160 acagttctag gatagccttc aaagccacag agaagtgctg tcttgaaaac caaaaattgt 5220 gctgggacct gtctctgctt tggttgcttc ccactccccc agagctggac tcttggtcaa 5280 cactgaatca gctgcaaaat aaactcctgg attcctctct tgtaacagga gcccgaagtc 5340 aggcgcccac ttgtcttctc gcaggattgc catagacttt ttctgtgtgc ccaccattcc 5400 agactgaagt agagatggca gtggcagaga ctgggaaggc tgcaacgaaa acaggaagtt 5460 attgcaccct gggaatagtc tggaaatgaa gcttcaaaac ttgcttcatg ttcagttgta 5520 cacagactca ctcccaggtt gactcacacg tgtaaatatt cctgactatg tctgcactgc 5580 ttttatctga tgcttccttc ccaaaatgcc aagtgtacaa ggtgagggaa tcacccttgg 5640 attcagagcc cagggtcgtc ctccttaacc tggacttgtc tttctccggc agcctctgac 5700 acccctcccc ccattttctc tatcagaagg tctgagcaga gttggggcac gctcatgtcc 5760 tgatacactc cttgtcttcc tgaagatcta acttctgacc cagaaagatg gctaaggtgg 5820 tgaagtgttt gacatgaaga cttggtctta agaactggag caggggaaaa aagtcggatg 5880 tggcagcatg tacccgaaat cccagaactg gggaggtaga gacggatgag tgcccggggc 5940 tagctggctg ctcagccagc ctagctgaat tgccaaattc caactcctat tgaaaaacct 6000 ttaccaaaca aacaaacaaa caaataataa caacaacaac aacaacaaac taccccatac 6060 aaggtgggcg gctcttggct cttgaggaat gactcaccca aacccaaagc ttgccacagc 6120 tgttctctgg cctaaatggg gtgggggtgg ggcagagaca gagacagaga gagacatgac 6180 ttcctgggct gggctgtgtg ctctaggcca ccaggaactt tcctgtcttg ctctctgtct 6240 ggcacagcca gagcaccagc acccagcagg tgcacacacc tccctccgtg cttcttgagc 6300 aaacacaggt gccttggtct gtctattgaa ccggagtaag ttcttgcaga tgtatgcatg 6360 gaaacaacat tgtcctggtt ttatttctac tgttgtgata aaaaccgggg aactccagga 6420 agcagctgag gcagaggcaa atgcaaggaa tgctgcctcc tagcttgctc cccatggctt 6480 gccgggcctg ctttctgcaa gcccttctct ccccattggc atgcctgaca tgaacagcgt 6540 ttgaaatgct ctcaaatgtc actttcaaag aaggcttctc tgatcttgct aactaaatca 6600 gaccatgttt caccgtgcat tatctttctg ctgtctgtct gtctgtctgt ctgtctatct 6660 gtctatcatc tatcaatcat ctatctatct atcttctatt tatctaccta tcattcaatc 6720 atctatcttc taactagtta tcatttattt atttgtttac ttactttttt tatttgagac 6780 agtatttctc tgagtgacag ccttggctgt cctggaaccc attctgtaac caggctgtcc 6840 tcaaactcac agagatccaa ctgcctctgc ctctctggtg ctggggttaa agacgtgcac 6900 caccaacgcc ccgctctatc atctatttat gtacttatta ttcagtcatt atctatcctc 6960 taactatcca tcatctgtct atccatcatc tatctatcta tctatctatc tatctatcta 7020 tctatcatcc atctataatc aattg 7045 <211>6473 <212>DNA <213>Cricetulus griseus <400>3 (SEQ ID NO: 13) agtgaacaca gatcaatctt ctctaagctg cttgagcctg tgttttcccg ttatacacag 60 gtaattggtg tgctgttaaa agctacttag aataaatgaa gaagaaaggg agaaggaggc 120 agaggagaag gagcaaagaa agaaggaaag ggggagggag ggagaggagg gagggaggga 180 gggagggagg gagaggaggg gagggggaga aagaagagaa ggagagatct tttccccact 240 gactatctca ggaaattacc acaggtggaa gggggtacta attaaggaat agctgtaagt 300 aacattattt ttattcgtag aacctcataa ctcttaagat gtgcttttta cccttttctg 360 ccttttagca caaataaact ccaacatgaa aattatccac tgtgcgtgtg aaaataccta 420 cacagagttc tgaatcattt gccaaattca agccccaatt tttatttcca ttttgactga 480 gagcaagatg ttccttttag gggatggaag cgtctgggtt tcccacactg aatgactcaa 540 ctcgaatgtt gcctcattaa cattctcgat ttttccgtaa tctctgctcc atgcattcaa 600 gataactgtg cctatcacaa atggcttttt agcagctcca ctctttctgg ggatgtggtg 660 gcccttccag tagctgccac cacggattgt cttcaatttc tcacttgttt ttgagttgag 720 tgtcagcctg acccctgggc atggccgcac atgactcagg caaagtgaga gtttcatcac 780 taaacgtggc tctgtttgct atgtctgttt tccctctaag agcaggttat tcaaatacca 840 tctggctgag gtcaagttgc ctcagagccc acagaatctc tacccaggtc cctgttggat 900 ccctaaaaac tcagtcatgc tgtaatctcc tctgaact gtgcaatgcc tgcaggctgt 960 cagcccagct ctctccttct gctcctgtc ctcctaggac cccatgcctc ctcaacgtc 1020 cacgtgtttc ttgctcctcc accacggttg ccaagccaaa atcgggtgg gcggggaggac 1080 attttcccaa gtgcctgttt ccctttttt ccttttgaca ccccagataa atcatctttc 1140 ccaatccaac acagccccac tgtgtctttg gggacttcat ccaatcacc aggaatgtat 1200 ccttagaaac aaaaatgca aacccagaac accaggac attaaagaa atttcactg 1260 gtgaggtcac aagtagtaga gactcttgt taacgggcag aactttcac ggacccagca 1320 tgctactgtg gcagttctgc aacaagctga aaatgccttt cccgaccacc caagccagtg 1380 ccacacaag gccaccttag ggtgtgcaca ggatgtcact aggcttggc ggaactcagg 1440 aaggagtctg aatttctcc cgttctctcc ttccctctc atccctc ttagctctg 1500 tctctcttc ctctctctcg tccccccct tcctccctcc cttcctgtg cagggccaca 1560 gatggaccgg gagacctca gcatgtcaa tcacaac gctctaccac tcaccac 1620 cctcgcctgc attgttacta ctactattat tatcttgata caggtctcca cattgagctc 1680 accctcacag tctccacatt gagctcaccc tcacagtctc cacattgagc tcaccctcac 1740 agtctccaca ttgagctcac cctcacagtc tccacattga gctcaccctc acagtctcca 1800 cattgagctc accctgtggc tctggcaaac cttgaattct ctcattcctc ctgcctcagc 1860 ctctggggtc gtggggatta gccaaaccca cttgaggttt tcttcaatca gcaaattctt 1920 agcgttcaat taacacacac tcataactcc agtactttgg aaaccggaac aggagaattt 1980 ctgtgagctg gaggctagct tggactacag tatgagaccc tgtctctaaa taaatacaca 2040 aagaaatctc accaagggcc tccctctctc agcaagctct aactgtggtg ggagttctgg 2100 gttgttccag ttaacgggct cagaactcta ctgcccagca catcagcccc tagacacagg 2160 tggctctcta catgtgaaca tgcagtcaca gaaatgaaat aaagtgaaaa ttttatttct 2220 tcagttgtat agcctcttcc gtgtgggctg tagttactgt cttgaatagg ataggctcag 2280 aatccttggt gctggaacca agagtttgat tccattagac gacagggaat ataatgccca 2340 atagggcatt cctctcccg gtcactagcg gtgcactttc tccgaatctt tgtcatgttg 2400 aattagaaaa gttagtattt tcctccatcc cttcccctcc tcccctcctc ccctctccc 2460 ctcctcccct cctccctccg tctccccgcc cctcccctcc ccctctgatc ctcccccatc 2520 tatcaaatcc aagaattcca gtaaaagag gaaaaacaatc gaagtgattt cgttgattgt 2580 cagttccacc aaagcaagac ttgactttag ttccgcgttt cggttcccgg catgcaccac 2640 agccagcgag caccgtggaa ggatgctagc acggtcctcc ccccgccccc actagctgtc 2700 ttcagctccc footgagg caaccgcact ccagattctc aatggagagt gtttacacaa 2760 tcgttgcggg tttgtgtgag cgcgcccgct tccagagaca cttcttcttt ttcttttttc 2820 catttcatcc cagtggcaac gcagagtgcc agatcattca ggccgtttgc agggcaagcc 2880 gtgggagctt ggcaagcaag gccccatttc ctagggaacc cgtgcctggc gcttcaggaa 2940 agcacgggaa cctggcactg tgactctgcg ggtattattt tgcagaactc tttattaaac 3000 gggagtttca agtccagctg gagacgacca ggcagcgcct ttaaccccag agtcacacac 3060 aggtgccttt tcttggggcc agattggggt tgtgtggcag acctgcgacc agcttgacaa 3120 ctcttctgcc aggccacaaa atggtgttgg ctgtaagagg tgacaccagg gacagggaag 3180 atcgctgcta ttctcctgag ctctccaaag acccacacca gtctgtcccc ctttcctcct 3240 gctcttcccc tgtatcgccc cctcaccatc tcccccaacg agactcttg catctcctcg 3300 gcacaaggat ttgaaatag atgcttgggg gtgagaagaa gaagagagaa agagagagaa 3360 ggaaggaagg atatatagat gatacagacg catacaggtg acatgtagct aatcattttt 3420 aattaaaaa taaattaaaa gcaaatcaag gatatatatg atacccttag agcaagtgtc 3480 tcacacac acaaacacac acacacaata tatatata tatatatatata tatatatatata 3540 tatatata ttacttgg aacaagtgtc cagaagggct ggggactcta aagtgcttgt 3600 caaagccagg ctcacatcag taatcttatc acctggtaga ctgagacagg aggattttga 3660 tgagttcagg cccagcctga gctgcagaat gtgattctat cccaaaaaag taaaataaaa 3720 taaattcaa aatacacgaa aagagtattt gctgaacaaa caagcctaaa gccctggatc 3780 ccttccccca tgtcctaaga aaataagtttt cttgaagctg gagggatggc tcagaggtta 3840 agagccccag ctgcacttgc ggaacactaa gacccagttc ccagacccca cactgtgggt 3900 cacaactgtc tcaaacgcca gctccggagg atccatgccc tctcctggcc tccaccggca 3960 ccaagaacac atacagtgcc catacattta tgcaagcaag gtattcacgc acataaaact 4020 aaaagaatat ttaataaaga tataacaaaa tagcatgaag cccagctggt acagaggttc 4080 aaactacatc ccaggttcat ccctctgcct ttgctctcag ttggcttggg taggctctt 4140 ctctgaactg gcgccctgcg ggttccacat tgagaccctc tcatttttaa acctacttct 4200 tctgggcggg gttaattgct gccagggctc aagccaacgc ttcctcttct ccacagcaat 4260 cttccaagtt tcacgagata accaggaact gctaagttca tgtgaacctt agtgaagaac 4320 ctgagtcttc ccatgtgatt ggtgtgtgca tgtgtgcata cacaaatgta tgtgtgtgct 4380 ctatgtgtgc ctatgtatgt gtgcatgcat gtgtgcatat acaaatgcat atatgtctat 4440 gtagtgtgcg tacacaaatg tatgtgtgtg ctcaatgtgt gcctatgtgt gtgtatgcat 4500 gtgtgcgtac acaatgcatg tgtgtggtgt ctgtgtgcct gtgtgtgtat gcatgtatgc 4560 atacacaaat gtatatgtgt ggtgtgtgaa tgtgtgccta tgtatgtgtg tgctgtgtgt 4620 gggtgtggta tgtgtgtgat gtgtggaggg gtgtgtatgt gtggtatgta taggtgatac 4680 gtttggggtg taatatgcgt atgtggtttg tgaaatgtag ttcgtgtgtg tgcatgtgtg 4740 cgtgcgtgcg tgcgtgcgtg cgtgtgtgtg tgtgtgtgtg tgtgtgtgtt ggatatagta 4800 tgtgtgaggt gtgtgtactc accatggcct ccctcacttg ggggagtgaa gtcagcagcc 4860 tggaccactc agggacatga gatactcaga cacatcttga tttccacccc tcttttcctg 4920 atcctccttc acgtgtcact ttcccaaaca ctggacaaca gtttgggggc atctgattcc 4980 actaatgaca gggacatcac atgtctccag agggaacacc ttctgtgtca catgtcatct 5040 gagaatgtag cagagtcaca gagaaatgtc acagaaacca aaatgcagag taccaaggta 5100 tagctaggca cagagcagag gggaagccgc tgaatttatt aaaaatgtca gaatcgtaaa 5160 agacagggga cagcggtggg gacattcagg gtccagtagc acacaggcag tccaaacctg 5220 atcactggaa ggtagtaggt aaggaaaggc tgcacacaga ttattcacac agtttataca 5280 tgtacacaga ttatcacat ggtttgtgta tgtgcacaga ttattcacac agtttataca 5340 tgtgtggctt cgtggtaact ttgagcttac tttcaattta aaaggatctc tctcacaagc 5400 tggggccggg aatggctgca gtcaacactc catcacttag tcacactgtg caaacagcac 5460 ctcctgactc atggtgactt gtagtaaaat gaagaggcca catttgcatc caagacagct 5520 catcagtacc tagtgaagaa tctgtccctg agtatttgca tgaatggacc cgggtccagg 5580 gcctggctgg gagtctccag gtgttgcagc cagaatgtca ttgtgttttt tcaggatccc 5640 5700 agtgattagg taacaaag ccttttaaaa accgagatca cccttgtcat gtccctggcc 5760 tcttagaaca agatccaagc ttttgctggt tgacaagtgg ggccatccag tgcgtctccg 5820 ttcctgctac ttcatctgga agacctctcc cactaacttg cccctgaccc ctcacaccctg 5880 ctgtttcctt tccacccgga agtgcttgtc taggctttca tggccatctg actgagcatc 5940 taggcctcag tccagtggtc cctcagctct ctctagtcac tgtactaatg gaaacggcca 6000 ctaactacat tttcaatatg gaagcctcct cctcaggaac ctccaagggc agaagcctcc 6060 agagaaccac tcctgacccc ctggagttct gagtgcttct ggccctctct gtgtctgcag 6120 gactattcac cacttgtgtt gaatggttca gtcctcacct cctctggcat gtgctcagtt 6180 ctcatctcat tggggagtcc ttcccaggtc actcttctct cctgtctttg aagtgttttt 6240 ttccttcatg gtatttctgt ctgggcacac acacagacac acatacacac acatacacac 6300 ccatgcagta tggcagatac atcacctatg tttcagattt ttattctacc atcacccaat 6360 acctgaatcc ccgaaaaagc cttagaaagc caggaatttg tgtatttttg tcagcactcc 6420 accccagcac ctgaagccaa gcctgactta atatttttgg ttttgtttct aga 6473 <211>7045 <212>DNA <213>Cricetulus griseus <400>4 (SEQ ID NO: 14) caattgatta tagatggatg atagatagat agatagatagat agatagatagat agatagatga 60 tggatagaca gatgatgat agttagaga tagataatga ctgaataata agtacataaa 120 tagatgatag agcggggcgt tggtggtgca cgtctttaac cccagcacca gagaggcaga 180 ggcagttgga tctctgtgag tttgaggaca gctgttac agaatggtt ccaggacagc 240 aaggctgtc actcagagaa atactgtctc aaaaaaaa agtaagtaaa caataata 300 aatgatact aggataga taggattg atgatagt agataatag agatagata 360 tgagatt tgagatga tgahagagagagagagagagagagagagagagagagagagagagaga 420 agataatgca cggtgaaca tggtctgatt tagttagca gatcagagaa gccttctttg 480 aaagtgacat ttgagagcat ttcaacgct gttcatgtca ggcatgccaa tggggagaga 540 agggcttgca gaaagcaggc ccggcaagcc atgggagca agctaggagg cagcattcct 600 tgcatttgcc tctgcctcag ctgcttcctg gagttccccg gttttatca caacagtaga 660 aaaaacca ggacaatgtt gtttccatgc atacatctgc aagaacttac tccggttca 720 tagacagacc aaggcacctg tgttgctca agaagcacgg agggaggtgtgcacctgc 780 tgggtgctgg tgctctggct gtgccagaca gagagcaaga caggaagtt cctggtggcc 840 900. 900. gtctctctct gtctctgtct ctgccccacc cccaccccat ttaggccaga gaacagctgt ggcaagcttt gggtttgggt gagtcattcc tcaagagcca agagccgccc accttgtatg gggtagtttg ttgttgttgt tgttgttatt 1020 atttgtttgt ttgtttgttt ggtaaaggtt tttcaatagg agttggaatt tggcaattca gctaggctgg ctgagcagcc agctagcccc gggcactcat ccgtctctac ctccccagtt 1140 ctgggatttc gggtacatgc tgccacatcc gacttttttc ccctgctcca gttcttaaga ccaagtcttc atgtcaaaca cttcaccacc ttagccatct ttctgggtca gaagttagat cttcaggaag acaaggagtg tatcaggaca tgagcgtgcc ccaactctgc tcagaccttc tgatagagaa aatgggggga ggggtgtcag aggctgccgg agaaagacaa gtccaggtta aggaggacga ccctgggctc tgaatccaag ggtgattccc tcaccttgta cacttggcat tttgggaagg aagcatcaga taaaagcagt gcagacatag tcaggaatat ttacacgtgt gagtcaacct gggagtgagt ctgtgtacaa ctgaacatga agcaagttt gaagcttcat 1560 ttccagacta ttcccagggt gcaataactt cctgttttcg ttgcagcctt cccagtctct 1620 gccactgcca tctctacttc agtctggaat ggtgggcaca cagaaaaagt ctatggcaat 1680 cctgcgagaa gacaagtggg cgcctgactt cgggctcctg ttacaagaga ggaatccagg 1740 agtttatttt gcagctgatt cagtgttgac caagagtcca gctctggggg agtgggaagc 1800 aaccaaagca gagacaggtc ccagcacaat ttttggttt caagacagca cttctctgtg 1860 gctttgaagg ctatcctaga actgttcttt gtatatcctt ccttgcaact agctcttata 1920 gaccaggctg gtcttgaact cacagagatc catctgcctc tgcctcccaa gtgctgggat 1980 taaaggcgtg cacctcggct gccaccaccc agctacatac ataatttaca samaaaaaaa 2040 taaaatactt taaagtgtta tagcagtttg aatgtaattg gccctgtcat ctcataggga 2100 gtggcactat taggaggtat ggctttgttg aaagaatat gtcactgtga gggtgggctg 2160 tgaggtttcc tatgctcagg gtaccagcca gtgtctcagc tgaggtcctg ttgcctgcaa 2220 gatgtaggac tctcatccct ttctccagca ccatgtctgt ctgcatgcca tcatgttccc 2280 agccatgatg acaatgtact aaaacctctg aaactgccac ccaactaaat gttttccttt 2340 ataagagttg ccatgctcat ggtgtctctt cacagcaata gaaaccctaa ctaagataag 2400 tgtattctcc cctactcccc atgatttaaa atttaggaag gcaggtaggc aggcaggcag 2460 gctggtatag tggttcattc tagcacctga gacctggaat gggaggattg tgagttagtt 2520 ctaggccatt ctggtgccta gaaaccagag ccgggggttg gcccaatgca gagcacttgc 2580 tctacgtatg gcccagcaca ataagtcaat ttcctcacct taaaggcttg acaatttaaa 2640 aacactggtt tttagttagt ccgtgtctgc tccacagatg gagacagcta atcacagatg 2700 catcaggggc cttcctgagt gctaaacatc aaacagcctt ctcccctcct gagcctttgt 2760 gtgcagaatg tgtccatcgc aagaagcaaa cagtcttgct tgcccaccaa cttccttcct 2820 gcatcagaag agctgggtgc aaactgcaag agtagcctca ccttagagat gggtcccatt 2880 gctctacatg ggagcattac cttccaagaa ggcaaaaatg tctcctggtt gagctttttt 2940 tgtcacctgt taaaggcaaa tcaacagaga ggctttgtct cacccactaa catcttggaa 3000 acaaatacca acgaacgctg gggaggatgt ggggaaagca gagccctcat gctctccgag 3060 ggaaaatcac acccactgtg gaacagtgtg gaaacctcaa agactgggat tacaagcagc 3120 acacaagcca gccacgctac tcctggtcac acaccacaaa gacgcttgca cattcacgct 3180 tacgctgcga acactagcaa cgttcccact gcctcctttg agccccgccc cccgcccctg 3240 ccccccgccc cgcccctgtg gtctatgttc ctcttcccta aagtcagctt ccacttctct 3300 gtctccatct tcgccccacc ctccctcctc gctacataat tgtctctatt ccatttctct 3360 gctttgaaac agctttttgc aaagcatcaa atctattgtc ctatgcccca aatcaacctc 3420 cagtttcaca agtgatacag gaaatcgttt tcctaattaa aaatcccccc tttgaccatt 3480 tattcccact cttggaacat cttccccttg aggaaagtta cagaatgagg tggctctcct 3540 cttcctattc gaggtgtttc cttcagactt tgtccgtgtc taatcttttt aactgttggc 3600 caggcctcca ccacggcaca gatgaactgt ggggttcatt tacctgaaac tctatggaag 3660 gatgtttatt tctccttcac tttagcaaat gataaagggc accattcact ctgtctattc 3720 tgcaggggcc attcctttct ctaggccaga tactgagaat tgctcccaga atcaatgtgg 3780 tatacatatt tccccttcaa cattgatagg cattgatcac acacacacac acacacacac 3840 acacacacac acacagtagc acaaatgtat tcccctagcc cgcttccatc ttgccacagg 3900 actccagagt ggccctggat agcaagcttc ctgttttgtt tctctgttcc tgctgctttt 3960 ccaccctcca gtctatcttt tctaagtcct tctgccattg tcctcttccc aactgtcctg 4020 agatgcagtc attgtctggg attcagacct tctctctctg cccaagtgag tatattgacc 4080 cccacggttt gtacaaccat aacttcaggg agcccgacaa aaactgtttt atgagccaag 4140 tagtcccagg acttgagagg tagaggcggg aagatcagca gtttgaggcc agcctggaga 4200 gcataagagc cggtctcaaa acaacaatgg aaactagata ctaagtaaaa atcctggggt 4260 gtttcatcat gaatgtctgt tcttctagta ccacgctgaa ctccgtacac agctccagct 4320 gttacggctt tcttagaatc catactcttt tttttttttt tttttttttt ttttttttgg 4380 tttttcgaga cagggtttct ctgtggcttt ggaggctgtc ctggaactag ctcttataga 4440 ccaggctggt ctcgaactca cagagatcca cctgcctctg cctccagagt gctgggatta 4500 aaggcgtgcg ccaccaacac ccggcagaat ccatactctt tttaaaaaaa gatttatcaa 4560 tttactatgt atacagcttt ctgcctgcat gtatccatgc atgtcagaag atggcaccag 4620 gtcgcattac agatggttgt gagccaccat gtggttgctg ggaattgaac tcagaatgtc 4680 tagagaagca accagttctc ttaacctctg agccatctct ccggcccccca gaaatccata 4740 ttcttgagga ttttttacac cccccccacc aaaagacgta tatctaaatt ttaatgtgag 4800 aattcacatt ttcttaagag ttgaacatag atttagagga aaatcagatc ccacatgatt 4860 aacaaagcat gcttgtgggc aggtctgcta ccaagaggtg ggccgtagct tctagctcag 4920 acaaactcac tcccttctc gtggcctctt cgccctcaag tcagaaactc accctgtgat 4980 tctgccccag aagttgctct agagcacagt gcatccttcc gtcttcactc tgtggcttga 5040 attgtgtcca tcgcttatga ttacaacccc tcacagagca tcctaactgg tttctttgca 5100 tgcctatggg cactcctcca ttctagaaca cccttgccat caatactatg aaaggagggg 5160 tggaggagga agagcaggaa gaggaggggg aagcgaggga agaggaagac acggatggca 5220 atgaggaggg gggagcaccc aagtcctccc tggatgagag tctcactggg agacttaata 5280 ttaattataa atgcttggtc agcagctggg caggataagg ttaggcagga gaaccagact 5340 aaggactctg ggaagcagaa gggcagagtc agacaaggag aggaaacagg aagtacaagg 5400 taaagtcacg tggcagaatg tagataatag aaatgggttc atttaagttg gaagagttag 5460 ctagtaacaa gcctgagcta tcagccgagc atttataatt aatattgagc ctccatattg 5520 gttatctggg aattggcggg cagaaaaaaa aaagtctgcc tacaagtcaa tgtcatgtag 5580 ctcccaaagc caaggtacct ttgttcagtg cttgactgag ccagcattat aaatttctc 5640 cagatgtacc gaatcacatt tcatagcaac atgcagacat caagttttcc ctgaagctct 5700 aaccagctgg ttgcatgctg tccggagtct cagctataac ccagaagtga cctgggtcgg 5760 ggaagaggtg gtactttgcc ttctttgcac tctctgtgtt gcctcaccca ttcagcttca 5820 agcaatgtga ctgcctgacc ctgaggggcgt ttacaacgcc tgacccacag accacaagtc 5880 aaccagctgg tgtgctcacg atacctagtc tgaaccatag ccctgctccc accctgccctc 5940 catctccacc ctttctcac tgctcac agctggctag caagactgc ctcagacctg 6000 agcacaggct ccactccaca gccgtgactg ttcgagccac ttaaatcaa gagcgcttgt 6060 cttccgctca gtaaatctct cctcagctca ctgatgacgt tgactctc tagacagcac 6120 atttgggttt agacactgc tacttgagct cttcattcag tcctcagaa tacctcattt 6180 gggtcagatt cccaaagagg aagatagggt tcctggcaga cagacatgtc tcattccttt 6240 gaaatccttc agagaaatgc agtgactatg gcaccttctt aaaagcaca cacacaata 6300 acacacac acacacac acacacac acacacac atatccccct cactgtcatc 6360 cttgatatgt atatgatata tataaaatca ttgttttata ctgtgataat tgattatgaa 6420 taaaatttac taaaatgac aattaaaatt atgggggggg ctggagagat ggctcatcag 6480 ttaagagaac agttgctgct ctgcagaac acgagagttc agttcccagc acccacatca 6540 ggcagctcat aaccatgtgt ggtgtcagtt ccaggagatc tggtgccctc ttctggcctc 6600 ctccagcacc tgctacatgt ggttcacaca cacacacaca cacacacaca cacacacaca 6660 cacacacaca caaataaata taaagattat ttttttcaaa actgagttaa aaataggttc 6720 tatctgattc atactaaggc ttttcacagt ggttaagtct attagatatg tctagccata 6780 tcctttctcc cttctttctt gaggagaggc ttttaaagct acaagttaca gccttctttg 6840 caaataagag taccatttaa caggcctctg accaatgaga tgccagaatc ggttgcccag 6900 gagcttccca aacagtccat tatagggaaa ggtggtacaa accagtagat taggcatgtt 6960 ccacttccta agtgccgtgc caaataagga aatggcctca aatgtttgcc ttttatcttc 7020 acccacctct gaattgcacg ctagt 7045 <211>13515 <212>DNA <213>Cricetulus griseus <400>5 (Sequence number 15) tctagaaaca aaaccaaaaa tattaagtca ggcttggctt caggtgctgg ggtggagtgc 60 tgacaaaaat acacaaattc ctggctttct aaggcttttt cggggattca ggtattgggt 120 180. gatggtagaa taaaaatctg aaacataggt gatgtatctg ccatactgca tgggtgtgta 240. tgtgtgtgta tgtgtgtctg tgtgtgtgcc cagacagaaa taccatgaag gaaaaaaaca cttcaaagac aggagagaag agtgacctgg gaggactcc ccaatgagat gagaactgag 360. cacatgccag aggaggtgag gactgaacca ttcaacacaa gtggtgaata gtcctgcaga 420. cacagagagg gccagaagca ctcagaactc cagggggtca ggagtggttc tctggaggct tctgcccttg gaggttcctg aggaggaggc ttccatattg aaaatgtagt tagtggccgt 480 ttccattagt acagtgacta gagagagctg agggaccact ggactgaggc ctagatgctc agtcagatgg ccatgaaagc ctagacaagc acttccgggt ggaaaggaa cagcaggtgt gaggggtcag gggcaagtta gtgggagagg tcttccagat gaagtagcag gaacggagac 660 gcactggatg gccccacttg tcaaccagca aaagcttgga tcttgttcta agaggccagg 720 gacatgacaa gggtgatctc ggtttttaaa aggctttgtg ttacctaatc acttctatta 840. gtcagatact ttgtaacaca aatgagtact tggcctgtat tttagaaact tctgggatcc tgaaaaaaca caatgacatt ctggctgcaa cacctggaga ctcccagcca ggccctggac 900 ccgggtccat tcatgcaaat actcagggac agattcttca ctaggtactg atgagctgtc 960 ttggatgcaa atgtggcctc ttcattttac tacaagtcac catgagtcag gaggtgctgt 1020 ttgcacagtg tgactaagtg atggagtgtt gactgcagcc attcccggcc ccagcttgtg 1080 agagagatcc ttttaaattg aaagtaagct caaagttacc acgaagccac acatgtataa 1140 actgtgtgaa taatctgtgc acatacacaa accatgtgaa taatctgtgt acatgtataa 1200 actgtgtgaa taatctgtgt gcagcctttc cttacctact accttccagt gatcaggttt 1260 ggactgcctg tgtgctactg gaccctgaat gtccccaccg ctgtcccctg tcttttacga 1320 ttctgacatt tttaataaat tcagcggctt cccctctgct ctgtgcctag ctataccttg 1380 gtactctgca ttttggtttc tgtgacattt ctctgtgact ctgctacatt ctcagatgac 1440 atgtgacaca gaaggtgttc cctctggaga catgtgatgt ccctgtcatt agtggaatca 1500 gatgccccca aactgttgtc cagtgtttgg gaaagtgaca cgtgaaggag gatcaggaaa 1560 agaggggtgg aaatcaagat gtgtctgagt atctcatgtc cctgagtggt ccaggctgct 1620 gacttcactc ccccaagtga gggaggccat ggtgagtaca cacacctcac acatactata 1680 tccaacacac acacacacac acacacacac acgcacgcac gcacgcacgc acgcacacat 1740 gcacacacac gaactacatt tcacaaacca catacgcata ttacacccca aacgtatcac 1800 ctatacatac cacacataca cacccctcca cacatcacac acataccaca cccacacaca 1860 gcacacacat acataggcac acattcacac accacacata tacatttgtg tatgcataca 1920 tgcatacaca cacaggcaca cagacaccac acacatgcat tgtgtacgca cacatgcata 1980 cacacacata ggcacacatt gagcacacac atacatttgt gtacgcacac tacatagaca 2040 tatatgcatt tgtatatgca cacatgcatg cacacataca taggcacaca tagagcacac 2100 acatacattt gtgtatgcac acatgcacac accaatcaca tgggaagact caggttcttc 2160 actaaggttc acatgaactt agcagttcct ggttatctcg tgaaacttgg aagattgctg 2220 tggagaagag gaagcgttgg cttgagccct ggcagcaatt aaccccgccc agaagaagta 2280 ggtttaaaaa tgagagggtc tcaatgtgga acccgcaggg cgccagttca gagaagagac 2340 ctacccaagc caactgagag caaaggcaga gggatgaacc tgggatgtag tttgaacctc 2400 tgtaccagct gggcttcatg ctattttgtt atatctttat taaatattct tttagtttta 2460 tgtgcgtgaa taccttgctt gcataaatgt atgggcactg tatgtgttct tggtgccggt 2520 ggaggccagg agagggcatg gatcctccgg agctggcgtt tgagacagtt gtgacccaca 2580 gtgtggggtc tgggaactgg gtcttagtgt tccgcaagtg cagctggggc tcttaacctc 2640 tgagccatcc ctccagcttc aagaaactta ttttcttagg acatggggga agggatccag 2700 ggctttaggc ttgtttgttc agcaaatact cttttcgtgt attttgaatt ttattttatt 2760 ttactttttt gggatagaat cacattctgc agctcaggct gggcctgaac tcatcaaaat 2820 cctcctgtct cagtctacca ggtgataaga ttactgatgt gagcctggct ttgacaagca 2880 ctttagagtc cccagccctt ctggacactt gttccaagta taatatatat atatatatat 2940 atatatatat atatatatat atatattgtg tgtgtgtgtt tgtgtgtgta tgagacactt 3000 gctctaagg tatcatatat atccttgatt tgcttttaat ttatttttta attaaaatg 3060 attagctaca tgtcacctgt atgcgtctgt atcatctata tatccttcct tccttctctc 3120 tctttctctc ttcttcttct cacccccaag catctatttt caaatccttg tgccgaggag 3180 atgccaagag tctcgttggg ggagatggtg aggggcgat acaggggaag agcaggagga 3240 3300 gtccctggtg tcacctctta cagccaacac cattttgtgg cctggcagaa gagttgtcaa 3360 gctggtcgca ggtctgccac acaaccccaa tctggcccca agaaaaggca cctgtgtgtg 3420 actctggggt taaaggcgct gcctggtcgt ctccagctgg acttgaaact cccgtttaat 3480 aaagagttct gcaaataat acccgcagag tcacagtgcc aggttcccgt gctttcctga 3540 agcgccaggc acgggttccc taggaaatgg ggccttgctt gccaagctcc cacggcttgc 3600 cctgcaaacg gcctgaatga tctggcactc tgcgttgcca ctgggaatgaa atggaaaaaa 3660 gaaaagaag aagtgtctct ggaagcgggc gcgctcacac aaacccgcaa cgattgtgta 3720 aacactctcc attgagaatc tggagtgcgg ttgccctcta ctggggagct gaagacagct 3780 agtgggggcg gggggaggac cgtgctagca tccttccacg gtgctcgctg gctgtggtgc 3840 atgccgggaa ccgaaacgcg gaactaaagt caagtcttgc tttggtggaa ctgacaatca 3900 acgaaatcac ttcgattgtt ttcctctttt tactggaatt cttggatttg atagatgggg 3960 gaggatcaga gggggagggg aggggcgggg agacggaggg aggaggggag gaggggagga 4020 ggggaggagg ggaggagggg aagggatgga ggaaaatact aacttttcta attcaacatg 4080 acaaagattc ggagaaagtg caccgctagt gaccgggagg aggaatgccc tattgggcat 4140 tatattccct gtcgtctaat ggaatcaaac tcttggttcc agcaccaagg attctgagcc 4200 tatcctattc aagacagtaa ctacagccca cacggaagag gctatacaac tgaagaaata 4260 aaatttcac tttatttcat ttctgtgact gcatgttcac atgtagagag ccacctgtgt 4320 ctaggggctg atgtgctggg cagtagagtt ctgagcccgt taactggaac aacccagaac 4380 tcccaccaca gttagagctt gctgagagag ggaggccctt ggtgagattt ctttgtgtat 4440 ttatttagag acagggtctc atactgtagt ccaagctagc ctccagctca cagaaatttct 4500 cctgttccgg ttccaagt actggagtta tgagtgtgtg ttaattgaac gctaagaatt 4560 tgctgattga agaaaacctc aagtggtttt ggctaatccc cacgacccca gaggctgagg 4620 caggaggaat gagagaatttc aaggttgcc agagccacag ggtgagctca atgtggagac 4680 tgtgaggtg agctcaatgt ggagactgtg agggtgagct caatgtggag actgtgaggg 4740 tgagctcaat gtggagactg tgaggtgag ctcaatgtgg agactgtgag ggtgagctca 4800 atgtggagac ctgtatcaag ataatatag tagtagtac atgcaggcg agggtgtggt 4860 tgagtgtag agcagttagt tgattgaca tgcttgaggt ctcccggtcc atctgtggcc 4920 ctgcaacagg agggaggga gggagggggg gagagagagagagagag agahagaagc 4980 taggatagggg atgagag gagggaa acgggaa attcagactc cttcctgagt 5040 tccgccaacg cctagtgaca tcctgtgcac accctaggt ggccttgtg tgcactgc 5100 ttgggtggtc gggaaaggca tttcagctt gttgcagaac tgccacagta gcatgctggg 5160 tccgtgaaag tttctgcccg ttaacaagaa gtctctacta cttgtgacct caccagtgaa 5220 aatttcttta attgtctcct ggtgttctgg gttttgcatt tttgtttcta aggatacatt 5280 cctgggtgat gtcatgaagt ccccaaagac acagtggggc tgtgttggat tgggaaagat 5340 gatttatctg gggtgtcaaa aggaaaagaa gggaaacagg cacttgggaa aatgtcctcc 5400 cgcccacccg aattttggct tggcaaccgt ggtggaggag caagaaacac gtggacgttt 5460 gaggaggcat ggggtcctag gaggacagga agcagaagga gagagctggg ctgacagcct 5520 gcaggcattg cacagtttca gaaggagatt acagcatgac tgagttttta gggatccaac 5580 agggacctgg gtagagattc tgtgggctct gaggcaactt gacctcagcc agatggtatt 5640 tgaataacct gctcttagag ggaaaacaga catagcaaac agagccacgt ttagtgatga 5700 aactctcact ttgcctgagt catgtgcggc catgcccagg ggtcaggctg acactcaact 5760 caaaaacaag tgagaaattg aagacaatcc gtggtggcag ctactggaag ggccaccaca 5820 tccccagaaa gagtggagct gctaaaaagc catttgtgat aggcacagtt atcttgaatg 5880 catggagcag agattacgga aaaatcgaga atgttaatga ggcaacattc gagttgagtc 5940 attcagtgtg ggaaacccag acgcttccat cccctaaaag gaacatcttg ctctcagtca 6000 aaatggaaat aaaaattggg gcttgaattt ggcaaatgat tcagaactct gtgtaggtat 6060 tttcacacgc acagtggata attttcatgt tggagtttat ttgtgctaaa aggcagaaaa 6120 gggtaaaaag cacatcttaa gagttatgag gttctacgaa taaaaataat gttacttaca 6180 gctattcctt aattagtacc cccttccacc tgtggtaatt tcctgagata gtcagtgggg 6240 aaaagatctc tccttctctt ctttctcccc ctcccctcct ctccctccct ccctccctcc 6300 ctccctcctc tccctccctc cccctttcct tctttctttg ctccttctcc tctgcctcct 6360 tctccctttc ttcttcattt attctaagta gcttttaaca gcacaccaat tacctgtgta 6420 taacgggaaa acacaggctc aagcagctta gagaagattg atctgtgttc actagcgtgc 6480 aattcagagg tgggtgaaga taaaaggcaa acatttgagg ccatttcctt atttggcacg 6540 gcacttagga agtggaacat gcctaatcta ctggtttgta ccacctttcc ctataatgga 6600 ctgtttggga agctcctggg caaccgattc tggcatctca tggtcagag gcctgttaaa 6660 tggtactctt atttgcaag aaggctgtaa cttgtagctt taaaagccctc tcctcaagaa 6720 agaagggaga aaggatatgg ctagacatat ctaatagact taaccactgt gaaagcctt 6780 agtatgaatc agatagaacc tattttac tcagttttga aaaaaataat ctttatattt 6840 atttgtgtgt gtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgt gaaccacatg 6900 tagcaggtgc tggaggc cagagaggg caccagatct cctggactg acaccacaca 6960 tggttatgag ctgcctgatg tgggtgctgg gaactgaact ctcgtgttct gcaagagcag 7020 caactgttct cttaactgat gagccatctc tccagccccc cccataattt taattgttca 7080 ttttagtaaa ttttattcat atcattat cacagtataa aacaatgatt ttatatat 7140 catatacata tcaggatga cagtgagggg gatatgtgtg tgtgtgtgtg tgtgtgtgtg 7200 tgtgtgtgtg tgtgttattt gtgtgtgtgc ttttaagaa ggtgccatag tcactgcatt 7260 tctctgaagg atttcaagg atgagacat gtctgtctgc caggaccct atctctcctct 7320 ttgggaatct gacccaaatg aggtattctg aggaactgaa tgaagagctc aagtagcagt 7380 gtcttaaacc caaatgtgct gtctagagaa agtcaacgtc atcagtgagc tgaggagaga 7440 tttactgagc ggaagacaag cgctctttga tttaagtggc tcgaacagtc acggctgtgg 7500 agtggagcct gtgctcaggt ctgaggcagt ctttgctagc cagctgtgat gagcagtgaa 7560 gaaagggtgg agatggaggc agggtgggag cagggctatg gttcagacta ggtatcgtga 7620 gcacaccagc tggttgactt gtggtctgtg ggtcaggcgt tgtaaacgcc ctcagggtca 7680 ggcagtcaca ttgcttgaag ctgaatgggt gaggcaacac agagagtgca aagaaggcaa 7740 agtaccacct cttccccgac ccaggtcact tctgggttat agctgagact ccggacagca 7800 tgcaaccagc tggttagagc ttcagggaaa acttgatgtc tgcatgttgc tatgaaatgt 7860 gattcggtac atctggagaa aatttataat gctggctcag tcaagcactg aaaaggta 7920 ccttggcttt gggagctaca tgacattgac ttgtaggcag actttttttt ttctgcccgc 7980 caattcccag ataaccaata tggaggctca atattaatta taaatgctcg gctgatagct 8040 caggcttgtt actagctaac tcttccaact taaatgaacc catttctatt atctacattc 8100 tgccacgtga cttaccttg tacttcctgt ttcctctcct tgtctgactc tgcccttctg 8160 cttcccagag tccttagtct ggttctcctg cttaacctta tcctgcccag ctgctgacca 8220 agcatttata attaatatta agtctcccag tgagactctc atccagggag gacttgggtg 8280 ctccccctc ctcattgcca tccgtgctt cctcttccct cgcttcccc tctctctct 8340 gctctctc ctccacccct cctttcatag tattgatggc aagggtgttc tagaatggag 8400 gagtgcccat aggcatgcaa agaaaccagt taggatgctc tgtgaggggt tgtaatcata 8460 agcgatggac acaattcaag ccacagagtg aagacggaag gatgcactgt gctctagagc 8520 aacttctggg gcagaatcac agggtgagtt tctgacttga gggcgaagag gccacgagga 8580 agggagtgag tttgtctgag ctagaagcta cggcccacct cttggtagca gacctgccca 8640 caagcatgct ttgttaatca tgtgggatct gattttcctc taaatctatg ttcaactctt 8700 aagaaaatgt gaattctcac attaaaattt agatatacgt cttttggtgg ggggggtgta 8760 aaaaatcctc aagaatatgg atttctgggg gccggagaga tggctcagag gttaagagaa 8820 ctggttgctc ttctagacat tctgagttca attcccagca accacatggt ggctcacaac 8880 catctgtaat gcgacctggt gccatcttct gacatgcatg gatacatgca ggcagaaagc 8940 tgtatacata gtaaattgat aaatcttttt ttaaaaagag tatggattct gccgggtgtt 9000 ggtggcgcac gcctttaatc ccagcactct ggaggcagag gcaggtggat ctctgtgagt 9060 tcgagaccag cctggtctat aagagctagt tccaggacag cctccaaagc cacagagaaa 9120 ccctgtctcg aaaaaccaaa aaaaaaaaa aaaaaaaaa aaaaaaaga gtatggattc 9180 taagaaagcc gtaacagctg gagctgtgta cggagttcag cgtggtacta gaagaacaga 9240 cattcatgat gaaacacccc aggattttta cttagtatct agtttccatt gttgttttga 9300 gaccggctct tatgctctcc aggctggcct caaactgctg atcttcccgc ctctaccct 9360 caagtcctgg gactacttgg ctcataaaac agttttgtc gggctccctg aagttatggt 9420 tgtacaaacc gtgggggtca atactcac ttgggcagag agagaaggtc tgaatcccag 9480 acaatgactg catctcagga cagttgggaa gaggacaatg gcagaaggac ttagaaaaga 9540 tagactggag ggtggaaaag cagcaggaac agagaaacaa aacaggaagc ttgctatcca 9600 gggccactct ggagtcctgt ggcaagatgg aagcgggcta ggggaataca tttgtgctac 9660 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgat caatgcctat caatgttgaa 9720 ggggaaatat gtataccaca ttgattctgg gagcaattct cagtatctgg cctagagaaa 9780 ggaatggccc ctgcagaata gacagagtga atggtgccct ttatcatttg ctaaagtgaa 9840 ggagaaataa acatccttcc atagagtttc aggtaaatga accccacagt tcatctgtgc 9900 cgtggtggag gcctggccaa cagttaaaaa gattagacac ggacaaagtc tgaaggaaac 9960 acctcgaata ggaagaggag agccacctca ttctgtaact ttcctcaagg ggaagatgtt 10020 ccaagagtgg gaataaatgg tcaaaggggg gatttttaat taggaaaacg atttcctgta 10080 tcacttgtga aactggaggt tgatttgggg cataggacaa tagatttgat gctttgcaaa 10140 aagctgtttc aaagcagaga aatggaatag agacaattat gtagcgagga gggagggtgg 10200 ggcgaagatg gagacagaga agtggaagct gactttaggg aagagaaca tagaccacag 10260 gggcggggcg gggggcaggg gcggggggcg gggctcaaag gaggcagtgg gaacgttgct 10320 agtgttcgca gcgtaagcgt gaatgtgcaa gcgtctttgt ggtgtgtgac caggagtagc 10380 gtggctggct tgtgtgctgc ttgtaatccc agtctttgag gtttccacac tgttccacag 10440 tgggtgtgat tttccctcgg agagcatgag ggctctgctt tccccacatc ctccccagcg 10500 ttcgttggta tttgtttcca agatgttagt gggtgagaca aagcctctct gttgatttgc 10560 ctttaacagg tgacaaaaaa agctcaacca ggagacattt ttgcctttt ggaaggtaat 10620 gctcccatgt agagcaatgg gacccatctc taaggtgagg ctactcttgc agtttgcacc 10680 cagctcttct gatgcaggaa ggaagttggt gggcaagcaa gactgtttgc ttcttgcgat 10740 ggacacattc tgcacaaa ggctcaggag gggagaaggc tgtttgatgt ttagcactca 10800 ggaaggcccc tgatgcatct gtgattagct gtctccatct gtggagcaga cacggactaa 10860 ctaaaaacca gtgtttttaa attgtcaagc ctttaaggtg aggaaattga cttatgtgc 10920 tgggccatac gtagagcaag tgctctgcat tgggccaacc cccggctctg gtttctaggc 10980 accagaatgg cctagaacta actcacaatc ctcccattcc aggtctcagg tgctagaatg 11040 aaccactata ccagcctgcc tgcctgccta cctgccttcc taaattttaa atcatgggga 11100 gtaggggaga atacacttat cttagttagg gtttctattg ctgtgaagag acaccatgag 11160 catggcaact cttataaagg aaaacattta gttgggtggc agtttcagag gttttagtac 11220 attgtcatca tggctgggaa catgatggca tgcacacaga catggtgctg gagaaaggga 11280 tgagagtcct acatcttgca ggcaacagga cctcagctga gacactggct ggtaccctga 11340 catagaggaaa cctcacagcc caccctcaca gtgacatatt tccttcaaca aagccatacc 11400 tcctaatagt gccactccct atgagatgac agggccaatt acattcaaac tgctataaca 11460 ctttaaagta ttttattttt attattgtaa attatgtatg tagctgggtg gtggcagccg 11520 aggtgcacgc ctttaatccc agcacttggg aggcagaggc agatggatct ctgtgagttc 11580 aagaccagcc tggtctataa gagctagttg caaggaagga tatacaaaga acagttctag 11640 gatagccttc aaagccacag agaagtgctg tcttgaaaac caaaaattgt gctgggacct 11700 gtctctgctt tggttgcttc ccactccccc agagctggac tcttggtcaa cactgaatca 11760 gctgcaaaat aaactcctgg attcctctct tgtaacagga gcccgaagtc aggcgcccac 11820 ttgtcttctc gcaggattgc catagacttt ttctgtgtgc ccaccattcc agactgaagt 11880 11940 gggaatagtc tggaaatgaa gcttcaaaac ttgcttcatg ttcagttgta cacagactca 12000 ctcccaggtt gactcacacg tgtaaatatt cctgactatg tctgcactgc tttatctga 12060 tgcttcttc ccaaaatgcc aagtgtacaa ggtgagggaa tcacccttgg attcagagcc 12120 cagggtcgtc ctccttaacc tggacttgtc tttctccggc agcctctgac acccctcccc 12180 ccattttctc tatcagaagg tctgagcaga gttggggcac gctcatgtcc tgatacactc 12240 cttgtcttcc tgaagatcta acttctgacc cagaaagatg gctaaggtgg tgaagtgttt 12300 gacatgaaga cttggtctta agaactggag caggggaaaa aagtcggatg tggcagcatg 12360 tacccgaaat cccagaactg gggaggtaga gacggatgag tgcccggggc tagctggctg 12420 ctcagccagc ctagctgaat tgccaaattc caactcctat tgaaaaacct ttaccaaaca 12480 aacaaacaaa caaataataa caacaacaac aacaacaaac taccccatac aaggtgggcg 12540 gctcttggct cttgaggaat gactcaccca aacccaaagc ttgccacagc tgttctctgg 12600 cctaaatggg gtgggggtgg ggcagagaca gagacagaga gagacatgac ttcctgggct 12660 gggctgtgtg ctctaggcca ccaggaactt tcctgtcttg ctctctgtct ggcacagcca 12720 gagcaccagc acccagcagg tgcacacacc tccctccgtg cttcttgagc aaacacaggt 12780 gccttggtct gtctattgaa ccggagtaag ttcttgcaga tgtatgcatg gaaacaacat 12840 tgtcctggtt ttatttctac tgttgtgata aaaaccgggg aactccagga agcagctgag 12900 gcagaggcaa atgcaaggaa tgctgcctcc tagcttgctc cccatggctt gccgggcctg 12960 ctttctgcaa gcccttctct ccccattggc atgcctgaca tgaacagcgt ttgaaatgct 13020 ctcaaatgtc actttcaaag aaggcttctc tgatcttgct aactaaatca gaccatgttt 13080 caccgtgcat tatctttctg ctgtctgtct gtctgtctgt ctgtctatct gtctatcatc 13140 tatcaatcat ctatctatct atcttctatt tatctaccta tcattcaatc atctatcttc 13200 taactagtta tcatttattt atttgtttac ttactttttt tatttgagac agtatttctc 13260 tgagtgacag ccttggctgt cctggaaccc attctgtaac caggctgtcc tcaaactcac 13320 agagatccaa ctgcctctgc ctctctggtg ctggggttaa agacgtgcac caccaacgcc 13380 ccgctctatc atctatttat gtacttatta ttcagtcatt atctatcctc taactatcca 13440 tcatctgtct atccatcatc tatctatcta tctatctatc tatctatcta tctatcatcc 13500 atctataatc aattg 13515 <211>14553 <212>DNA <213>Mus musculus <400>6 (SEQ ID NO: 16) cttgaagaac acatgttttc caagagggag cacccatgtt ggaatgacaa tgtagttagt 60 gctcctctcc tgtaggttag tgctcctttg ctataggtaa gtgctcctct cctataggtc 120 agtgctcctc tcctataggt tagtgctcct ctcctatagg ttagtgctcc tctctcag 180 gttagtgctc ctctgctcta ggttagtcct gctctcctat agtacctaga gagctagggc 240 aaatgggcta ggcccgaagt gcagagacaa acagctatgg aagactgggt aagcacttcc 300 aagctacgaa agagcagtgt gaagggtcag ggcttgtgca gttagtaggg gagatcttcc 360 agttgaagaa agaagaac tgagagccac tgggtatcat cctcctgcgc catgccttcc 420 tggatactgc catgctccca ccttgatgat aatggaatga acctctgaac ctgtaagcca 480 gccccaatga aatattgttt ttatgagagt tgccttggtc atgctgtctg ttcacagcag 540 taaaacccta ataaggcag aagttggtac foottttgct gtgatagacc tgaccatgct 600 ttcctttgaa agaatgtgga tttggtgact ttggatttgc aacacagtgg aatgctttaa 660 atggagatta atgggtcatc aattcctagt aggaatatgg aagactttgt tgctgggagt 720 atttgaactg tgttgacctg gcctaagaga tttcaaagga gaagaatttc agaatgtggc 780 ataaagacag tttttgtggt attttggtga agaatgtggc tactttttgc ccttgtctga 840 aaagtctgcc tgagactaaa gtgaagagaa tcagattaat tgcattgaca agggaagttt 900 gtggctgcgc tatctggaaa cttacagcca gcctcttgga cctcgggtga cttacgcaaa 960 tactcaggga cagagatgct tgactctgta ctgatgagtt gtcttggatg caaatatggg 1020 ctcttcattt gactacatgt cacgatgagt caggagctgc tctctccaga gtgtgacaaa 1080 gcgaggggat gctgacggta gctgttctag ctttgaaggt aagcctgcac ttatgctaaa 1140 gtcacacata cacgagccgg gtggagaacc tgtctgtgtg gagacacctt tcattacctg 1200 tggcatccag cctctcaagc ttggactgcc tgtgtgctcc tggactctgg aggtcccact 1260 gctctgtcct ctgctgctta tgatactgac attttaaaag aatccagtgg ttcccccctg 1320 tactcggtgt ctacttctac ctggatgttc ctcatttatg ttctgtgaca cttctctgtg 1380 actctgctgc attcctgggt gacatgtgga caccctgtcc ctttgcagac catgatgtca 1440 ctgtcactag tggaatcaga tgccccaagt gttgtcctgt gtttgggaac gtgacaggca 1500 gtacagaagc agaagaggaa gggtgaaaac ggaaatgtca cagcagcatc tgatgtgtgc 1560 ctcagtcacg catgctgctg attggaacta ctcagcatga gagagggcca tggtgaatac 1620 acaaccctat acacactgtg tccatttctc tctctctctt acacagagag agagggagga 1680 gggggagggg gaggcggagg gggaggggga gggagaggga gtgggagagg gagagggaga 1740 gggagaggga gagggagagg gagagggaga gggagagttt aatgtctgtg aagagatacc 1800 atgaccaaag caactcttat aaaggacaac atttaattgg ggctggctta caggttcaga 1860 aattcagtcc attctcacca tggtgggaag catgcaggta gatgtggtgc tggaggaacc 1920 aagagttcta tatcctgatc tgaaggcagc caggagaaga ctgcctcttc tgcacagggc 1980 agagcttgag catagaacat caaagccctt ccccacactt cctccaacaa ggtcatacat 2040 acttcaacaa agacacacct cctaacggtg ccactccctg tggaccaacc atttaaacgc 2100 atgagtctat gagggtcaaa gctcttcaaa ccaccacact catgtacaca cacacacaca 2160 cacacacaca ctctcataca cacacacaca cacactcaca cacacacaca cacacacaca 2220 cacacacaca ccacacacac acacacacac agagttctat tttgcactgt ttcactgtca 2280 caaggttcta cttatctcag acacactgcc aggaattgtg tgggaagact ttcagtttct 2340 ttgggttcac atggacttag cagttcttgg tgatcctgaa agatttctgc agaaagaagc 2400 caaagtgttg agcccaaggc ctggccacac attagtcctg tctagatgaa caggggttta 2460 aaaataaggg ggcatcaagg tgaagccagc aggggctgac ttagagagga gacccaccca 2520 agccaactgc tcgaagtcaa aagcgatgaa tccccatatc cagctgtgcc cggtgctgtc 2580 ttgctacatc tttagtaaat gttcttttag ttgtatgcgt atgaatattt tgcttgcata 2640 tatttgtgta caccataggt gttcctaggg cctatggagg ccagaagagg gcatcagatc 2700 ctttggaact ggaattatag acacttgtta cccatagagt agattgtggg aaatgagcct 2760 ttagtcttcg agagcggcca gtgctcttaa cctttggtcg tttctccagg tctttgagac 2820 tttattttct tggacatcag gacaggatcc agggctttga gcttgtttct tcagccagct 2880 ttcttttcat gtatattaaa ttttatgtta ttttgctttc tttttcccca agacagaatc 2940 acactctata tagctcaggc tgggtttgaa ttcagtttcc ctgtctcagt ctaccgggta 3000 atatgattac agatgtgagt ctgactttgg tatcaaagtc cccagccctt ctggatatgt 3060 gttttaagga tatcagatat atccttgatt tgctttgaat tttcttttta gttacaacat 3120 aattagttcc gtgtcacctg aatatgtgta tgtcacctac atagtcttcc ttcttctctt 3180 cttccctctc ccaccttccc aggtacctgt ctgtcttcat atccttgtgc tgagagtctt 3240 gttgagggag atgatgaccg agacagagcc actggggaag ggagatgggc tagtgcaggt 3300 cttcagagag gagctcgtga atattgtagc ccctttagtc cctggcatgt cctcttgtat 3360 agccaccgcc atgctgtggc ctggcagaag tgaataagtt gtccagctgt tgacaggcct 3420 gccctccaga cccagtctga tcccaagaaa gggcatctgt gtctgtctct gaggccgtaa 3480 gtgctgcctg gttgtctcca gcttgacttg acactccctc cttaataaga gtaccacaga 3540 acagggtctg cagagtccct gggccaggtc cctgtgctgt cctggaatgc caggcgtgaa 3600 tttcctgtga agtaggactt tgctcgccaa gctcccacgg cttgcccttc agatagccag 3660 aattatctgg taccctgcat tgccgttca tacgcagt atcactgga gcgcgcgcgc 3720 gcacacacac acacacac acacacac acacacac tccatcttta 3780 aaccccacccc cccagcaacg gcggtgtaaa cactctccat caggaagctg aaacgcagtt 3840 gccctctgct ggggagatga agcagcttg ctgggggcga ggaccgtgct agcaccttc 3900 cctggtgcac acgggctctg gtgcatgacg ggaacggaaa cgcggaacta aagtcagtcc 3960 tgctttttttttttttttttttttttttttttttttttttttttttgggcgttggtg 4020 gtggactgag tgacaatcag tgaatcact taggtttttttctctctt cgttggtttt 4080 gatgacggt gggagaggggt cagagagaa gggagggat gggagagag ggaggaggga 4140 ggggcgggag gcggggggcg aggaaacgt gctactct ccaatcctac agaaaagg 4200 tttggagaaa gccgcactga gtgacccagc agaggaatc caggaatgtc cgctggaatc 4260 tgactgttga ttccagcgcc atgcagagaa tctaggctgg taggaacatt ctttgtccta 4320 tccgacataa taactccaac siacacggaa aagaaggct atacaagtga agaatggca 4380 ttttcacttt catgactata caatcactc caggtagtaa cacgtgtcta gcacagcggt 4440 tctcaacctg gggtcacga tcccactt ttctgcat cagacatttt tacgttgtta 4500 ttcataacag tagcaaatt gcagctatga agtaacaatg aaatgcattt atggtgcgtg 4560 tgtgtgtgtg tgggggta tcaccttaac atttactgta agaaggttga gatactgct 4620 ccagcagcta gtgtgttgga cttaggttct gggtatatta ccagcaatag ccaaccagaa 4680 tcccaccca ccacagcatt gaggccccat gcagggcttg ctgggagagg cactgataag 4740 acttctttat gtatttatt agagacgaat actcattagg taggccaagc tagcgtcaa 4800 ctcatggcaa ttctcctcct ccagttctct aagtactgga ctcaggagtg tgttgccatc 4860 atacagta aggatttatt gactgaagaa atctcaagt ggctttggtt aatccctact 4920 acgccagagg ctgaggcagg aggcgcgcaa ggtcaggct tgcctgggct acatagag 4980 tgagctcaat ttgacactt ggtgcggtgt tagtagtaat agtaagatg aaggtgtggc 5040 tcaggtgggg ccggtgatgzgacacttg gggtctcctg gtccatctgc agctgtgcaa 5100 caggaagagc ggagaatgag aggaagaga gaaaagacag aatgagagag agggaggaag 5160 agaaaaaag gaaaagagag aggaaaggaa aaaggaaaat gaggaaagcg agaaagaaga 5220 5280 agagggagga agagagaaaa aggaaaagag agagaaaagg aaaaaggaaa atgaggaaag 5340 cgagaaagaa gaaatgagaa agaggaaagg gagaaagaaa tgagagagag aaaagaaaag 5400 acagaatgcg agaagagggag gaagagagaa aaaggaaaag agaagagaag ggaaaaagga 5460 5520 agaaaagaaa agacagaatg cgagagagg aggaagagag aaaaaggaaa agagagagga 5580 agggaaaaag gaaaatgagg aaagcgagaa agaagaaatg agaaagagga aagggagaaaa 5640 gaaatgagag agaaaaaga aaagacagaa tgcgagagag ggaggaagag agaaaaagga 5700 5760 gaaagggaga aagaaatgag cgagataaaa gacagaattt gagagaggga ggaagaaata 5820 ggaaaagaga ggaaaggatg gagaaaagag agaaagaaag agagatgaaa gagagaaagg 5880 agaaatgaaa tgagagagag agagagacac aaagagccag agagagaaga aaaaagggga 5940 aagagaaaga gaaagaggaa ggctcctctt ggacacatct tcctttatct ttccctgggg 6000 accgccaaag cctggtggca tactgtacat tctgtacact gttcattcaa aacaggctct 6060 gtcttaaaga tggtctgagc ggtcagaaaa gggtattgtt aacttgtttg caaaactgcc 6120 tcaggagagt gctgagtgcg tgaaagttgc tgcccgttaa ggagaagtct ctactacttg 6180 tgatctcacc atcgaaaatt tctttaattg tctcctggtg ttctgggttt tgcagttttg 6240 tttctaagga tacattcttg ggtgatgtca caaagtcccc aaagacacgg tggagctgtg 6300 ttagatgggg aaagacagtc tgctgaggat ttatctggaa ctgtcagaag gaaaagaagg 6360 taaatggggc acttgggaaa gtggcctcta gtttgacttc tggcttagca aaggttgtgg 6420 ggagataagg catacacagt agttagcagg aggcaacagg gtcctgggag gacgcgaggc 6480 agaaggagag gctgggctga cagcatgcaa tcattgcata gtctccaaag gagattgcaa 6540 catggctgag ttttcagagg tcctacagag cccgtggtag agattctgtg ggttctgaga 6600 caacttgact ttagccagat ggtatttgag taatctggga gagagaaaac agctacagca 6660 aacagggcca catttagtga cgaaactctc actttgactg ttgagtcatt tgcagtgggc 6720 cctgaggtca ggctggccct cagctcaaaa acaagcgagg aactgaagca attactcaga 6780 taatccacag ccacagccac tggaaagggc cacatcccca gagacagcac agcaggggtg 6840 ggggtggggc tatgagaaag ttagtgattg tagcagttat ctagaatgtg cggagcagag 6900 gaggttacac aaaaacctag aatgtcattc aatgtgggaa accgagaggc tcccaagccc 6960 taaaaggaac agtttgcttt cagccaaaat ggaaataaaa tttggggctt aaatctggca 7020 aatgattcag accttctgtg taggtgtctt taaatgcaca gcagattgat tttcatgttg 7080 gagtttattt gaactaaaag acagaaatgg tgaaaagcac acctgaagaa attgagatgc 7140 tatgaataaa atcatttact tacagctatc acttaattag tacctccttc caccttgctg 7200 atttattggg ctagtcaagg aagaaaagat cttccctcct ccttctctcc tcctccccct 7260 cctctcctcc tcccctcccc tccttgacct tcctctcctc cttttccctc ctccccctct 7320 tcttctcttc accccctcct cccctcccct cctctgtact cctccccttt cctcccaatc 7380 tcttttttct cccccttctt ctctttctcc cccctcctct tccctcctct tcctccctcc 7440 ctccctcctc ctcctcatcc tcctcttcct cttcatcctc ttctccttcc tccctctcct 7500 cctcctcctt ttccagccct acctaccttc cctttcttct tcatttattc aaagtagctt 7560 tgaacagcac tactcggttt agttgtgtat aaaaggaaaa tgcaggtcca agcagcttgg 7620 ggaagattgc tttttgctct ctggaggcag atgatgacag ttcaagatca ttccttttgc 7680 tccatgtcac aggaaggggg acatgccgaa tctaccagtt tgcagccacc tacacaggat 7740 ccaccttcac ttctaaggaa atgtttggga agctacctac caaccacttc tggcatctca 7800 tgggctagag gactcttaaa tggcactctt atttgtttaa taaaggaggt tgtgacgtgt 7860 agttttaaat cccttccaca caacaattgc tactctctga ccaaaaaaga agggagacag 7920 gatacggcta ggtgtctagt agactttacc actttgaaaa gccttaatat aaatcaggta 7980 gatacatct tttaacttat tcttgtaaag acaaaaacaa aactttattt ttatttgtgt 8040 gtatgcttgt gtgtgtgtgc ctgtgtgtat accacatgtc gctggtgccg gagaacacca 8100 gaagagggga cctgatctcc tggagctaaa gctatccatg gttctgagct gcctgatgtg 8160 ggtgctggga acagaactct ggtcttctgc aagagcaaca agcctcctct taactacgaa 8220 tctcctcccc atccccccaa atacatttaa ttattcattt tagcagcttt atttcgtaac 8280 tacttatcac agcataaaac aaggatttta tatatattac atgcaatcga ggataagagt 8340 tgaggggaga tgcgtgtgct ccttctgggt gtctgtgcttt ttgaagaatg taagcagtgc 8400 acaagggacc gaggcgtgcc tgtctgccag gagctgtctt cttcccttgg actctgagct 8460 gagtgcagtg ctccgaagaa gtaaaagacg acctcatgaa gcaatgtctt caacccaaac 8520 atgctgtcca gacaaagtcc agcttcatta gtgctctgag gagagactta ctgagcctca 8580 ggaaagcccc cctcagcatg gcgaaagtcc actttgattg aagtgactcg aaagccatgg 8640 cagtgcggcg gcggccgcgt ggagcttgtg ctcgagtcgg aagcggcatc tttgtcaggc 8700 ggctgtgatt agcacgggga ggcaggactg gagtgaagga agagttgggg gcggggctta 8760 gcgctctggt ctcctaagct gtagtcagcg cctcaagatt tgtaacctgc cttctgcctt 8820 cccagccagg cagtcaagtg gctccaagct gaagactgca aagtgcccct aaccttttgg 8880 ttatagcgag gctgaagaca ccgtgctctt tcatgaaagc cggatgtctg aaatccgatt 8940 tgataaatat ggataaaacg tataacgctc gatcaatcga atcgaaggag ctcacgattg 9000 gcaccacggc tttggggaca acagagtact gactcgttgg gaggacttgg atacttcccc 9060 tcctcttcca tctcttcccc tttcctcact tcctcctcct tccttctcca ttttctccct 9120 cttcactgtt tcttactatt tttacaaaag attttattta tttatttatt tatttattta 9180 tttatttatt tatttattta tttatttaat gtatgcgagt acactgtagc tgtcttcaga 9240 cacaccagaa gagggcgtca agttccatta gagatggttt cgagccacca tgtggttgct 9300 ggggcctctg gaaggaccgc cagtgctctt aacccctgag ccatttctcc agtacccttc 9360 tcaccgtttc tcttcaatct tcttcctctt ccttctccac tttccttgtc ttcttggttt 9420 cattatcttt ctccctttct tcctctctc cccttcttcc tcctccactg tagttttcct 9480 tccctactct tttcctgcct cccctctcct cccctcat tccccctcct cttctcct 9540 tctccctcct cctccttcct tctccctctc cctctcccc tctccctctct cccttctccc 9600 cctcctcttc ctctttctcc ttctccaccc ctcctgtcac agtatcaatg gcaagggtgt 9660 tctagaatgg aggagtgtcc cctaggcact aacgaaagcc agttaggatg ctctgagacg 9720 ggtacaattc agggagggcc gtggggatgg aagggttgtg ctgcgattca ttctggagca 9780 acccccaggc agaatcatga ggttggttcc ggattcgcag ggcacaattc agaagaggaa 9840 ggtttcagga aggacgagtt tgtctgagat aggagttaca tctgatgtct tggcagcaga 9900 gccactgtac aagcgtgctt tattaaccac gtgggattaa atcttctttt aaatttattt 9960 tcaactctta aggaaacgtg aactttcaca ttcaaattta gacttgcagc tcttatgggg 10020 aaaaaaaggg gatcttaaga atattaagca taggcggctg gagagatggc tcagcggtta 10080 agagcactct ctgctctccc agaggtcctg agttcaattc ctagcaacca cataatagtt 10140 aacaacgtc tttaatgaat tctaatgccc tcttctggtg tgtctgaaga cagttacagt 10200 gtactcatat aaataaaata aagaaattta aaaaaatgaa tattaggcat agattcctgg 10260 atcctaagaa agccatcaga gctggagcca tgtgtgggat cctgcttggt gctggagggg 10320 cagagttcat gcccccgggg ttttactta ttcacatt ttcatcgttg ttttgaaaca 10380 gggtcttgtg tggtccaggc tggccttgaa ctcatctttc agcctctacc tcacaggttc 10440 tgggattact tggttcctaa aagtatctcc gtcaagctcc ctggtgttat ggctgtgcca 10500 accaggaggg tctatacact cgctcaggta gagggagaag atccgaatct ctgacaggga 10560 ctgctgcctc tcggggcaaa tggagtgaag gacagcggca gaaggattta ggaaagatgg 10620 acgggagagt ggaaatgctg cagaagccag aaaacaaagc aggaagcctg ctgtccagtg 10680 gggctcaaga gcggagggat gcgagggc tgcgcaggaa catttagcgt ctgcgtctat 10740 gggggtaggg gcggggtgcc agcacctagt cacctgaagg ggaaatgctt gcccagggag 10800 caggtctcag tagctgacct agagaaagga gcggccccta cagaggagac acgggtcact 10860 gtttgttaaa gtgaaggaga aataaatatt ctttcaaaga atcttaggtg agcccagttc 10920 atctgcgctg tggaggcctg gggaacagtt aaaaagaccc tgacacacac ccaaggcaaa 10980 caagcaacac acggctcctt ccgtaagggt ccatgattct ctgaagaatc agccccggaa 11040 tcagccccgg aatcaggtag tccgtaaaca caatgagtgt tttactctgc agaagtccag 11100 cctgctggcg tctcccatta ccaaataga gggatagtca cgtgagctca ccggctcgat 11160 ttaaggcacg tggttttcca gggtagatga gctttggctt ctggaaccat tatggggcac 11220 gaaggatgga gccaggatttt tttttttttt ttttttttc tattagcaat tgatttgctt 11280 gggcttggct ggacttgccc agttcttagg cccagctttc ttaactgccg atctgaagtc 11340 tgtcatggag tcagcctagc cttctcactt cccttcagct cgaataggaa gaggaggtgc 11400 acaccagatg gtctgagagc agggataaat ggtgtgcctt tgtctttcag tatttcgtta 11460 ttttaagtag gaagatgctt ttctgtatta cattgcttgt gaaaccggaa gttgattcgg 11520 ggcacaggac aatggatttg gtgttttgca aggactgttt cagaagagag aggagtggaa 11580 gggtggttag agtgaggagt ggggtggac gggatggggg aagagaa agggccagac 11640 aggctaggta gggctgagag gaggcggtgg gaacttcttg agttagcgca gcagtaaact 11700 tggatgtgcg tgtatctttg tgatatatga cccggagccg tgtagctggc tccgatagta 11760 ctgctaatgt cagtgtcggg gggggggggt cccatactgt tccacagggg ctgcacattc 11820 ccatcgagag caggagggct cctctctcca tacatcctcg ccagcattcc ttgttgtttc 11880 tgtgatgaca gggggtggga tgaaatctct ctgttggttt gagagaccgt gaagaagctc 11940 aaccccagga cattttgcag tcttggaagg cagtgcctcc atgtggagcc gtggagccca 12000 tctctgagtc caggtcactc ttgcagttcg cactcagctc ttcagatgca ggagagacgt 12060 tggtgggaaa gcaagattgt ttgcttgttg agatagacac attctccaca caaaggctca 12120 cgtggggcaa aggctgattg acgtacagcg ttcaggaacg cctgtggtag agctatgatt 12180 agctgtctcc atctatgaag caagaaaga gttataaaaa aaatcaatgt tttcaaattg 12240 tcaaactttt aacccgacag caagcgctct gtccctgggc taatccctag ccctggtttc ttgagatggg gtcttttgtg cactagactg gcctagaact cacgatctta gtgttccagc ctcccagctg ctgggatgag ccgctataac cagtctgcct gccttcctaa attttaagtg atgggaagtg ggggagaata cagtttaaag tatgcagatc tgagagcagg aacctggcaa agccaagggg ccggagttac aggcggctaa catggggtgct gggaactgac ccaggtcctt gagaggagca gtgtgtactc ttgaccaaac aggtccgtct ctccagtccc cgtagtatta aaaataggta ctacgggcat ggtggtgcac acctttaatc ccagcactag ggaggcagag gcaggtggat ttctgagttt gaggccagcc tggtctacaa aatgagttcc aggacagcca cggctataca gagaaccct gtcttgaaaa caaaacaaca acaaaatagg tactacaaag cgatgtaatt gtgctcaaac atgcaaaccg aggggactgt atgcataaga aagagaaaga cggccacact ggttctatct gggtgacagg aaatcagtat ttttattttt cacattcatt ttttgttgt tgttgttgac acagtgattt ttctcaaa aacattattt cttttatagt 12960 tcccctgagg agctgttttt aaagccgtgc ttgaaaac cattgaagga gcagaggcag 13020 ggagactcct gtgtggcagt cggtgaagca ggccctgc agcaggctg gccctggact 13080 tgggagtctc ttccctccc tcctgtgctc aaatagcaaa tgtcaggctt caatgtagct 13140 agaaggttct agaatgatta agttccaag gctgaagagc ttccctttt gccttcact 13200 tccctggaga gtcgttgtg tgttccggag tctgcaggt gccttttgtg atgcggttgg 13260 ttcatctcgg gagattccgc ctggaggacc caagttcaag ccctgcctga gctacagagt 13320 gactttcagg tcttctgcgc aattcagtga gacccagtct acaataaa agtaaaaga 13380 aggctgtgga tggaactcgg tggtagagtt ctggttttac tcctagagg agggag 13440 Gaggagggagggagggagggagggagggagggagggagggagggg 13500 aggagggg ctgachagaagagagagggagggagggagggagggagggaagg 13560 gagggggggggggggagggagggagggagggagggagggagggagggagggagggagg 13620 ggaaagaaga gaagggtaag aagaaactgt tccaatggtc tgggccacag agtgatggcc 13680 ttttgtggtg atcagctgta atccttgatt tgacacaacc tagaatctgg gaagcgagtt 13740 tctgtgaagg agcattcaca ctggctggcc tgtgggcgtg catgtgggag actgtcataa 13800 ttaggttcat taatacagga agtcccagcc cactacaaat ggcttcgttc catacccaag 13860 agatgctaac tgtagacggt tggagaaagc aagcaagctg tggatacccc acgctctttc 13920 acctcggctc ctggggggtg ggtgcactgt gtctcttggt attttaaagt cctgccttga 13980 cgtccctgct gtgacagact gtaactggaa ttgtgagctt tagtccttta gttttctacg 14040 ttggtttttc tcaggatatt ttatcgcagt aacagaaaca agaccaggac acttgatctc 14100 ctctgatcaa cactgaagag ttacaaaaca ggctgaggaa acaaactttc ttctccctct 14160 cccccttctg tccctccct tccttctcgc tccctccctt gccccctctc tccctgtctc 14220 tgtctctgtc tctgtctctg tctctgtctc tgtctctgcc tctcccctcc cctcccctcc 14280 ctctgtctct gtctctgtct ctgtctctgt ctctgtctct gtctctgtcc ctttctcctc 14340 tatctcctaa atggctggag gccatgctag ctcaatgttg aactttgaac acgtatttag 14400 gaaatctttg ttcttaacag ttctgaagtg ctgaagtggt ggtttagtct ctcggcctga 14460 caagctcact tcctctcact ctgtcttaat gaccaaatct gccatttccc taaaacagca 14520 caggctccag ctccaggttg ctccggagcg gag 14553 CHO stable site 2 sequence - US Patent No. 9,816,110 <211>4001 <212>DNA <213>Cricetulus griseus <400>1 (SEQ ID NO: 17) ccaagatgcc catcaactga ttaatagatg ataaaattat tgtacatttc agtgtaatat 60 tattcagttt ttaagaaaaa tgaaattatg taataagcat gtaaatggat atatcttgaa 120 acaaccattc cccattatat tacctaaaca ttgaaagtcc aaaatcatat gatcttttta 180 gtggatctac taatcttttg ctatatgtat tttattgaac tacccatgga tgtgagataa 240 ttggtaacaa cagcacatgg gagagcatgg gatcattcaa ggaagattag agagaatgca 300 ttttttagga gatatggag gagcaataga aaggattaaa tgaggttact gatgaagtg 360 atggttagag aaggcaat gaggagggat aactaccact taggggcttt tgaaaaagac 420 atagagaaaa tactattgta gaaacttccct atattggtg tatagttata tacaccaaag 480 agctcagatg gagttaccct ataatggaaa tattaactac ttttcac tgtgataaaa 540 catcctgaac agagcacat agattgggaa gcatttactt tggctcag ttctaacggg 600 aaaaatttc aatgaatg aatgaatg tcagcaaca gcagtagca tggcctgaga 660 agcaggtgag agctcacatc tgaagtgta agaatgtagc aggagaaca aactgcaaat 720 gaccagaaaa tgcttttgga tcagagcccca taccctctg actgacttct ccagaaattc 780 tgaacaaata aaactcccca aacagagcca taactgaagg tccagtgtct gagactacta 840 ggggtatttc ttattcaac cactacaatg gggtgggggg agcaatcctc caagtaggca 900 ctacacacag acaaaaaa acttagtaa ctggaatgga ttgacttatt tgaattactt 960 gccagtggag ctacatagag cacaattatt gtatttaat tacccttta gatcttacaa 1020 aacttgacag taagatcata ttgctaaaga aaccacatat ttgaatcagg gaacatggtg 1080 atatctagtt gttcttcaac tggaacttc atgctttctg cccagcattc atgttgctgg 1140 aaagagcaat gtacactacc agtgtagaa ttaaatcatc atcttatca agatgtggat 1200 cctataagtt acataaaaa ttagcctgat aagatatccc caccagaaga atattcacat 1260 aaatgctatg ggagcaacaa gctattttct aaatttagctt taatcctatt ctacaagaga 1320 gatccatat ctagaatagt tatagggatc aagaacccat ggcttgattg gtcataggcc 1380 caatgggaga tcctaatatt attgttctac aaaatgaaaa taactcctaa tgacttgttg 1440 ctgcagtaat aagttagtat gttgctcaac tctcacaga gaagttttgt cttacaataa 1500 atggcaatta aagcagcccc acaagattta tatcataccg atctcctcat ggcctatgca 1560 tctagaagct aggaacaaa gaggacccta aggagacat acatggtccc cctggagaag 1620 gggaaggggg cagacctcc aaagctatt gggaggcatgg gggagggggg agggagttag 1680 aagaagaga agggataa agggaggagagaggacaag aggagagagg aagatctagt 1740 chaaggaggaggaggaggaggaggaggagagagaggaggaggaggc cttgtatgtt 1800 taaatagaaa actggcacta gggaattgtc caagatcca caagtccaa ctaataatct 1860 aagcaatagt cgagaggcta ccttaaaagc ctttctctga taatgagat gatgactacc 1920 ttatatacca tcctagagcc ttcatccagt agctgatgga agcagaagca gatactaca 1980 gctaacact gagctagttg cagacaggga ggagtgatga gcaagtcaa gaccaggctg 2040 gagaaacaca cagaaacagc agacctgaa aaaatgttgc acatggaccc cagactgata 2100 gctgggagtc cagcatagga ctttctaga aaccctgaat gaggatatca gtttggaggt 2160 ctggttaatc tatggggaca ctggtagtgg atcatattt atccctagtt catgactgga 2220 atttgggtac ccattccaca tggaggattt ctctgtcagc ctagacacat gggggaggtt 2280 ctaggtcctg ctccaaataa tgtgttagac tttgagac tcccttgaga agactcaccc 2340 tccctgggga gcagaaaggg gatgggatga gggttggtga gggacaggag aggagggg 2400 ggtgagggaa ctgggattga caagtaaatg atgcttgttt ctaatttaaa tgataaagg 2460 aaaagtaaaa gaagaaaaga aaacaggcca aaagattata aaagacagag gtggtgggtg 2520 actataaaga aacactatta tctaaataaa aacatgtcag aagcacacat gaacttatag 2580 tgttatgaa agtatgtata ataactacat aatctcaagc caagaaaaaa atatcatctt 2640 tcagtgatga aggtgatttt atttctccca gaattaaagc caaagaccta atgaaagtaa 2700 ttatcttcaa aaggttgaaa atacatactt tgcaatacac agatctgcct agaaatctca 2760 tgttcacaat acacatgatg ctcaattgaa ttccattcaa tgttacagtt tagataaaca 2820 gtttgtagat aaactcacaa tgtatcattt ctttttattt tttgaccaaa cagcttctca 2880 tctgttattc agaataattc ctcgatggca ggatatccat cccaattggg ggaaggggag 2940 aatttgaaga aaacctagac cacatacata tttgccattg ggaaacaaag tctaaaatga 3000 tgttgttcac atcttctcta ctagtcctct ccccgtccca aagaaccttg gtatatgtgc 3060 ctcattttac agagagagga aagcaggaac tgagcatccc ttacttgcca tcctcaaccc 3120 aaaatttgca tcattgctca gctctgccct tctcatatga cagttacaag tcaaggcttc 3180 caaagtccct ctgtcatgtt tgtgtcaat agttataca gatgacttca tgtcttcata 3240 tctaatgtct tatatagatt atattaaac atgttattt ctctaaccac attttaatt 3300 aatttaaaaa tccattaatt gtgtcttaa atgcagaca gagtgctgag acacaata 3360 agcctgatga tctgaatttg aactcacac ccaccacatg gagaatcaac ttccaaaat 3420 tttcctatta cttcacact cacattg spider ataataatga acaaaatgaa 3480 atgaataaaaattaagtc tctgtaggta atgctactgt gcagcaaag taaaatggc 3540 agcttaagct tgctttatgg ttacacttta ccatcttcca ttaattataa ggacttcaat 3600 catggcagaa ctatgctgtt attgtctcag tgtacctaa ccaggtgttc cagatgttct 3660 taatgtggac acctaaacta tttgatattt gggttaagat ctttccctct ttcagaagaa 3720 acctcaggac agagggaatc ttgtctttta attttgagtc tgtagacttt ttccattca 3780 atatacatg aacaagtga tgagaaat taatcaaag gtgggaattg caatgatatt 3840 aggttcaata ttaagcttca atattacat ggaatcgcct gttatacact gagtgttttgg 3900 caataaggga tttttagaag aaggagtttt tattctcaac aggttcctta agtttagctc 3960 aaataaatct aagcaatcca ctctagaatt aaatagtttc c 4001 <211> 14931 <212> DNA <213> Cricetulus griseus <220> <221> misc_feature <222> (2176)..(2239) <223> n is a, c, g, t, or a missing nucleotide <400> 4 (SEQ ID NO:18) catgtacact tatgcaagta tgatatggcc caacacagta ttttacacca atttttatct 60 ataaaatata catgtacatc aaaatatatt attaataata acatcattat tctttctttc 120 caagtaataa acacatacac tgaaattttg gttcttgtgg ataattttaa tgaaacagga 180 aatgcaaatt tatcttagca tgtttacttc actttctttg catagataac cagtaatcac 240 attgatggat catgtagtga aatgtatttt taggtatcta aggaattttg gcttcgtttt 300 gtgcttgttg acactgaatt ctattcctaa caacagtgtg taaggattct gtctgatttc 360 ttttaccagt atttgtccat ttgcattttc tttattattc atggctgctg ttctagaaag 420 tggaaggtag tgtgtcaagt ctgtttaca tgttccctg atgatcagtg tcttacacc 480 tctctgagta catgttggcc aatgtcgttt ctagacccat ctattcttgc ttgacttatc 540 ctggtacatg cctgccaaga aatttctct catcctttct gtctctcac tgatttactt 600 gatgtgtgga ttcacattg atcatatgga atagagat acaatttct ttattcacag 660 tttggaagac ttcaatctc atagatcatc attatttt gctactgttc cctatgctat 720 ggtgaaattt ccatttgaat aattgcttaa acaattaca agaaagaatc tatttttact 780 tgcaataact tccattcag aacatttact acactgttac tatatccaa aactagtttt 840 atatatcatg tgagaaatga ctaattcata atttggccat gatatttt tcagaaacag 900 aaaagtgac caatacatac acatgctat aaatattaag acttcagcaa atttaatatt 960 tattcatgat atcacataaa attcatttat tatgtttat ttaaatgtgt ttttaaaaca 1020 gtggtatcac taaatattaa gttagatgtg tttagtgct taatgaattt atattttaga 1080 atgttataag ttgtatatag tcaaatatgt aaaattttt atttttagg tctttctcat 1140 taaggtattt taattttggg tccctttcc agagtgactc tagctcatga tgagttgaca 1200 taaaaactaa acagtacaaa atgtacattg cattcagtat tgcacttgat ctttgcactg 1260 aagtttgagt cagttcatac atttagtact tgggaagtac attaagctaa ctttcattgc 1320 tctggcaaaa tgctcgataa gataagagtc tattgtggaa agccatggca gcaggaaagt 1380 aagactgctg atgatgttta atccatagtc aagacgcaga aggagatgaa tgctggtatc 1440 caacatttt tgctgttcat tttctctaga accctagtcc ataaagatgt atgacttgca 1500 ttcaaaatgc gtccccttca gttgttcaac ttttctgtaa atacctttc aggcatgtct 1560 agaagattgt ttcgcaaata cttctcaatc cattcaagtt gatagtgcag attaatcact 1620 gcagaataaa agcctgtaac ttggctcacg tgccaaggaa tatgcacact cctgacacat 1680 caataagtaa atcaaagtgt agcttttgcc tttaacattg ccagacttat gtaatgttct 1740 gcacgttctt cctccatcac ttttattct aatggtgttt ccttgacatt gaatcacgct 1800 gtggaagctg cttagaatta acattgaaat ctactgatat attatgatg cagcaattta 1860 gatttactat tttacttaga attttttata attgagagaa tataatattt tcacagttat 1920 ctatctgctg taatagagg attttaaaaa aaatctctat aacttttttt tacaacacac 1980 agtaaaatta agttaaaatt tataaagtc actatgttga tttcaaagtg tgctacgccc 2040 acggtggtca cgcaggtgta gcagaagatg ccactaaggt gggctaaggc cgatgggttg 2100 gggtctgcgc tccctggaga tgagccccag gcggttccct ggcaatcagc tgcgatcatg 2160 atgcccgatg agccannnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn 2220 nnnnnnnnn nnnnnnnnc tgggtgactt tatggaaaga atttgataga tttcatgatg 2280 tagaagaatt ttattaggct tattttacag gagactaaga ccctgggacc taaagatatc 2340 tgggtcctga gaatcaggaa atgggtagag acgtggttga tggtatgaga cagattttag 2400 agaactctta gatcatgggc aatgaccgca atctgatgct tagatagat catctataaa 2460 caattatgct gttctttttc tttctgttgt atgatctgat gatgtagccc ccttgccaag 2520 ttccctgatc ccccttgcca agttccctga ttgtaacagt atataagcat tgcttgagag 2580 catattcaac tacattgagt gtgtctgtct gtcattcct cgccgattcc tgatttctcc 2640 ttgagccttt tccctgttc tccctcggtc ggtggtctc acgagaggcg gtccgtggca 2700 aaagtgtata atgttctaa aacatttgaa ctctaaaaca tgcaaatga aaattaaaa 2760 taaaaaca tgaaaatta atatattag ctgctaaag ttaaacaata ctatatata 2820 ttttgttatt agaattcaa atcacattag ttggatttta tttgacatt gcattctttc 2880 aaataatt tcataaaaaagtttcccc atgatagtag aaataata catatgtatc 2940 tatctattta tttaactaca catatatagc atttgtttca actaaaataa atgaatgagc 3000 aaagcaccta agtaattggt gtctatatta tttatgaagc caatagtttc aataaatta 3060 tcatgcataa ggaggtattg caatgttaa accttttg aaacagatat tcccagttac 3120 agaaattata atttctaatc tttcctataa gtagaatgat gataattaat ataggccatt 3180 tgtaaataat gttcagatta aaatattctc tattcacta gagaagaatg atattaatg 3240 tattatattt tattcccat ttgtttgca tattcctct attackccctca gcagtttaaa 3300 tttgttcac catatgtgtg tgtgtttgta tcttaatat ggcactaaaa ttagaataat 3360 daddyaa tctttagg aaagattatt gatttatttt atgttgatag gaaatatct 3420 tttaattgtc ciegaatact ttttctcta ttttaggact gatcagaccc aggactaata 3480 ttttatatgt actaattcta tgtaccaaaa tatgttatta tctcatgaat tctgtctcaa 3540 tattgaggta aaaaatag tccatcatga actttaaaat taaaataatg attattaat 3600 ttttattcat atttgttg tatgaatggt tatacatcac atgtgtgcct ggtgactgtg 3660 aatgtcagga gaggtag aagccactgg aattggaata agagatata ttgagatgt 3720 tatgtgggtg ctgagaatta gacgcaagcc atcttcaaga atagccagca tactatacca 3780 ctgagtaatc cattcatccc tcaatatta tctttgtaga cagtaaatat atttctaaac 3840 tataaatgac cagaaaaatt aatgtattat taatgaagac attcatctca tgtgacacac 3900 ttcacctgtc taaatcagta acacctctc cactattaa gatttctaa gtgcatgaca 3960 cttactattt ctaaagctgt ccaatggggg ccagtcccca gtcagcaccc agtgagataa 4020 tccatgaatg catttatatc ttaggaaaaa ttcttatcta tgtagtattt agaacatttt 4080 catgtgaggg gataaacaag gaagcacaga tgctttctga tagaaacttt ctctttaatt 4140 catctagaaa aaaaaaacct ctcaggaaaa tctctcttgc tctcctccca atgctctatt 4200 cagcatcttc tccctactta attctagatc tttttctcta tgcctccttg ctgctgccct 4260 gctggctctg ctctatgcct ccccatgtca cttttctttg ctatctcacc gttaccttct 4320 ctgcctcact ctctgccttc ttctctgctt ctcacatggc caggctctgg acaattatag 4380 ttatatgtta cattctcata acacatgata tgtcacatag tttctctcag gctagggata 4440 tcacaatgac tggccaatga gcaagtggcc ttgcatgtag ctctaagttg gtgatggttc 4500 ccagacagta agtagccatt tggttgaaat ttgaggttgg gtagtacatg aagactgaat 4560 tttcttcaaa ctctggcctt gaaatagtaa aacaacacct atgaaaatga cgacctgtat 4620 ttgtctttag aggcaaccac atattgtctg cagggcctgc tttgaatttg ctctgaagtt 4680 agcttgtttg tgtaaaagga agaatcctat atcagcctga gaaatgtaaa atatcctagc 4740 atttcaagtc atcaaaatta tatggagt ataaatcatc cttctgacta ttcatagtca 4800 tattgtgtc caccaagtat aaaacacact accaagggc tgtggaaaaa atcgccataa 4860 ctgttcttat tagggaggca tagcagtggt acctgaggaa gttacagcaa caccactca 4920 tccagtcaat aacccatgg ctttgccact tggaggtacc caataatgtt tggctttgcc 4980 gagtaggact cacaaatt cagaggggtca atttttaat gctggttgtc actgctgaac 5040 agtcccattg ccctctgcat aattccacat tggaaagctt tttacactga ttgccaatca 5100 ttaaacagcc tactcagcat aaacaggtat gatattc tgcattttgt tacattacta 5160 gatgaatttcc tattctctcc tacaatagtg gaactgaaaa aagatacaca atcatactac 5220 ccctctacta atcttatgac ttatatcatt tcaatttca gaccataatg caactattg 5280 accaaaacat gtgagatga aaatagaa tgtagaata tattacatat aaaaagaaaa 5340 ggcggactta tttgtttta ttcttagca tgcatagca tacatgatt gaggtttata 5400 tataaaggg acaataatc ttcaagaac tacccctac tgaattaaaa tattaaagaa 5460 ggtcacacat ttactcaaat atattagact actgggcaaa tagacatgaa aagtagagtt 5520 atattgagg taggccttct gtgaaatgtc taggaatt atgtttcata cagtgtgtaa 5580 ccaagtggga atcatatcag aaagcagtca aaagcttata ttacaagtaa cagatgcttg 5640 gttatatgac ctcccagagc ttgactgtct atacacaaaa agtgtgtta ataaaactgt 5700 aatttgggct atgttttttt aaatggctc accacatga aaggaaggga atgagcatgt 5760 catggatgct tagagatt gcttccagca agagaattg agctttggct cttattacag 5820 aaacatgaca aggtgtgagt tttatttat agaaattata tatatttta agctggggac 5880 taaaaattttt attgaaaaaaaaaagg gataggcatg tactgaagc aaaaatagga 5940 tgtcaatgct gtaatgttat tttggacc aaatagtat ttcctataga atgacaatg 6000 atcttaggtt attattctc aataagatga caagttcaca agatatccta gttcattaaa 6060 atcgttttag tcatttaata gagtgctgtg atagattaca caaggaaag cacttacgat 6120 gagaaataat gatatccaca attattttct taattcttag aacattcta ttgttatatc 6180 tcaatctcag aagccactta ttgctttatt attgaaacat atgaattgt aagttatata 6240 ttgtctatgg tgacattca aagaacatgt gacgtacagt gtagcacaga taagaacat 6300 aactgcagct gatcagtaa ctaaacttac atacattaaa tctgccatgt tggcacagt 6360 gtgtgcacta ccaaggatg tactaatgct cacgacactc ccctatgtca ccctttgttc 6420 atcattacat cataggtcta tttgtttgc ttttgaatc tagaccagt cttttgtgtc 6480 tttccaagca cagagctcat taatttacct catagacttg tttaacttct tctggttcat 6540 aattgaata gaatactca ctactaatta tgtgagaccc tgccagtacc atagcacatg 6600 gataatttt acataaaca tgcatacaag windowgatt cagactgaac atgaattttta 6660 gagaaatcag gaggagtat atgggagtgg tggagtgag actagagaaa tgtattaaa 6720 ctataatctc atacaagc tctaagc aaaaaacatg aaacattgtc attcaagtga 6780 aacatcagtc ttcaattgg aagatattt ttactaggaa atgtctggt agatggttat 6840 tacttagaaaaaaaat tagaaaacgg taacttta taaaaagaat atacaatga 6900 gactacatga aaagttctta actaatgaaa caataatctt gaactttt tcttaaagt 6960 ttaatca taaccatcat ggaattcaa atttaaacta tttacatatt acccctgaaa 7020 taataactaa tacctaa aaatatata aaaaaaat ggcaatgcat gccatcatgg 7080 atttgggaga gagaatgttc attgcagttc tgaatggata ctggtgccac cacggtgaaa 7140 atctctgtat aggtccttcc aaagctgaa atagacata tcacagacc tgccacat 7200 ttttcaagca ataccaccaa ggactctacc tgactgcaga gatactct cataaaatat 7260 tattgttgat ctattcataa tattctggaaa atagaacag ccagatgcc catcaactga 7320 ttaatagatg aaaattat tgtacatttc agtgtatatat tattcagtttt ttaagaaaaa 7380 tgaaattatg taataagcat gtaaatggat atatcttgaa acaaccattc cccattatat 7440 tacctaaca ttgaagtcc aaaatcatat gatcttttta gtggatctac taatctttg 7500 ctatatgtat tttattgaac tacccatgga tgtgagataa ttggtaacaa cagcacatgg 7560 gagagcatgg gatcattca ggagattag agagaatgca ttttgga gatatggag 7620 gagcataga aaggattaaa tgaggttact gatgaagtg atggttagag aaggcaat 7680 gaggagggat aactagcact taggcctttt tgaaaaagac atagagaaaa tactattgta 7740 gaaacttccct atattggtg tatagttata tacaccaag agctcagatg gagttaccct 7800 ataatggaaa tattaactac ttttcac tgtgataaaa catcctgaac agagcaacat 7860 agattgggaa gcatttactt tggctcag ttctaacgggg aaaaatttc atgatgaaag 7920 aatgaatatg tcagcaaca gcagtagcaa tggctgaga agcaggtgag agctcacacatc 7980 ttgaagtgta agaatgtagc aggagaaca aactgcaat gaccagaaaa tgctttttgga 8040 tcagagcccca taccccctg actgacttct ccagaaattc tgacaata aaactcccca 8100 aacagagcca taactgaagg tccagtgtct gagactacta ggggtatttc ttattcaac 8160 cactacaatg gggtgggggg agcaatccctc caagtaggca ctacacacag acaaaaaa 8220 accttagtaa ctggaatga ttgacttatt tgaattactt gccagtggag ctacatagag 8280 cacaattatt gtatttaat tacccttta gatcttaca aacttgacag windowcata 8340 ttgctaaaga aaccacatat ttgaatcagg gaacatggtg atatctagtt gttcttcaac 8400 tggaaacttc atgctttctg cccaccattc atgttgctg aaagagcaat gtacactacc 8460 agtgtagaaa ttaaatcatc aatcttatca agtgtggat cctataagtt acataaaaa 8520 ttagcctgat aagatatccc caccagaga atattcacat aaatgctatg ggagcacaa 8580 gctattttct aaattagctt taatcctatt ctacaagaga gatccatat ctagaatagt 8640 tatagggatc aagaacccat ggcttgattg gtcataggcc caatgggaga tcctaatatt 8700 attgttctac aaaatgaaaa taactcctaa tgacttgttg ctgcagtaat aagttagtat 8760 gttgctcaac tctcacaaga gaagttttgt cttacaataa atggcaatta aagcagcccc 8820 acaagattta tatcataccg atctcctcat ggcctatgca tctagagct aggaacaaa 8880 gaggacccta agagagacat acatggtccc cctggagaag gggaaggggg caaccctcc 8940 aaagctaatt gggagcatgg gggagggg agggagttag agggaga agggataaa 9000 agggagggagggagggagggagggagggagggagggagggagggagggagg 9060 caagaaaaga gatacatag taggaggagc cttgtatgtt taaatagaa actggcacta 9120 gggaattgtc caagatcca caagtcca ctaataatct aagcaatgt cgagaggcta 9180 ccttaaaagc ctttctctga taatgagat gatgactacc ttatatacca tcctagagcc 9240 ttcatccagt agctgatgga agcagaagca gatactaca gctaacact gagctagttg 9300 cagahaggga ggagtgatga gcaagtcaa gaccaggctg gagaacaca cagaaacagc 9360 agacctgaaa aaatgttgc acatggaccc cagactgata gctgggagtc cagcatagga 9420 cttttctga aaccctgaat gaggatatca gtttggaggt ctggttaatc tatggggaca 9480 ctggtagtgg atcatattt atccctagtt catgactgga atttgggtac ccattccaca 9540 tggaggaatt ctctgtcagc ctagacacat gggggaggtt ctaggtcctg ctccaaataa 9600 tgtgttagac tttgagac tcccttgaga agactcaccc tccctgggga gcagaaaggg 9660 gatgggatga gggttggtga gggacaggag aggagggg gggtgagggaa ctgggattga 9720 caagtaatg atgcttgttt ctaatttaaa tgaataagg aaagtaaa gagaaaaga 9780 aaacaggcca aaagattata aaagacagag gtggtgggtg actataaaga aacactatta 9840 tctaaataaa aatatgtcag aagcacacat gaacttatag tgttatgaa agtatgtata 9900 ataactacat aatctcaagc caagaaaaaa atatcatctt tcagtgatga aggtgatttt 9960 atttctccca gaattaaagc caaagaccta atgaaagtaa ttatcttcaa aaggttgaaa 10020 atacatactt tgcaatacac agatctgcct agaaatctca tgttcacaat acacatgatg 10080 ctcaattgaa ttccattcaa tgttacagtt tagataaaca gtttgtagat aaactcacaa 10140 tgtatcattt ctttttattt tttgaccaaa cagcttctca tctgttattc agaataattc 10200 ctcgatggca ggatatccat cccaattggg ggaaggggag aatttgaaga aaacctagac 10260 cacatacata tttgccattg ggaaacaaag tctaaaatga tgttgttcac atcttctcta 10320 ctagtcctct ccccgtccca aagaaccttg gtatatgtgc ctcattttac agagagagga 10380 aagcaggaac tgagcatccc ttacttgcca tcctcaaccc aaaatttgca tcattgctca 10440 gctctgccct tctcatatga cagttacaag tcaaggcttc caaagtccct ctgtcatgtt 10500 tggtgtcaat agtttataca gatgacttca tgtcttcata tctaatgtct tatatagatt 10560 atattaaac atgttattt ctctaaccac atttttaattt atttaaaaa tccattaatt 10620 gtgtctataa atgcagaca gagtgctgag acacatata agcctgatga tctgaatttg 10680 aaactcacac ccaccacatg gagaatcaac ttccaaaat tttcctatta cttccacact 10740 tacaccattg tashaacaca attaatga aaaaatgaa atgaataa aattaagtc 10800 tctgtaggta atgctactgt gcagcaaag taaaatggc agcttaagct tgctttatgg 10860 ttacacttta ccatcttcca ttattataa ggacttcaat catggcagaa ctatgctgtt 10920 attgtctcag tgtaacctaa ccaggtgttc cagatgttct taatgtggac acctaaacta 10980 tttgatattt gggttaagat ctttccctct ttcagagaa acctcaggac agagggaatc 11040 ttgtctttta attttgagtc tgtagacttt ttccattca atatacatg aaacaagtga 11100 tgaagaaat taatcaaag gtgggaatttg caatgatatt aggttcaata ttaagcttca 11160 atattacat ggaatcgcct gttatacact gagtgttttgg caataggga ttttagag 11220 aaggagtttt tattctcaac aggttcctta agttttagctc aaataatct aagcaatcca 11280 ctctagaattt aaatagtttc ctaagggcac agctatgaat agagctcaat ttacatataa 11340 aattttgttc accatttg accattczag accattaaaaaaaaat 11400 ttagatgtca atatcaagtg atagttcat ctcctttttt atatatatc acctaaatca 11460 ccattttctc agaaaaatct ggctgaagt tctgtctgga acttcacat gaaaaatatg 11520 cacagcttgc tattataat cctagttgat ttttaagatt catgtctggt gtctgactca 11580 gaggggccag aggctagaca atatttt gatcttcat tgtgaagatt tttaatgatt 11640 attttatat aaatacaa gatgatgat atgtaactt tgtacagttc atagacgctg 11700 aactactttg tgcttaaaat gttagttccc tatcataaat gataggtgat aagtgtagt 11760 ttaatacttt ccctctgagc tatattcatg tactgagaa ttattaaa catgaaaga 11820 ctgtgtttat agtctcagct cctgagaact ggtcaacct taggcaggtg atgccagga 11880 gcaacgtttt tctctacag aggatgcttt gctgccaagc aacctggttg tgtggaatg 11940 ttccttttt aatcaagttt aaagggtctt catcatgctg tgctccaca tattttcagg 12000 ttagagcttg gtccttggag tattacttt taccagaaaa ttcatagtat tctttcaata 12060 actaacaact aaacttttcg aaaaaaga attggaattt caatttaaa gcctgagtaa 12120 aattcttgtg aatcaggata ttttattt agtcttatct tttaaaagt tattttatt 12180 tttaaaaaat tataatatac ttcatatt tccctccttc acttttcttt acaacactt 12240 ctatagatca ccatgtgtttt ttttttac atttatggcc tctttctgtt cattgttatt 12300 acatacaaat agtcttgcct atagagaac accacaattt gttacctgat aacaaattat 12360 cacccttaa aacctacaa ctattgaat tactgaaag actatactta tagtaaa 12420 gatatagtg tgtgcacata tatagataca catatatgta ggatttttaa ttttagattt 12480 tagacatca attattat atgactgaga aactagacac tataaatgag cattcagtat 12540 tcaaccgt gattttagat attgtcacaa tgacagaaaa tttcttata gaaaatttta 12600 agttttgtga ttgctctgtg cacttagtga agtctcacag aaaagaatc atagtatttt 12660 tagtttata taaaaagtac atattaa atggttggc aaaaacac atttgagcat 12720 tttcctatt tactatcaag tagtatcatt ttgaaataat atttgacta gtttcaaaaa 12780 tgaaaacaaa atttaacta atgcctaat ctagcctgat aacatttta tgaatgaat 12840 tattcaatag tgttatcaat taggggcca aaacttttcc taaaataaa cttttaattt 12900 tttccattt tttttaaat tagaacaaa attgttttac atgtaaatca gagttttccctc 12960 accctcccct tctccctgtc cctcactaac accctactg tcccatacca tttctgctcc 13020 ccagggagggg tgaggctc catggggaaa ctcaggagtc tgtctatcct tcggatagg 13080 gcctaggccc tcaccattt gtctaggcta aggctcacaa agtttactcc tatgcttg 13140 ataagtactg atctaca agagacacca tagattcct aggcttccctc actgacacccc 13200 atgttcatgg ggtctggaac aatcatatgc tagtttccta ggtatcagtc tggggaccat 13260 gagctccccc ttgttcaggt caactgtttc tgtgggtttc accaccctgg tcttgactgc 13320 tttgctcatc actcctccct ttctgtaact gggttccagt acaattccgt gtttagctgt 13380 gggtgtctac ttctactttc atcagcttct gggatggagc ctctaggata gcatacaatt 13440 agtcatcatc tcattatcag ggaagggcat ttaaagtagc ctctccattg ttgcttggat 13500 tgttagttgg tgtcatcttt gtagatctct ggacatttcc ctagtgccag atactcttt 13560 aaacctacaa gactacctct attatggtat ctctttcttt gctctcgtct attcttccag 13620 acaaaatctt cctgctccct tatattttcc tctcccctcc tctctcccc ttctcattct 13680 cctagatcca tcttcccttc ccccatgctc ccaagagaga tgttgctcag gagatctttgt 13740 tccttaaccc ttttcttggg gatctgtctc tcttagggtt gtccttgttt ctagcttct 13800 ctggaagtgt ggattgtaag ctggtaatca tttgctccat gtctaaaatc catatatgag 13860 tgatgtttgt cttttttgga ctgggttacc tcactcaaaa tggttctttc catatgtctg 13920 tggattcaa tagcacaac aacatacagt atcttggggc aacactacc aacaagtga 13980 aagaccagta tagcagaac tttgagtttta aagaaaaaaagaaga taccagaaaa 14040 tggaagatc tcccatgctc ttgaatagc agaatcaca tagtaaaat ggcaatcttg 14100 ccaaaatcca tctacagact caatgcaatc cccattaaat accagcacac ttctcacag 14160 acctgaaga atatactta actttatatg gagaaaaaa agacccagga taggccaaac 14220 aaccctgtac atgaggca cttccagagg catccccatc cctgacttca agctctatta 14280 taggtaata atcctgaaaa cagcttggta atggcacaaa atagacagg tagaccaatg 14340 gattgagtt gaaaaccctg atattaaccc acatacttat gaacacctga ctttgacaaa 14400 gaagctagg ttatacaatg tagaagaa agcatctca acaaatcgtg ctggcataac 14460 tggatgctgg catgtagaag actgcagata gatccatgtc taatgccatg cacaaactt 14520 aagtccaaat ggatcaaaa cctcacata aatccagcca cactgaacct catagaagag 14580 aaagtgggaa gtatccttga aataattggt acaggacc acatcttgaa cttaacacca 14640 gtagcacaga caatcagatc aataatcaat aaatgggacc tcctgaaact gagaagcttc 14700 tgtaaggcaa tggataagtc aacaggacaa aatggcagcc cacggaatgg gaaaagatat 14760 tcaccaatcc tatatctgac agagggctgc tctctatttg caaagaacac aataagctag 14820 tttttaaaac accaattaat ccgattataa agttgggtag agaactaaat aaagaattgt 14880 taacagagca atctaacttg gcagaaagac acataagaaa gtgctcacca t 14931

[0130] It should be understood that the description, specific examples, and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the invention. Various changes and modifications within the invention, including combining the embodiments in whole or in part, will become apparent to those skilled in the art from the discussion, disclosure, and data contained herein and are therefore considered part of the invention.

[0131] The present invention may be embodied in other specific forms without departing from its spirit or essential attributes, and therefore, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims

1. 1. A method for controlling transcription of a polynucleotide of interest in a cell, said method comprising: I. Maintaining cells in a medium that lacks an effective amount of a ligand for both the activator and the repressor, wherein the cells: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding the activator; and (C) a second operator; and (D) a polynucleotide encoding the repressor, maintaining that transcription of said polynucleotide of interest is inhibited in the absence of said ligands of both said activator and said repressor; II. Controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the ligand of both the activator and the repressor.

2. 2. The method of claim 1, wherein the second operator is operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest.

3. The method of claim 1, wherein the activator binds to the first operator in the presence of the ligand and enables transcription of the polynucleotide of interest.

4. 1. A method for controlling transcription of a polynucleotide of interest in a cell, said method comprising: I. Maintaining cells in a medium that does not have an effective amount of a ligand of an activator (activator ligand) and that has an effective amount of a ligand of a repressor (repressor ligand), wherein the cells: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding the activator; and (C) a second operator; and (D) a polynucleotide encoding the repressor, maintaining transcription of said polynucleotide of interest is inhibited in the absence of said activator ligand and in the presence of said repressor ligand; II. Controlling the cells to transcribe the polynucleotide of interest by maintaining the cells in a medium having an effective amount of the activator ligand and not having an effective amount of the repressor ligand.

5. 5. The method of claim 4, wherein the second operator is operably linked and positioned 3' to the promoter and 5' to the polynucleotide of interest.

6. 1. A method for controlling transcription of a polynucleotide of interest in a cell, the method comprising maintaining a cell in a medium that does not have an effective amount of a first ligand of a first regulatory fusion protein (RFP) and has an effective amount of a second ligand of a second RFP, wherein the cell: (A) a promoter; (B) an Arc operator; and and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B), and (C) are operably linked, and transcription of the rtTA polynucleotide is controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr), such that rtTA is capable of controlling the transcription of a polynucleotide of interest.

7. 7. The method of claim 6, wherein the first ligand is selected from the group consisting of tetracycline and doxycycline.

8. 7. The method of claim 6, wherein the second ligand is selected from the group consisting of estrogen, estradiol (E2), tamoxifen, and 4-hydroxytamoxifen (OHT).

9. A cell capable of regulated transcription of at least one polynucleotide of interest, said cell comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding an activator; and (C) a second operator; and (D) a polynucleotide encoding a repressor, A cell wherein transcription of said polynucleotide of interest is inhibited in the absence of ligands of both said activator and said repressor and is permitted in the presence of said ligands of both said activator and said repressor.

10. A cell capable of regulated transcription of a polynucleotide of interest, said cell comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding an activator; and (C) a second operator; and (D) a polynucleotide encoding a repressor, A cell in which transcription of the target polynucleotide is inhibited in the absence of an effective amount of an activator ligand and in the presence of an effective amount of a repressor ligand, and is permitted in the presence of an effective amount of the activator ligand and in the absence of an effective amount of the repressor ligand.

11. The cell of claim 10 , wherein the second operator is operably linked and positioned 3′ to the promoter and 5′ to the polynucleotide of interest.

12. The cell of claim 10 , wherein the activator binds to the first operator in the presence of the activator ligand, allowing transcription of the polynucleotide of interest.

13. A cell capable of regulated transcription of a polynucleotide of interest, said cell comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), wherein the first RFP is: (1) a transcriptional activation domain fused to a first DNA-binding domain; and (2) a polynucleotide comprising a ligand-binding domain, wherein the first ligand can bind to the ligand-binding domain of the first RFP, and the DNA-binding domain of the first RFP can bind to the operator located 5' when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP, which is different from the first RFP, wherein the second RFP is (1) a DNA-binding domain, and (2) a ligand-binding domain, wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and the second RFP is capable of binding to the second operator in the presence of the second ligand; and a polynucleotide, wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand, and is permitted in the presence of an effective amount of the first ligand and in the absence of an effective amount of the second ligand.

14. The cell of claim 13 , wherein the second operator is operably linked and positioned 3′ to the promoter and 5′ to a polynucleotide sequence encoding a protein of interest.

15. The cell of claim 13, wherein the second operator is operably linked to the polynucleotide sequence encoding the first RFP, and the second RFP is capable of controlling the transcription of the polynucleotide encoding the first RFP.

16. A cell capable of regulated transcription of a polynucleotide of interest, said cell comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5' to the promoter; (B) a polynucleotide encoding a regulatory fusion protein (RFP), said RFP comprising: (1) a transcription activation domain fused to a DNA binding domain, and (2) a polynucleotide comprising a ligand-binding domain, wherein the ligand can bind to the ligand-binding domain of the RFP, and the DNA-binding domain of the RFP can bind to the first operator when in the presence of the ligand; (C) a second operator; and (D) a polynucleotide encoding a repressor protein, wherein the repressor protein can bind to the second operator only in the absence of the ligand; A cell in which transcription of the target polynucleotide is inhibited in the absence of an effective amount of the ligand of both the activator and the repressor, and is permitted in the presence of an effective amount of the ligand of both the activator and the repressor.

17. The cell of claim 16 , wherein the second operator is operably linked and positioned 3′ to the promoter and 5′ to the polynucleotide of interest.

18. A cell capable of regulated transcription of a polynucleotide of interest, said cell comprising: (A) a promoter; (B) an Arc operator; and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B), and (C) are operably linked, and transcription of the rtTA polynucleotide is controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr); A cell, wherein rtTA is capable of controlling said transcription of a polynucleotide of interest.

19. A bioreactor comprising: (i) the cells of claim 9; and (ii) a culture medium.

20. A bioreactor comprising: (i) cells described in claim 10; and (ii) a culture medium.

21. A bioreactor comprising: (i) cells described in claim 13; and (ii) a culture medium.

22. A bioreactor comprising: (i) cells described in claim 16; and (ii) a culture medium.

23. A bioreactor comprising: (i) cells described in claim 18; and (ii) a culture medium.

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