# Patent

## Title
Adeno-associated virus (AAV) producer cell line and related methods

## Abstract

The present disclosure relates to a mammalian cell line for producing adeno-associated virus (AAV), suitably including nucleic acids encoding helper genes and AAV genes, under the control of derepressible promoters. The disclosure also relates to isolated nucleic acid molecules that encode such genes, as well as methods of using the mammalian cells for producing AAVs.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent Application No. 62/783,589, filed Dec. 21, 2018, and 62/866,092, filed Jun. 25, 2019, the disclosures of each of which are incorporated by reference herein in their entireties.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 17, 2019, is named 0132-0049US1 SL.txt and is 364,567 bytes in size.
FIELD OF THE INVENTION
The present disclosure relates to mammalian cell lines for producing adeno-associated virus (AAV). The cells suitably include nucleic acids encoding helper genes and AAV genes, under the control of derepressible promoters. The disclosure also relates to isolated nucleic acid molecules that encode such genes, as well as methods of using the mammalian cells for producing AAVs.
BACKGROUND OF THE INVENTION
The safety profile and long-term expression capacity make adeno-associated virus (AAV) an excellent viral vector for gene therapy in humans. The wildtype AAV genome is composed of a 4.7 kb single-stranded DNA that includes regulatory genes for replication (Rep) and structural genes for Capsid (Cap), flanked by inverted terminal repeats (ITR) for virus replication and packaging. As a dependent virus, AAV replication in host cells requires the coinfection of helper viruses, such as Adenovirus (Ad) and Herpes Simplex virus. Alternatively, the expression of cloned helper genes can also support AAV replication. For instance, recombinant AAV can be produced in HEK293 cells by the co-transfection of three plasmids: pHelper plasmids expressing E2A, E4Orf6 and VA from Adenovirus, pRep-Cap plasmids for Rep and Cap proteins, and AAV transfer plasmids carrying the desired gene of interest (GOI).
Currently, AAV manufacturing relies on several bridging platforms. Besides the triple trasnfection in HEK293 cells noted above, AAV can be produced by co-infection of two baculoviruses expressing Rep-Cap and the GOI, respectively, into insect cells. However, these baculoviruses are unstable at higher passage and are time-consuming to prepare (see, e.g., Urabe et al., “Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors,” Human Gene Therapy 13:1935-1943 (2002)). HeLa packaging cells with stably integrated Rep-Cap and GOI have also been developed. These systems, nevertheless, still require the wildtype Adenovirus as a helper virus, which poses risks of contamination of replicative adenovirus in AAV products (see, e.g., Robert et al., “Manufacturing of recombinant adeno-associated viruses using mammalian expression platforms,” Biotechnology Journal 12: 1600193 (1-16) (2017).
What are needed, are cell lines and related methods for production of AAV that are easily scalable to large volume production, to provide reproducible and stable results, while limiting contamination and reducing cost.
SUMMARY OF THE INVENTION
In some embodiments, provided herein is a mammalian cell for producing an adeno-associated virus (AAV), comprising a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter, a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter, and a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters.
In additional embodiments, provided herein is a mammalian cell for producing an adeno-associated virus (AAV), comprising a nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
In further embodiments, provided herein is an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
In still further embodiments, provided herein is a method of producing an adeno-associated virus (AAV) in a mammalian cell comprising transfecting the mammalian cell with an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters, treating the mammalian cell with a binding partner of the repressor element, activating the first, second and third derepressible promoters, producing the AAV, and harvesting the AAV.
In further embodiments, provided herein is a method of treatment with an adeno-associated virus (AAV) comprising: transfecting the mammalian cell with an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters, treating the mammalian cell with a binding partner of the repressor element, activating the first, second and third derepressible promoters, producing the AAV, harvesting the AAV and administering the AAV to a mammalian patient.
In still further embodiments, provided herein is a method of producing an adeno-associated virus (AAV) comprising: transfecting a mammalian cell stably expressing one or more nucleic acids encoding TetR and/or TetR-KRAB with a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA under control of a first derepressible promoter, a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, and, optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter, treating the mammalian cell with a binding partner of the TetR and/or TetR-KRAB, activating the first, second, and third derepressible promoters, producing the AAV, and harvesting the AAV.
In still further embodiments, provided herein is a method for producing an adeno-associated virus (AAV), comprising: stably transfecting a mammalian cell with a nucleic acid encoding a TetR and/or TetR-KRAB repressor, chicken hypersensitive site-4 (cHS4) sequences flanking the TetR and/or TetR-KRAB repressor, and a selection gene, transfecting the stably transfected mammalian cell with: a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA, under control of a first derepressible promoter; a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter; and optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter; treating the mammalian cell with a binding partner of the TetR; activating the first, second and third derepressible promoters; producing the AAV; and harvesting the AAV.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic of the use of derepressible promoters to control the expression of helper and VA genes in accordance with embodiments hereof.
FIGS. 2A and 2B show exemplary nucleic acid molecules for production of helper and VA genes in accordance with embodiments hereof.
FIG. 3 shows the results of induction of helper and AAV genes in accordance with embodiments hereof.
FIGS. 4A-4C shows schematics of derepressible constructs for expression of AAV genes in accordance with embodiments hereof.
FIG. 5 shows exemplary derepressible p5 promoters in accordance with embodiments hereof.
FIG. 6 shows exemplary derepressible p19 promoters in accordance with embodiments hereof.
FIG. 7A-7B shows exemplary derepressible p19 promoters, including an artificial intron, in accordance with embodiments hereof.
FIGS. 8A-8B show the results of Rep-Cap expression and AAV titers using the Rep-Cap vectors, in accordance with embodiments hereof.
FIGS. 9A-9B show additional results of Rep-Cap expression and AAV titers using the Helpers and Rep-Cap vectors, in accordance with embodiments hereof.
FIGS. 10A-10D show exemplary nucleic acid constructs encoding helper, AAV and VA genes, in accordance with embodiments hereof.
FIGS. 11A-11E show exemplary nucleic acid constructs encoding helper, AAV, gene of interest, and Rep-Cap vectors, in accordance with embodiments hereof.
FIGS. 12A-12B show exemplary nucleic acid constructs encoding TetR and TetR-KRAB, in accordance with embodiments hereof.



DETAILED DESCRIPTION OF THE INVENTION
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the method/device being employed to determine the value. Typically the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variability depending on the situation.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, system, host cells, expression vectors, and/or composition of the invention. Furthermore, compositions, systems, cells, and/or nucleic acids of the invention can be used to achieve any of the methods as described herein.
Adeno-associated virus (AAV) has emerged as the vector of choice for gene therapy in over 120 clinical trials worldwide. The fast-growing demand of recombinant AAV requires highly efficient and robust manufacturing platforms. However, current methods for AAV production, including transient transfection and helper virus systems, are extremely costly and lab-intensive. Described herein is a plasmid/helper virus-free AAV producer cell line, and methods of use thereof, that provides efficient AAV manufacturing for a long-term solution at significantly reduced cost. The AAV producer cell line described herein represents a next generation platform for both clinical and commercial AAV manufacturing.
Thus, in embodiments, provided herein is a mammalian cell for producing an adeno-associated virus (AAV).
As used herein, the term “mammalian cell” includes cells from any member of the order Mammalia, such as, for example, human cells, mouse cells, rat cells, monkey cells, hamster cells, and the like. In some embodiments, the cell is a mouse cell, a human cell, a Chinese hamster ovary (CHO) cell, a CHOK1 cell, a CHO-DXB11 cell, a CHO-DG44 cell, a CHOK1SV cell including all variants (e.g. POTELLIGENT®, Lonza, Slough, UK), a CHOK1SV GS-KO (glutamine synthetase knockout) cell including all variants (e.g., XCEEDTM Lonza, Slough, UK). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK293, a HeLa cell, or a HT1080 cell.
Mammalian cells include mammalian cell cultures which can be either adherent cultures or suspension cultures. Adherent cultures refer to cells that are grown on a substrate surface, for example a plastic plate, dish or other suitable cell culture growth platform, and may be anchorage dependent. Suspension cultures refer to cells that can be maintained in, for example, culture flasks or large suspension vats, which allows for a large surface area for gas and nutrient exchange. Suspension cell cultures often utilize a stirring or agitation mechanism to provide appropriate mixing. Media and conditions for maintaining cells in suspension are generally known in the art. An exemplary suspension cell culture includes human HEK293 clonal cells.
As used herein, the term “adeno-associated virus (AAV)” refers to a small sized, replicative-defective nonenveloped virus containing a single stranded DNA of the family Parvoviridae and the genus Dependoparvovirus. Over 10 adeno-associated virus serotypes have been identified so far, with serotype AAV2 being the best characterized. Other non-limiting examples of AAV serotypes are ANC80, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In addition to these serotypes, AAV pseudotypes have been developed. An AAV pseudotype contains the capsid of a first serotype and the genome of a second serotype (e.g. the pseudotype AAV2/5 would correspond to an AAV with the genome of serotype AAV2 and the capsid of AAV5).
As referred to herein, the term “adenovirus” refers to a nonenveloped virus with an icosahedral nucleocapsid containing a double stranded DNA of the family Adenoviridae. Over 50 adenoviral subtypes have been isolated from humans and many additional subtypes have been isolated from other mammals and birds. Birds. See, e.g., Ishibashi et al., “Adenoviruses of animals,” In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562 (1984); Strauss, “Adenovirus infections in humans,” In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984). These subtypes belong to the family Adenoviridae, which is currently divided into two genera, namely Mastadenovirus and Aviadenovirus. All adenoviruses are morphologically and structurally similar. In humans, however, adenoviruses show diverging immunological properties and are, therefore, divided into serotypes. Two human serotypes of adenovirus, namely AV2 and AV5, have been studied intensively and have provided the majority of general information about adenoviruses.
In embodiments, the mammalian cell provided herein suitably includes a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter, a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter, and a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters.
In exemplary embodiments, the nucleic acid molecules encoding the various components for producing an AAV are contained within the mammalian cell in separate nucleic acid molecules, for example separate plasmids or vectors. In other embodiments, the nucleic acid molecules encoding the various components for producing an AAV are included on the same plasmid or vector. In further embodiments, certain of the components are contained on the same nucleic acid molecule (e.g., helper genes and AAV gens), while other genes are contained on separate nucleic acid molecules (e.g., gene encoding the repressor element).
A “nucleic acid,” “nucleic acid molecule,” or “oligonucleotide” means a polymeric compound comprising covalently linked nucleotides. The term “nucleic acid” includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single- or double-stranded. DNA includes, but is not limited to, complimentary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA, or MIRNA.
In the various embodiments described herein, the nucleic acid molecules are capable of encoding the various genes. That is the nucleic acid molecules, when transcribed, produce mRNA for the genes described herein, which is then translated to the desired or required proteins.
As described herein, suitably the mammalian cells include a nucleic acid molecule encoding a viral helper gene. Viral helper genes include various adenoviral virus genes, herpes virus genes and bocavirus genes (see, e.g., Guido et al., “Human bocavirus: Current knowledge and future challenges,” World J. Gateroenterol 22:8684-8697, the disclosure of which is incorporated by reference herein in its entirety). In exemplary embodiments, the viral helper gene is an adenovirus helper gene. As referred to herein, the term “adenovirus helper gene” or “AV helper gene” refers to a gene that is composed of one or more nucleic acid sequences derived from one or more adenovirus subtypes or serotypes that contributes to Adeno-associated virus replication and packaging. In some embodiments, the Adenovirus helper gene is E1A, E1B, E2A, E4 (including E4Orf6), VA, or a combination thereof or any other adenovirus helper gene. In exemplary embodiments, the adenovirus helper gene comprises both E2A and E4Orf6 genes. Suitably, an internal ribosome entry site (IRES) element is included between the E2A and E4Orf6 genes. The IRES element initiates translation of the E4Orf6 gene after the E2A gene in a single expression cassette, providing stability to the construct.
The various nucleic acid molecules encoding the various genes described herein are suitably under control of a derepressible promoter. As used herein “under control” refers to a gene being regulated by a “promoter,” “promoter sequence,” or “promoter region,” which refers to a DNA regulatory region/sequence capable of binding RNA polymerase and initiating transcription of a downstream coding or non-coding gene sequence. In other words, the promoter and the gene are in operable combination or operably linked. As referred to herein, the terms “in operable combination”, “in operable order” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a promoter capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
In some examples of the present disclosure, the promoter sequence includes the transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. In some embodiments, the promoter sequence includes a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive the gene expression, e.g., in the host cell or vectors of the present disclosure. In some embodiments, the promoter is not a leaky promoter, i.e., the promoter is not constitutively expressing any of the gene products as described herein. In other embodiments as described herein, the promoter is a constitutive promoter, which initiates mRNA synthesis independent of the influence of an external regulation.
Suitably, the promoters used to control the transcription of the various genes for producing the AAVs described herein are derepressible promoters. As used herein, a “derepressible promoter” refers to a structure that includes a functional promoter and additional elements or sequences capable of binding to a repressor element to cause repression of the functional promoter. “Repression” refers to the decrease or inhibition of the initiation of transcription of a downstream coding or non-coding gene sequence by a promoter. A “repressor element” refers to a protein or polypeptide that is capable of binding to a promoter (or near a promoter) so as to decrease or inhibit the activity of the promoter. A repressor element can interact with a substrate or binding partner of the repressor element, such that the repressor element undergoes a conformation change. This conformation change in the repressor element takes away the ability of the repressor element to decrease or inhibit the promoter, resulting in the “derepression” of the promoter, thereby allowing the promoter to proceed with the initiation of transcription. A “functional promoter” refers to a promoter, that absent the action of the repressor element, would be capable of initiation transcription. Various functional promoters that can be used in the practice of the present invention are known in the art, and include for example, PCMV, PH1, P19, P5, P40 and promoters of Adenovirus helper genes (e.g., E1A, E1B, E2A, E4Orf6, and VA).
Exemplary repressor elements and their corresponding binding partners that can be used as derepressible promoters are known in the art, and include systems such as the cumate gene-switch system (CuO operator, CymR repressor and cumate binding partner) (see, e.g., Mullick et al., “The cumate gene-switch: a system for regulated expression in mammalian cells,” BMC Biotechnology 6:43 (1-18) (2006), the disclosure of which is incorporated by reference herein in its entirety, including the disclosure of the derepressible promoter system described therein) and the TetO/TetR system described herein (see, e.g., Yao et al., “Tetracycline Repressor, tetR, rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells,” Human Gene Therapy 9:1939-1950 (1998), the disclosure of which is incorporated by reference herein in its entirety).
In exemplary embodiments, the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2). A schematic showing an exemplary depressible promoter system is provided in FIG. 1. A derepressible promoter including the PCMV promoter, and a derepressible promoter including the PH1 promoter, are shown, both including two TetO sequences (TetO2). As illustrated schematically, upon binding of two tetracycline repressor proteins (TetR—the repressor elements for the TetO2 sequences), to the TetO2 sequences, both the PCMV promoter and the PH1 promoter are repressed. That is, little or no transcription takes place from these promoters. Upon binding of a binding partner for TetR (suitably Doxycycline (Dox), the TetR proteins change conformation, release from the TetO2 sequences, and the functional promoters begin their normal transcription processes, as they would naturally. As illustrated schematically in FIG. 1, this results in the change of the overall system from an “off” position (where suitably no transcription is taking place from the PCMV promoter and the PH1 promoter), to an “on” position when Dox is added, allowing the PCMV promoter and the PH1 promoter to return to their natural state of transcribing the genes under their control.
For example, as shown in FIG. 1, the PCMV promoter with TetO2 sequences (suitably the pcDNA4/TO promotor; INVITROGEN®), is in an “off” position when bound by TetR. When Dox is added, the TetR changes conformation, release from the TetO2 sequences of the depressible promoter, and the PCMV promotor proceeds to transcribe the adenoviral helper genes (e.g., E2A and E4).
As described herein, and as illustrated in FIG. 1, the mammalian cell can further comprise a nucleic acid encoding a viral-associated (VA), non-coding RNA under control of a fourth derepressible promoter. As shown in FIG. 1, this derepressible promoter can include the functional promoter PFH, and the TetO2 sequences controlling the expression of the non-coding RNA (see, e.g., Wiederschain et al., “Single-vector inducible lentiviral RNAi system for oncology target validation, Cell Cycle 8:498-504 (2009), the disclosure of which is incorporated by reference herein in its entirety, including for the disclosure of the promoter system and sequence). As shown in FIG. 1, the PH1 promoter with TetO2 sequences, is in an “off” position when bound by TetR. When Dox is added, the TetR changes conformation, release from the TetO2 sequences of the depressible promoter, and the PH1 promotor proceeds to transcribe the VA 1 non-coding RNA.
FIG. 2A shows an exemplary nucleic acid molecule that can be utilized in the various mammalian cells and methods described herein. As illustrated, a CMV promoter is used upstream of both E2A and E4Orf6 genes, linked via a IRES element. The CMV promoter includes the CMV enhancer and the tet operator (TetO2), for control, via derepression. Also illustrated in FIG. 2A is an exemplary location for the H1 promotor, also including TetO2 sequences, controlling the expression of the VA non-coding RNA.
FIG. 1 and FIG. 2A also illustrate that, in embodiments, the mammalian cells can include the repressor element under control of a constitutive promoter. As described herein, suitably the repressor element that is encoded is a tetracycline repressor protein (TetR). As illustrated in FIG. 1 and FIG. 2A, a suitable promotor for expression of the repressor element is an hPGK promotor. By placing the repressor element under the control of a constitutive promotor, production of the repressor element, suitably TetR, is always active. That is, TetR is being produced upon introduction of the nucleic acid molecule into the mammalian cell. This provides tight control of the various derepressible promoters that are repressed by the TetR binding to the TetO2 sequences.
As shown in FIG. 2A, in exemplary embodiments, a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein, can be included. In FIG. 2A, this transcriptional repression domain is a Krueppel-associated box (KRAB) sequence, fused in frame to the C-terminus of TetR (see, e.g., Szulc et al., “A versatile tool for conditional gene expression and knockdown,” Nature Methods 3:109-116 (2006)). The use of a KRAB sequence, or other transcriptional repression domain, improves the repressive activity of the TetR when binding to TetO2, thereby minimizing the amount of leakage or basal gene expression prior to derepression (i.e., prior to the addition of Dox). FIG. 2B and FIG. 12A shows an exemplary nucleic acid molecule that lacks the KRAB sequence.
As shown in FIG. 12A, in exemplary embodiments, a nucleic acid encoding the tetracycline repressor protein, can be included or stably expressed within a mammalian cell. In FIG. 2A and FIG. 12B, this transcriptional repression domain is a Krueppel-associated box (KRAB) sequence, fused in frame to the C-terminus of TetR (see, e.g., Szulc et al., “A versatile tool for conditional gene expression and knockdown,” Nature Methods 3:109-116 (2006)). The use of a KRAB sequence, or other transcriptional repression domain, improves the repressive activity of the TetR when binding to TetO2, thereby minimizing the amount of leakage or basal gene expression prior to derepression (i.e., prior to the addition of Dox). FIG. 2B and FIG. 12A show an exemplary nucleic acid molecule that lacks the KRAB sequence.
In embodiments, the AAV gene that is encoded by the nucleic acid molecule comprises Rep and Cap genes. Other AAV genes that can be encoded by the nucleic acid molecules include any gene from any AAV serotype. In some embodiments, the AAV gene is Rep78, Rep68, Rep 52, Rep40, VP1, VP2, VP3, or a combination thereof. In some embodiments, the AAV gene is from adeno-associated virus type 2. In some embodiments, the AAV gene is from the adeno-associated virus Anc80.
As referred to herein, the term “Rep” gene refers to the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are collectively required for replicating the viral genome, or functional homologues thereof such as the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication. Thus, the rep coding region can include the genes encoding for AAV Rep78 and Rep68 (the “long forms of Rep”), and Rep52 and Rep40 (the “short forms of Rep”), or functional homologues thereof. The rep coding region, as used herein, can be derived from any viral serotype, such as the AAV serotypes described herein. The region need not include all wild-type genes but may be altered, (e.g., by insertion, deletion or substitution of nucleotides), so long as the rep genes present provide for sufficient integration functions when expressed in a suitable target cell. See, e.g. Muzyczka, N., Current Topics in Microbiol. and Immunol. 158:97-129 (1992); and Kotin, R. M., Human Gene Therapy 5:793-801 (1994).
As referred to herein, the term “Cap” gene refers to the art-recognized region of the AAV genome which encodes the capsid proteins of the virus. Illustrative (non-limiting) examples of these capsid proteins are the AAV capsid proteins VP1, VP2, and VP3. Cap genes used in this disclosure can come from any AAV serotype or a combination of AAV serotypes.
FIG. 4A shows the natural location and promoter drivers of Rep and Cap genes. As known in the art, for successful production of AAV, the ratio of Rep78 and Rep52 genes must be maintained at optimum levels. For example, as discussed in Li et al., “Role for Highly Regulated rep Gene Expression in Adeno-Associated Virus Vector Production,” Journal of Virology 71:5236-5243 (1997) (the disclosure of which is incorporated by reference herein in its entirety), control of the amount of Rep78 production can interference with DNA replication. In addition, Rep78 can be toxic if overly produced in mammalian cells. See, e.g., Clark et al., “Cell Lines for the Production of Recombinant Adeno-Associated Virus,” Human Gene Therapy 6:1329-1341 (1995), the disclosure of which is incorporated by reference herein in its entirety, discussing that elevate rep protein levels can be associated with cytotoxicity. Also, the location of the promoter for Rep52 expression (p19) is located within the coding region for Rep78. As described herein, various modifications have been made to the natural locations of the Rep genes and promoters to overcome these challenges.
In exemplary embodiments, the mammalian cell can include a nucleic acid encoding a Rep78 gene under control of the second derepressible promoter and a Rep52 gene under control of a third derepressible promoter. As shown in FIG. 4B, one way to achieve this arrangement is to remove the Rep52 gene from within the Rep78 gene, and place it downstream of the Rep78 and Cap genes. The Rep78 gene can be under the control of a derepressible promoter (p5), that includes the TetO2 sequences. In such embodiments, the natural p19 promoter within Rep78 is modified or mutated to be silenced. The removed Rep52 gene is also placed under the control of a derepressible promoter (p19) that includes TetO2 sequences.
FIG. 5 shows three potential locations for each of the TetO sequences, relative to the TATA box, rep-binding element (RBE) and initiator element (INR), for modification to the p5 promotor of Rep78. The wild-type p5 promotor is also illustrated schematically. FIG. 6 shows three potential locations for each of the TetO sequences, relative to the TATA boxes and the Spl transcription factor, of the p19 promoter for Rep52. The wild-type p19 promotor is also illustrated schematically. Additional locations of the TetO sequences are also encompassed herein and can readily be envisioned by those of ordinary skill in the art.
In still further embodiments, the Rep78 gene can be under control of a derepressible promoter, and the Rep52 gene can be under control of a derepressible promoter that is contained within an artificial intron. Such an embodiment is represented schematically in FIG. 4C. As illustrated, a derepressible p5 promoter (e.g., including TetO2 sequences as described herein) is placed upstream of a Rep78 gene. A derepressible p19 promoter (e.g., including TetO2 sequences) contained within an intron (designated In-i-p19), controls the expression of Rep52. A schematic of this embodiment is provided in FIG. 7A, where the two tet operator sequences are illustrated within a chimeric intron. This chimeric intron can be inserted at various locations relative to the components of the p19 promoter. For example, as shown in FIG. 7B, the intron sequence is suitably placed downstream of the TATA-2 of the p19 promoter. Spacing can be, for example, between about 1 and 25 base pairs downstream from the TATA-2 sequence.
As described herein, in exemplary embodiments, the Cap gene that is encoded by the nucleic acid molecules is suitably under the control of a native promoter. That is, the Cap gene does not necessarily have to be under the control of a derepressible promoter, though a derepressible promoter can be used if desired. In suitable embodiments, the Cap gene is under the control of a p40 promoter.
In exemplary embodiments, the nucleic acid molecules include two inverted terminal repeat (ITR) sequences. As known in the art, these ITR sequences (i.e., AAV2 ITR) are single stranded sequence of nucleotides, followed downstream by its reverse compliment. ITR sequences represent the minimal sequence required for replication, rescue, packaging and integration of the AAV genome. Suitably, these ITR sequences flank a gene of interest. Thus, in embodiments, the nucleic acid molecules further encode a gene of interest. This gene of interest can be, for example, a reporter gene, a selection gene, or a gene of therapeutic interest, for example.
For example, as illustrated in FIG. 10C, a gene of interest, such as the gene encoding green fluorescent protein (EGFP) is flanked by two ITR sequences.
A “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. “Gene” also refers to a nucleic acid fragment that can act as a regulatory sequence preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. In some embodiments, genes are integrated with multiple copies. In some embodiments, genes are integrated at predefined copy numbers.
As referred to herein, the term “gene of interest” or “GOT” is used to describe a heterologous gene. As referred to herein, the term “heterologous gene” or “HG” as it relates to nucleic acid sequences such as a coding sequence or a control sequence, denotes a nucleic acid sequence, e.g. a gene, that is not normally joined together, and/or are not normally associated with a particular cell. In some embodiments, a heterologous gene is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
As referred to herein, a “reporter gene” is a gene whose expression confers a phenotype upon a cell that can be easily identified and measured. In some embodiments, the reporter gene comprises a fluorescent protein gene. In some embodiments, the reporter gene comprises a selection gene.
As referred to herein, the term “selection gene” refers to the use of a gene which encodes an enzymatic activity that confers the ability to grow in medium lacking what would otherwise be an essential nutrient; in addition, a selection gene may confer resistance to an antibiotic or drug upon the cell in which the selection gene is expressed. A selection gene may be used to confer a particular phenotype upon a host cell. When a host cell must express a selection gene to grow in selective medium, the gene is said to be a positive selection gene. A selection gene can also be used to select against host cells containing a particular gene; a selection gene used in this manner is referred to as a negative selection gene.
As referred to herein, the term “gene of therapeutic interest” refers to any functionally relevant nucleotide sequence. Thus, the gene of therapeutic interest of the present disclosure can comprise any desired gene that encodes a protein that is defective or missing from a target cell genome or that encodes a non-native protein having a desired biological or therapeutic effect (e.g., an antiviral function), or the sequence can correspond to a molecule having an antisense or ribozyme function. Representative (non-limiting) examples of suitable genes of therapeutic interest include those used for the treatment of inflammatory diseases, autoimmune, chronic and infectious diseases, including such disorders as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholestemia; various blood disorders including various anemias, thalassemias and hemophilia; genetic defects such as cystic fibrosis, Gaucher's Disease, adenosine deaminase (ADA) deficiency, emphysema, etc. Several antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences around the translational initiation site (AUG codon) of an mRNA) that are useful in antisense therapy for cancer and for viral diseases have been described in the art and are also examples of suitable genes of therapeutic interest.
In some embodiments, the mammalian cells provided herein are substantially free of helper virus. As referred to herein, a “helper virus” is any non-AAV virus that is added to enable the replication and packaging of adeno-associated virus. Representative (non-limiting) examples of helper viruses are adenovirus and herpes virus. In some embodiments, the term substantially free of helper virus refers to a cell that has fewer than 100, fewer than 10, or fewer than 1 helper virus per cell. In some embodiments, the term substantially free of helper virus refers to a cell in which no helper viruses are present or to a population of cells in which no helper viruses are present using detection methods known to those skilled in the art. In some embodiments, no wild-type helper virus is in the cell. In some embodiments, the term wild-type virus refers to any complete-non-AAV virus that can replicate in the cell independently of any other virus.
The AAV producer cells described herein provide a long-term and cost-effective solution for large scale AAV manufacturing. As constitutive expression of either helper or Rep proteins can be cytotoxic, the strategies described herein allow for control of their expression by engineered, derepressible promoters.
In still further embodiments, provided herein is a mammalian cell for producing an adeno-associated virus (AAV), comprising, in a single nucleic acid molecule, sequences encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters. In such embodiments, this single nucleic acid molecule includes all of the various sequences, along with other required elements, to enable the production of an AAV within the cell.
FIGS. 10A and 10B show exemplary nucleic acid molecules including these various sequences that can be utilized in mammalian cells to produce AAVs.
As described herein, suitably the mammalian cells are mammalian cell cultures, and in embodiments, can be suspension cultures. As described herein, the use of suspension cell cultures allows for increased scalability and production of AAV.
As described herein and as shown in FIGS. 1, 2A-2B, and 10A-10B, suitably the single nucleic acid molecule includes an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Various constructs are described herein for encoding the Rep and Cap genes, including Rep78 and Rep52 genes. In embodiments, a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter (e.g., as shown in FIG. 4B, the Rep52 gene is separate from the Rep78 gene). In additional embodiments, for example as described herein with reference to FIGS. 4C and 7A-7B, a Rep78 gene can be placed under control of the second derepressible promoter and a Rep52 gene can be placed under control of a fourth derepressible promoter contained within an artificial intron. Suitably, the Cap gene is under control of a native promoter.
Various derepressible promoters are described herein, and in embodiments, the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2). In embodiments, the functional promoter of the first derepressible promoter (i.e., controlling the expression of the adenovirus helper gene comprising E2A and E4Orf6 genes) is a cytomegalovirus (CMV) promoter.
As described herein, in embodiments and as illustrated in FIGS. 1, 2A-2B, and 10A-10D, suitably the repressor element of the derepressible promoter is under control of a constitutive promoter, such that it is produced at all times to limit expression of other genes under the control of the derepressible promoters. Suitably, the repressor element that is encoded is a tetracycline repressor protein, for binding to TetO2 sequences to act as the derepressible promoter. In embodiments, for example as shown in FIGS. 2A, 10A and 10C, a nucleic acid encoding a transcriptional repression domain (e.g., a KRAB sequence) is included in frame with the nucleic acid encoding the tetracycline repressor protein. This transcriptional repression domain provides for improved repressive activity of the TetR when binding to TetO2, thereby minimizing the amount of leakage or basal gene expression prior to derepression.
Exemplary mammalian cells that can be used in the embodiments and methods described herein are described throughout, and include for example, Chinese hamster ovary (CHO) cells, as well as human cells, including human embryonic kidney (HEK, such as HEK293) cells.
As described herein, suitably the mammalian cells further include a nucleic acid molecule encoding a gene of interest (GOI). As shown in FIGS. 10C-10D, suitably the GOI is included between two ITR sequences.
Also provided herein is an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
As used herein an “isolated nucleic acid molecule” includes vectors and plasmids that can contain the isolated nucleic acid molecule, as well as similar structures where the isolated nucleic acid molecule can be manipulated, stored, shipped, and ultimately utilized in various cell transfection systems. The isolated nucleic acid molecules described herein can be used for production of AAVs as described herein, but can also be utilized in various non-AAV producing cell lines (including transient transfection systems). The isolated nucleic acid molecules described herein suitably further include various additional elements and sequences as required to allow for use in the cellular systems, including mammalian cells, described herein.
For example, as shown in FIGS. 10A-10B, two plasmid constructs are shown. As indicated, in embodiments, an internal ribosome entry site (IRES) element can be included between the E2A and E4Orf6 genes. As described herein with reference to FIG. 4B in embodiments, a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter (i.e., separated from the Rep52 gene).
In further embodiments, as shown with reference to FIGS. 10A-10B, a Rep78 gene is under control of a derepressible promoter (ip5 promoter as shown, and various iterations described in FIG. 5) and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron, illustrated as ip19, and described with reference to FIGS. 7A-7B. The isolated nucleic acid further suitably includes the Cap gene under control of a native promoter (i.e., p40).
As described herein, various derepressible promoters can be included in the isolated nucleic acid molecules, and suitably include a functional promoter and two tetracycline operator sequences (TetO2). As shown in FIGS. 10A and 10B, with regard to the helper genes E2 and E4, suitably this functional promoter is a CMV promoter, which includes the TetO2 sequences. The derepressible promoter for use with the viral-associated, non-coding RNA suitably includes an H1 promoter, as well as the TetO2 sequences.
As described herein and as shown in FIGS. 10A and 10B, the repressor element (e.g., a tetracycline repressor protein), is suitably under the control of a constitutive promoter, for example, a hPGK promoter. As shown in FIGS. 10A and 12B, the isolated nucleic acid molecules can further include a nucleic acid encoding a transcriptional repression domain (e.g., a KRAB sequence) in frame with the nucleic acid encoding the tetracycline repressor protein. As shown in FIGS. 10C-10D and FIG. 11B, the isolated nucleic acid molecules can further include a gene of interest (GOI, e.g., a GFP), suitably between the two ITR sequences.
As described herein and as shown in FIGS. 12A-12B, the repressor element is suitably flanked by an insulator, for example, a chicken hypersensitive site-4 (cHS4) sequence.
In embodiments, a nucleic acid encoding a transcriptional repression domain (such as KRAB) is included in frame with the nucleic acid encoding the tetracycline repressor protein (e.g., TetR-KRAB).
Additional genetic and sequence elements for inclusion in the isolated nucleic acid molecules described herein are known in the art and can be found illustrated schematically in FIGS. 10A-10D and FIGS. 11A-11E.
The terms “sequence identity” or “% identity” in the context of nucleic acid sequences described herein refers to the percentage of residues in the compared sequences that are the same when the sequences are aligned over a specified comparison window. A comparison window can be a segment of at least 10 to over 1000 residues in which the sequences can be aligned and compared. Methods of alignment for determination of sequence identity are well-known can be performed using publicly available databases such as BLAST (blast.ncbi.nlm.nih.gov/Blast. CGI.).
In some embodiments, nucleic acid molecules have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with a reference nucleic acid molecule, respectively (or a fragment of the reference polypeptide or nucleic acid molecule). In certain embodiments of the disclosure, polypeptides or nucleic acid molecules have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% or 100% sequence identity with a reference nucleic acid molecule, respectively (or a fragment of the reference nucleic acid molecule). In some embodiments, nucleic acid molecules have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity with a reference nucleic acid molecule, respectively.
A “vector” or “expression vector” is a replicon, such as a plasmid, phage, virus, or cosmid, to which a nucleic acid molecule described herein may be attached to bring about the replication and/or expression of the attached nucleic acid molecule in a cell. “Vector” includes episomal (e.g., plasmids) and non-episomal vectors. The term “vector” includes both viral and nonviral means for introducing a nucleic acid molecule into a cell in vitro, in vivo, or ex vivo. The term vector may include synthetic vectors. Vectors may be introduced into the desired host cells by well-known methods, including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors can comprise various regulatory elements including promoters.
Also provided herein is a method of producing an adeno-associated virus (AAV) in a mammalian cell. Suitably, the methods described herein include transfecting the mammalian cell with an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
“Transfection” as used herein means the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. A “transfected” cell comprises an exogenous nucleic acid molecule inside the cell and a “transformed” cell is one in which the exogenous nucleic acid molecule within the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can be integrated into the host cell's genomic DNA and/or can be maintained by the cell, temporarily or for a prolonged period of time, extra-chromosomally. Host cells or organisms that express exogenous nucleic acid molecules or fragments are referred to as “recombinant,” “transformed,” or “transgenic” organisms. A number of transfection techniques are generally known in the art. See, e.g., Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene 13:197 (1981). Such techniques can be used to introduce one or more exogenous DNA moieties, such as an AAV vector cassette, AAV helper constructs, and other nucleic acid molecules, into suitable host cells.
Various methods of transfecting the mammalian cells with the isolated nucleic acid molecules described herein (i.e., vectors), are known in the art and include various chemical and physical methods, for example, electroporation, cell injection, calcium phosphate exposure, liposome or polymer-based carrier systems, etc.
In exemplary embodiments, a vectors such as the PIGGYBACTM transposon can be used for the stable integration of these nucleic acid molecules, which allows one-step insertion of large nucleic acid sequences in multiple copies randomly in a cell genome. The system consists of a PIGGYBACTM Vector and the Super PIGGYBACTM Transposase which recognizes transposon-specific inverted terminal repeats (ITRs) and efficiently integrates the ITRs and intervening DNA into the genome at TTAA sites. The Super PIGGYBACTM Transposase is delivered to the cell via the Super PIGGYBACTM Transposase Expression Vector, which is co-transfected with one or more PIGGYBACTM Vectors.
The methods further include treating the mammalian cell with a binding partner of the repressor element. As described herein, in the presence of a repressor element, the functional promoters of the derepressible promoters controlling the transcription of the various genes encoded by the nucleic acid molecules, are repressed. That is, the genes are not actively being transcribed, and are instead awaiting derepression. As noted herein, the repressor element of the derepressible promoters is suitably under control of constitutive promoter, such that the repressor element is being produced soon after transfection of the nucleic acid molecule into the mammalian cell. Upon treatment with the binding partner of the repressor element, the repressor element binds to the binding partner, changes conformation, and no longer represses the derepressible promoter. This results in the activating of the first, second and third (and additional as needed) derepressible promoters (i.e., the functional promoters of the derepressible promoters) within the mammalian cell.
Following the activation, the various elements are transcribed and translated within the mammalian cell, resulting in the production of the AAV. The AAV is then harvested using methods known in the art.
While the methods described herein can be utilized in any mammalian cell, including a mammalian cell culture, suitably the mammalian cell culture is a suspension culture, including a human cell such as an HEK suspension cell culture.
As described throughout the nucleic acid molecules can further include an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes. Exemplary constructs related to the Rep78 and Rep 52 genes are described herein, including where a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter. In additional embodiments of the methods, a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron.
As described herein, the use of an artificial intron allows for removal of the fourth derepressible promoter following activating the derepressible promoters and prior to the producing the AAV. As described herein, the derepressible promoter within the intron ensures the repression of Rep52 gene expression before activation, while still allowing for the expression of the Rep78 protein following the removal of the intron during mRNA splicing. In exemplary embodiments of the methods, the Cap gene is under control of a native promoter, such as p40.
In embodiments, the functional promoter of the derepressible promoter controlling the expression of the helper genes is a cytomegalovirus (CMV) promoter. Suitably, the repressor element that is encoded in the methods described herein is a tetracycline repressor protein, and suitably the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2). In embodiments, the repressor element is under control of a constitutive promoter, such as hPGK, for example when the repressor element that is encoded is a tetracycline repressor protein.
As described herein, in embodiments utilizing the TetR and/or TetR-KRAB repressor element, treating the cells with doxycycline changes the conformation of the TetR and activates the transcription of the various genes.
Various mammalian cells can be utilized in the methods described herein, including human cells such as human embryonic kidney (HEK) cells, or other mammalian cells including Chinese hamster ovary (CHO) cells.
As described throughout, in embodiments, the AAV comprises a nucleic acid molecule encoding a gene of interest. This GOI can be a reporter gene, a selection gene, or any other gene of interest, including a gene of therapeutic interest.
The methods of producing the AAVs can be used in a continuous manufacturing system. In exemplary embodiments, the use of a suspension cell culture allows for the production of large volumes of AAV, with high productivity and prolonged culture conditions to allow for multiple harvests of AAV for each batch of starting cells.
Production methods can utilize any suitable reactor(s) including but not limited to stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. As used herein, “reactor” can include a fermenter or fermentation unit, or any other reaction vessel and the term “reactor” is used interchangeably with “fermenter.” The term fermenter or fermentation refers to both microbial and mammalian cultures. For example, in some aspects, an example bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing. Example reactor units, such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors in each unit and/or a facility may contain multiple units having a single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi fed-batch, fed-batch, perfusion, and/or a continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume between about 100 mL and about 50,000 L. Non-limiting examples include a volume of 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Additionally, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material including metal alloys such as stainless steel (e.g., 316 L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
In embodiments and unless stated otherwise herein, the devices, facilities, and methods described herein can also include any suitable unit operation and/or equipment not otherwise mentioned, such as operations and/or equipment for separation, purification, and isolation of such products. Any suitable facility and environment can be used, such as traditional stick-built facilities, modular, mobile and temporary facilities, or any other suitable construction, facility, and/or layout. For example, in some embodiments modular clean-rooms can be used. Additionally and unless otherwise stated, the devices, systems, and methods described herein can be housed and/or performed in a single location or facility or alternatively be housed and/or performed at separate or multiple locations and/or facilities.
In further embodiments, provided herein is a method of treatment with an adeno-associated virus (AAV) comprising: transfecting the mammalian cell with an isolated nucleic acid molecule encoding: an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters, treating the mammalian cell with a binding partner of the repressor element, activating the first, second and third derepressible promoters, producing the AAV, harvesting the AAV, and administering the AAV to a mammalian patient.
Suitably, the methods are used to treat a human patient with a gene of interest, including a gene of therapeutic interest. Administration to a human patient can include, for example, inhalation, injection, or intravenous administration, as well as other administration methods known in the art.
The methods of producing the AAV and the use of various derepressible promoters are described herein.
In further embodiments, a method of producing an adeno-associated virus (AAV) is provided and includes transfecting a mammalian cell stably expressing one or more nucleic acids encoding TetR and/or TetR-KRAB with: a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA, under control of a first derepressible promoter, a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter; treating the mammalian cell with a binding partner of the TetR; activating the first, second and third derepressible promoters; producing the AAV; and harvesting the AAV.
As described herein, suitably the mammalian cell is a mammalian cell culture, including a suspension culture
As described herein, suitably the mammalian cell is a Chinese hamster ovary (CHO) cell or a human cell including a human embryonic kidney (HEK) cell.
As described herein, using 2 or 3 different nucleic acids or transposons to transfect mammalian cells offers advantages over combining the nucleic acids in one plasmid, including the ability to optimize the ratio of separate AAV-producing and packaging components and to exert temporal control over expression of each transposon.
As described herein, suitably the nucleic acid encoding the E2A and E4Orf genes further comprises an internal ribosome entry site (IRES) element between the E2A and E4Orf genes.
As described herein, suitably the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
As described herein, suitably the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
In some embodiments, the mammalian cell used to produce adeno-associated virus (AAV) is stably expressing one or more nucleic acids encoding TetR and/or TetR-KRAB under control of a constitutive promoter.
As described herein, stable expression of a TetR and/or TetR-KRAB in a mammalian cell (prior to insertion of nucleic acids carrying the components of the virus, suitably via transposons) maximizes the repression of potentially cytotoxic AAV genes introduced by transfection and confers increased temporal control over the derepressible promoter elements.
In some embodiments, a KRAB repressor domain is fused in frame with the TetR.
As described herein, fusing a KRAB repressor domain in frame with the TetR ensures that “leaky” expression of the potentially cytotoxic AAV-packaging nucleic acids is less likely to occur. A KRAB repressor domain fused in frame with the TetR repressor domain provides an additional mechanism to accomplish high levels of repression of derepressible promoters.
In some embodiments, the mammalian cell produces a sufficient amounts of TetR. As used herein, a “sufficient” of TetR is defined as the level of amount and/or activity of a repressor (e.g., TetR) to stop expression and/or activity of a repressible and/or derepressible element (i.e., prior to addition of doxycycline).
As described herein, producing sufficient amounts of TetR is necessary for repression of the derepressible promoters under normal conditions (e.g., before addition of doxycycline). If sufficient amounts of TetR are not produced by the mammalian cell, the cell may prematurely transcribe and translate potentially cytotoxic AAV-packaging nucleic acids.
In further embodiments, the mammalian cell further comprises stable expression of one or more nucleic acids encoding chicken hypersensitive site-4 (cHS4) flanking the TetR and/or TetR-KRAB repressor sequences. As described herein, expression of cHS4 sequences flanking the TetR and/or TetR-KRAB repressor sequences prevent the silencing of TetR expression and further improve the stability of integrated repressors in the mammalian cell genome.
In further embodiments, the treating comprises treating with doxycycline to remove repression of derepressible promoters.
In further embodiments, each of the first, second and third nucleic acids are flanked by transposon-specific inverted terminal repeats (ITRs).
In further embodiments, a method for producing an adeno-associated virus (AAV) comprises stably transfecting a mammalian cell with: a nucleic acid encoding a TetR and/or TetR-KRAB repressor, chicken hypersensitive site-4 (cHS4) sequences flanking the TetR and/or TetR-KRAB repressor, and a selection gene; transfecting the stably transfected mammalian cell with a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA, under control of a first derepressible promoter, a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, and optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter; treating the mammalian cell with a binding partner of the TetR; activating the first, second and third derepressible promoters; producing the AAV; and harvesting the AAV.
In some embodiments, the mammalian cell further comprises a selection gene, such asa zeocin resistance gene. Additional selection genes include other antibiotic resistance genes, such as kanamycin and geneticin resistance genes. As described herein, expression of a zeocin resistance gene allows for efficient selection of correctly integrated repressors in the mammalian cell genome.
Additional Exemplary Embodiments
Embodiment 1 is a mammalian cell for producing an adeno-associated virus (AAV), comprising a nucleic acid molecule encoding a viral helper gene under control of a first derepressible promoter, a nucleic acid molecule encoding an AAV gene under control of a second derepressible promoter, and a nucleic acid molecule encoding a repressor element of the first and the second derepressible promoters.
Embodiment 2 includes the mammalian cell of embodiment 1, wherein the mammalian cell is a mammalian cell culture.
Embodiment 3 includes the mammalian cell of embodiment 2, wherein the mammalian cell culture is a suspension culture.
Embodiment 4 includes the mammalian cell of any one of embodiments 1-3, wherein the viral helper gene is an adenovirus helper gene.
Embodiment 5 includes the mammalian cell of embodiment 4, wherein the adenovirus helper gene comprises E2A and E4Orf6 genes.
Embodiment 6 includes the mammalian cell of embodiment 5, further comprising an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 7 includes the mammalian cell of any one of embodiments 1-6, wherein the AAV gene comprises Rep and Cap genes.
Embodiment 8 includes the mammalian cell of embodiment 7, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a third derepressible promoter.
Embodiment 9 includes the mammalian cell of embodiment 7, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a third derepressible promoter contained within an artificial intron.
Embodiment 10 includes the mammalian cell of any one of embodiments 8 or 9, wherein the Cap gene is under control of a native promoter.
Embodiment 11 includes the mammalian cell of any one of embodiments 1-10, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 12 includes the mammalian cell of embodiment 11, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 13 includes the mammalian cell of any one of embodiments 1-12, wherein the repressor element is under control of a constitutive promoter.
Embodiment 14 includes the mammalian cell of any one of embodiments 1-13, wherein the repressor element is a tetracycline repressor protein.
Embodiment 15 includes the mammalian cell of embodiment 14, further comprising a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.
Embodiment 16 includes the mammalian cell of any one of embodiments 1-15, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
Embodiment 17 includes the mammalian cell of any one of embodiments 1-15, wherein the mammalian cell is a human cell.
Embodiment 18 includes the mammalian cell of embodiment 17, wherein the human cell is a human embryonic kidney (HEK) cell.
Embodiment 19 includes the mammalian cell of any one of embodiments 1-18, further comprising a nucleic acid molecule including two inverted terminal repeat (ITR) sequences.
Embodiment 20 includes the mammalian cell of any one of embodiments 1-19, further comprising a nucleic acid molecule encoding a gene of interest.
Embodiment 21 includes the mammalian cell of any one of embodiments 1-20, further comprising a nucleic acid encoding a viral-associated, non-coding RNA under control of a fourth derepressible promoter.
Embodiment 22 is a mammalian cell for producing an adeno-associated virus (AAV), comprising a nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
Embodiment 23 includes mammalian cell of embodiment 22, wherein the mammalian cell is a mammalian cell culture.
Embodiment 24 includes the mammalian cell of embodiment 23, wherein the mammalian cell culture is a suspension culture.
Embodiment 25 includes the mammalian cell of any one of embodiments 22-24, further comprising an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 26 includes the mammalian cell of any one of embodiments 22-25, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter.
Embodiment 27 includes the mammalian cell of any one of embodiments 22-25, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron.
Embodiment 28 includes the mammalian cell of any one of embodiments 26 or 27, wherein the Cap gene is under control of a native promoter.
Embodiment 29 includes the mammalian cell of any one of embodiments 22-28, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 30 includes the mammalian cell of embodiment 29, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 31 includes the mammalian cell of any one of embodiments 22-30, wherein the repressor element is under control of a constitutive promoter.
Embodiment 32 includes the mammalian cell of any one of embodiments 22-31, wherein the repressor element is a tetracycline repressor protein.
Embodiment 33 includes the mammalian cell of embodiment 25, further comprising a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.
Embodiment 34 includes the mammalian cell of any one of embodiments 22-33, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
Embodiment 35 includes the mammalian cell of any one of embodiments 22-33, wherein the mammalian cell is a human cell.
Embodiment 36 includes the mammalian cell embodiment 35, wherein the human cell is a human embryonic kidney (HEK) cell.
Embodiment 37 includes the mammalian cell of any one of embodiments 22-36, further comprising a nucleic acid molecule encoding a gene of interest.
Embodiment 38 is an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters.
Embodiment 39 includes the isolated nucleic acid of embodiment 38, further comprising an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 40 includes the isolated nucleic acid of any of embodiments 38-39, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter.
Embodiment 41 includes the isolated nucleic acid of any of embodiments 38-39 wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron.
Embodiment 42 includes the isolated nucleic acid of any of embodiments 40 or 41, wherein the Cap gene is under control of a native promoter.
Embodiment 43 includes the isolated nucleic acid of any of embodiments 38-42, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 44 includes the isolated nucleic acid of embodiment 43, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 45 includes the isolated nucleic acid of any of embodiments 38-44, wherein the repressor element is under control of a constitutive promoter.
Embodiment 46 includes the isolated nucleic acid of any of embodiments 38-45, wherein the repressor element is a tetracycline repressor protein.
Embodiment 47 includes the isolated nucleic acid of embodiment 46, further comprising a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.
Embodiment 48 includes the isolated nucleic acid of any of embodiments 38-47, further comprising a gene of interest.
Embodiment 49 is a method of producing an adeno-associated virus (AAV) in a mammalian cell comprising transfecting the mammalian cell with an isolated nucleic acid molecule encoding an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences, and a repressor element of the first, second and third derepressible promoters, treating the mammalian cell with a binding partner of the repressor element, activating the first, second and third derepressible promoters, producing the AAV, and harvesting the AAV.
Embodiment 50 includes the method of embodiment 49, wherein the mammalian cell is a mammalian cell culture.
Embodiment 51 includes the method of embodiment 50, wherein the mammalian cell culture is a suspension culture.
Embodiment 52 includes the method of any of embodiments 49-51, further comprising an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 53 includes the method of any of embodiments 49-52, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter.
Embodiment 54 includes the method of any of embodiments 49-52, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron.
Embodiment 55 includes the method of embodiment 54, wherein the fourth derepressible promoter is removed following the activating the derepressible promoters and prior to the producing the AAV.
Embodiment 56 includes the method of any of embodiments 53 or 54, wherein the Cap gene is under control of a native promoter.
Embodiment 57 includes the method of any of embodiments 49-56, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 58 includes the method of embodiment 57, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 59 includes the method of any of embodiments 49-58, wherein the repressor element is under control of a constitutive promoter.
Embodiment 60 includes the method of any of embodiments 49-59, wherein the repressor element that is a tetracycline repressor protein.
Embodiment 61 includes the method of embodiment 60, wherein the nucleic acid further comprises a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.
Embodiment 62 includes the method of any of embodiments 60 or 61, wherein the treating comprises treating with doxycycline.
Embodiment 63 includes the method of any of embodiments 49-62, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
Embodiment 64 includes the method of any of embodiments 49-63, wherein the mammalian cell is a human cell.
Embodiment 65 includes the method of embodiment 64, wherein the human cell is a human embryonic kidney (HEK) cell.
Embodiment 66 includes the method of any of embodiments 49-65, wherein the AAV comprises a nucleic acid molecule encoding a gene of interest.
Embodiment 67 includes the method of embodiment 66, wherein the AAV comprises a gene of therapeutic interest.
Embodiment 68 is a method of treatment with an adeno-associated virus (AAV) comprising transfecting the mammalian cell with an isolated nucleic acid molecule encoding, an adenovirus helper gene comprising E2A and E4Orf6 genes under control of a first derepressible promoter, an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, a viral-associated, non-coding RNA under control of a third depressible promoter, two inverted terminal repeat (ITR) sequences; and a repressor element of the first, second and third derepressible promoters; treating the mammalian cell with a binding partner of the repressor element; activating the first, second and third derepressible promoters; producing the AAV; harvesting the AAV; and administering the AAV to a mammalian patient.
Embodiment 69 includes the method of embodiment 68, wherein the mammalian cell is a mammalian cell culture.
Embodiment 70 includes the method of embodiment 69, wherein the mammalian cell culture is a suspension culture.
Embodiment 71 includes the method of any of embodiments 68-70, further comprising an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 72 includes the method of any of embodiments 68-71, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter.
Embodiment 73 includes the method of any of embodiments 68-72, wherein a Rep78 gene is under control of the second derepressible promoter and a Rep52 gene is under control of a fourth derepressible promoter contained within an artificial intron.
Embodiment 74 includes the method of embodiment 73, wherein the fourth derepressible promoter is removed following the activating the derepressible promoters and prior to the producing the AAV.
Embodiment 75 includes the method of any of embodiments 73 or 74, wherein the Cap gene is under control of a native promoter.
Embodiment 76 includes the method of any of embodiments 68-75, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 77 includes the method of embodiment 76, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 78 includes the method of any of embodiments 68-77, wherein the repressor element is under control of a constitutive promoter.
Embodiment 79 includes the method of any of embodiments 68-78, wherein the repressor element that is a tetracycline repressor protein.
Embodiment 80 includes the method of embodiment 79, wherein the nucleic acid further comprises a nucleic acid encoding a transcriptional repression domain in frame with the nucleic acid encoding the tetracycline repressor protein.
Embodiment 81 includes the method of any of embodiments 79 or 80, wherein the treating comprises treating with doxycycline.
Embodiment 82 includes the method of any of embodiments 68-81, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
Embodiment 83 includes the method of any of embodiments 68-82, wherein the mammalian cell is a human cell.
Embodiment 84 includes the method of embodiment 83, wherein the human cell is a human embryonic kidney (HEK) cell.
Embodiment 85 includes the method of any of embodiments 68-84, wherein the AAV comprises a nucleic acid molecule encoding a gene of interest.
Embodiment 86 includes the method of embodiment 87, wherein the AAV comprises a gene of therapeutic interest.
Embodiment 87 includes the method of any of embodiments 68-86, wherein the administering comprises inhalation, injection or intravenous administration.
Embodiment 88 is a method of producing an adeno-associated (AAV) virus, comprising: transfecting a mammalian cell stably expressing one or more nucleic acids encoding TetR and/or TetR with a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA under control of a first derepressible promoter, a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter, and, optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter; treating the mammalian cell with a binding partner of the TetR and/or TetR-KRAB; activating the first, second and third derepressible promoters; producing the AAV; and harvesting the AAV.
Embodiment 89 includes the method of embodiment 88, wherein the mammalian cell is a mammalian cell culture.
Embodiment 90 includes the method of any of embodiments 88-89, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
Embodiment 91 includes the method of any of embodiments 88-90, wherein the mammalian cell is a human cell.
Embodiment 92 includes the method of embodiment 91, wherein the human cell is a human embryonic kidney (HEK) cell.
Embodiment 93 includes the method of embodiment 89, wherein the mammalian cell culture is a suspension culture.
Embodiment 94 includes the method of any of embodiments 88-90, wherein the mammalian cell further comprises an internal ribosome entry site (IRES) element between the E2A and E4Orf6 genes.
Embodiment 95 includes the method of any of embodiments 88-91, wherein each of the derepressible promoters comprise a functional promoter and two tetracycline operator sequences (TetO2).
Embodiment 96 includes the method of embodiment 92, wherein the functional promoter of the first derepressible promoter is a cytomegalovirus (CMV) promoter.
Embodiment 97 includes the method of any of embodiments 88-96, wherein the stably expressed TetR and/or TetR-KRAB is under control of a constitutive promoter.
Embodiment 98 includes the method of embodiment 97, wherein the nucleic acid encoding the TetR-KRAB comprises KRAB fused in frame with the TetR.
Embodiment 99 includes the method of any one of embodiments 88-98, wherein the treating comprises treating with doxycycline.
Embodiment 100 includes the method of any one of embodiments 88-99, wherein each of the first, second and third nucleic acids are flanked by transposon-specific inverted terminal repeats (ITRs).
Embodiment 101 includes the method of any of embodiments 88-100, wherein the mammalian cell expresses sufficient amounts of TetR and/or TetR-KRAB.
Embodiment 102 includes the method of any of embodiments 88-101, wherein the AAV comprises a gene of therapeutic interest.
Embodiment 103 includes the method of any of embodiments 88-102, wherein the mammalian cell further comprises stable expression of one or more nucleic acids encoding chicken hypersensitive site-4 (cHS4) flanking the TetR and/or TetR-KRAB repressor sequences.
Embodiment 104 includes the method of any of embodiments 88-103, wherein the mammalian cell further comprises a zeocin resistance gene.
Embodiment 105 is a method for producing an adeno-associated virus (AAV), comprising: stably transfecting a mammalian cell with: a nucleic acid encoding a TetR and/or TetR-KRAB repressor; chicken hypersensitive site-4 (cHS4) sequences flanking the TetR and/or TetR-KRAB repressor; and a selection gene; transfecting the stably transfected mammalian cell with: a first nucleic acid encoding an adenovirus helper gene comprising an E2A gene, a E4Orf gene and a viral-associated non-coding RNA, under control of a first derepressible promoter; a second nucleic acid encoding an AAV gene comprising Rep and Cap genes under control of a second derepressible promoter; and optionally, a third nucleic acid encoding a gene of interest under control of a third derepressible promoter; treating the stably transfected mammalian cell with a binding partner of the TetR; activating the first, second and third derepressible promoters; producing the AAV; and harvesting the AAV.
Embodiment 106 includes the method of embodiment 105, wherein the stably transfected mammalian cell produces a sufficient amounts of TetR.
Embodiment 107 includes the method of any of embodiments 106 and 107, wherein the KRAB repressor domain is fused in frame with the TetR.
EXAMPLES
Example 1: Design and Validation of Derepressible Helper Genes
To drive the expression of E2A and E4Orf6 genes, a derepressible promoter from the pcDNA4/TO vector (INVITROGEN) was utilized. This promoter includes the complete CMV promoter with an insertion of two Tetracycline operator sequences (TetO2) between the TATA box and transcriptional start site (TSS). In the presence of tetracycline repressor protein (TetR), transcription initiation was blocked by the binding of TetR onto the TetO2 sites. When Tetracycline or Doxycycline is added into the medium, it binds to TetR and changes its conformation. This leads to the release of TetR and derepression/activation of the CMV promoter and results in induced gene expression (FIG. 1 showing OFF and ON conformations of TetR/TetO2 derepressible promoter system).
To simplify the design and improve the stability, an Internal Ribosome Entry Site (IRES) element was used to initiate the translation of E4Orf6 after E2A in a single expression cassette, driven by a single inducible CMV promoter (FIG. 1).
To induce the VA I non-coding RNA, an H1 promoter with TetO2 insertion was applied (see, e.g., Wiederschain et al., “Single-vector inducible lentiviral RNAi system for oncology target validation, Cell Cycle 8:498-504 (2009)). Similarly, the addition of Doxycycline releases TetR and turns on VA I expression (FIG. 1).
As mentioned, a TetR gene expression cassette is included for the control of the derepressible promoters (see FIG. 1). A constitutive human PGK promoter is used to drive the expression of TetR, followed by an IRES that directs the expression of puromycin N-acetyltransferase used to select the transposon integrated cells (see FIG. 2A).
To reduce the potential leaky expression from the derepressible promoters, an enhanced version of TetR was also included (see, e.g., Szulc et al., “A versatile tool for conditional gene expression and knockdown,” Nature Methods 3:109-116 (2006)). Briefly, a strong repressive domain of KRAB was fused in-frame to the C-terminal of original TetR, which improves its repressive activity and minimizes basal gene expression before induction. An SV40 Nuclear Localization Signal (NLS) was inserted as well to facilitate the nuclear entry of the larger TetR-KRAB fusion protein (FIG. 2A, FIGS. 12A-12B).
The sequence of the pcDNA3.1-E2A-E4-VA-TetR vector shown in FIG. 2A is provided below:








iHelper 1/pcDNA3.1-E2A-E4-VA-TetR (11, 986 bp)



(SEQ ID NO: 1)



GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATC



 


TGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCG


 


CTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAAT


 


TGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCC


 


AGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGG


 


TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC


 


CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT


 


CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG


 


TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT


 


GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG


 


GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC


 


GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA


 


GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT


 


TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC


 


GGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACT


 


GGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAA


 


ACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGCTT


 


CGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAG


 


TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA


 


CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA


 


ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG


 


GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA


 


AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG


 


TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA


 


TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACT


 


CACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACC


 


AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATG


 


GGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGAT


 


AGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAG


 


ATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCG


 


ATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagaggagc


 


agcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtg


 


tcgtccccgtccccgtcgccgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtat


 


cgagtccgaggacgaggaagactcatcacaagacgcgctggtgccgcgcacacccagcccgcggc


 


catcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaag


 


cccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgct


 


acaaatggtgggtttcagcaacccaccggtgctaatcaagcatggcaaaggaggtaagcgcacag


 


tgcggcggctgaatgaagacgacccagtggcgcgtggtatgcggacgcaagaggaagaggaagag


 


cccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtg


 


ggagaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaagg


 


cgaacttcaaactactgcctgaccaagtggaagctctggCggccgtatgcaagacctggctgaac


 


gaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtgacgatgatggggcg


 


attcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggct


 


gcgcgttgtggctgcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcatt


 


atgataaataaggagcacgtgattgaaatggatgtgacgagcgaaaacgggcagcgcgcgctgaa


 


ggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatctcca


 


acaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtct


 


tgcggcatgttcttctctgaaggcgcaaaggctcaggtggcttttaagcagatcaaggcttttat


 


gcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgccactacggtgcgagt


 


gcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgcc


 


ctgagcaacgcggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgca


 


ccacccggcgctgatagtgttccagtgctgcaaccctgtgtatcgcaactcgcgcgcgcagggcg


 


gaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatggtgcgc


 


agcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcac


 


taaacaccagtatcgcaacgtgtccctgccagtggcgcatagcgatgcgcggcagaacccctttg


 


atttttaacccgggagttctagggatctgcccctctccctcccccccccctaacgttactggccg


 


aagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtctt


 


ttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcc


 


cctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttc


 


ttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggt


 


gcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccac


 


gttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggct


 


gaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgcttta


 


catgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctt


 


tgaaaaacacgatgataaggatccaccggaggccaccatgactacgtccggcgttccatttggca


 


tgacactacgaccaacacgatctcggttgtctcggcgcactccgtacagtagggatcgtctacct


 


ccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacac


 


tttgacaatgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattc


 


aggaatgggttgttccctgggatatggttctaacgcgggaggagcttgtaatcctgaggaagtgt


 


atgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacga


 


gtcctgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcagg


 


ttttggccagctggtttaggatggtggtggatggcgccatgtttaatcagaggtttatatggtac


 


cgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttatgagggg


 


tcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatga


 


gctttggatacagcgccttgcactgtgggattttgaacaatattgtggtgctgtgctgcagttac


 


tgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaaggcgccttatgctgcg


 


ggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggc


 


ggcagcagtttattcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactct


 


acccccatgtagGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTG


 


TGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGT


 


GCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCA


 


TTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC


 


ATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGG


 


TATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCCGCaatatttgc


 


atgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttata


 


agttccctatcagtgatagagaaccggtgggcactcttccgtggtctggtggataaattcgcaag


 


ggtatcatggcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatgcggtt


 


accgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattc


 


cactttggccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgca


 


gggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacat


 


tcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacg


 


cttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaac


 


ggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgcc


 


gaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaa


 


ggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttc


 


cgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacc


 


tctctccccagggggatctgtgagtttggggacccttgattgttctttctttttcgctattgtaa


 


aattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtcccttg


 


tatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtc


 


tcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaa


 


tttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaa


 


ggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatg


 


ataaggtagaatatttctgcatataaattctggctggcgtggaaatattcttattggtagaaaca


 


actacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatg


 


aggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttcttttt


 


cctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaat


 


acgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaacagcgcat


 


tagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggt


 


gtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacgccttagccat


 


tgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttt


 


tacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacat


 


ttaggtacacggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgcca


 


acaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggggcattttactttaggtt


 


gcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagt


 


atgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttctt


 


attcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccccaa


 


aaaagaagagaaaggtcgacggcggtggtgctttgtctcctcagcactctgctgtcactcaagga


 


agtatcatcaagaacaaggagggcatggatgctaagtcactaactgcctggtcccggacactggt


 


gaccttcaaggatgtatttgtggacttcaccagggaggagtggaagctgctggacactgctcagc


 


agatcgtgtacagaaatgtgatgctggagaactataagaacctggtttccttgggttatcagctt


 


actaagccagatgtgatcctccggttggagaagggagaagagccctggctggtggagagagaaat


 


tcaccaagagacccatcctgattcagagactgcatttgaaatcaaatcatcagtttaagcgtaca


 


gcggctcccgggagttctagggatctgcccctctccctcccccccccctaacgttactggccgaa


 


gccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtctttt


 


ggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccc


 


tctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttctt


 


gaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgc


 


ctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgt


 


tgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctga


 


aggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttaca


 


tgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttg


 


aaaaacacgatgataaggatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctc


 


gccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgc


 


cacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcc


 


tcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtc


 


tggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccga


 


gttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggccca


 


aggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggc


 


agcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagac


 


ctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgagg


 


tgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaCCGCGTCTGGAACAA


 


TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTA


 


TTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTAT


 


CATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCT


 


GTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGT


 


GTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTC


 


CGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTT


 


GCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG


 


GGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGC


 


GGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCG


 


GCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCG


 


GATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGACCAAGCGACGCCCAACCTGC


 


CATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCG


 


TTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCT


 


TCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCA


 


TCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAA


 


ACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCG


 


TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA


 


ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA


 


CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC


 


CAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG


 


CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG


 


GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC


 


AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC


 


GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA


 


CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC


 


CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG


 


TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC


 


TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC


 


AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC


 


ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT


 


GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG


 


AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG


 


TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCA


 


GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG


 


GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC


 


AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTA


 


AAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC


 


TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAG


 


ATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG


 


AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG


 


CCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT


 


GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA


 


TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG


 


TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAG


 


CTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG


 


GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG


 


TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTT


 


GCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAA


 


GTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG


 


TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTA


 


CTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAG


 


GGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT


 


TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAG


 


AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC






The sequence of the pcDNA3.1-E2A-E4-VA-TetR-V2 vector shown in FIG. 2B is provided below:








iHelper2/ pcDNA3.1-E2A-E4-VA-TetR-V2 (11,641 bp)



(SEQ ID NO: 2)



GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATC



 


TGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCG


 


CTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAAT


 


TGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCC


 


AGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGG


 


TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGC


 


CCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT


 


CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG


 


TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT


 


GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG


 


GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC


 


GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA


 


GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT


 


TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC


 


GGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACT


 


GGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAA


 


ACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGCTT


 


CGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAG


 


TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA


 


CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA


 


ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG


 


GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA


 


AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAG


 


TACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA


 


TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACT


 


CACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACC


 


AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATG


 


GGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGAT


 


AGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAG


 


ATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCG


 


ATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagaggagc


 


agcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgccgc


 


cgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctggtgc


 


cgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaagcc


 


cgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaaccca


 


ccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcggacgc


 


aagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtgggagaa


 


gggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagtgg


 


aagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtgacga


 


tgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggctgca


 


ccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacgagc


 


gaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatctcc


 


aacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaaggcg


 


caaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgccac


 


tacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgcgg


 


aggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaaccctg


 


tgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatggtgc


 


gcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgtgtc


 


cctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaacccgggagttctagggatctgcccctctccctccccccccccta


 


acgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggccc


 


ggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaag


 


cagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctg


 


cggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaa


 


atggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacat


 


gctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataaggat


 


ccaccggaggccaccatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgtaca


 


gtagggatcgtctacctccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaatgc


 


acaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctaacgcggga


 


ggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacgagtcc


 


tgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcaggttttggccagctggtttaggatggtggtggatg


 


gcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttatgag


 


gggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgcactgt


 


gggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaaggcgc


 


cttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagtttattc


 


gcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtagGCGGCCGCTCGAGTCTAG


 


AGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC


 


TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTC


 


CTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC


 


TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG


 


GCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGG


 


GCTCTAGGGGGTATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCCGCa


 


atatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttataagttccctatcagtgatagagaa


 


ccggtgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccgtccg


 


ccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattccactttggcc


 


gcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaac


 


gcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggc


 


gacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagt


 


accctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcg


 


cgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttc


 


cgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatctgtgagttt


 


ggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaagatgtc


 


ccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttctgtaac


 


tttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaa


 


ggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggct


 


ggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgaggat


 


aaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtct


 


catcattttggcaaagaattgtaatacgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaacagcgcattaga


 


gctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaa


 


aaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaaga


 


ttttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacagaaaaaca


 


gtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggggcatttta


 


ctttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccattattacgac


 


aagctatcgaattatttgatcaccaaggtgcagagccagccttatattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgt


 


gaaagtgggtccccaaaaaagaagagaaaggtcgacggcggtggttcagtttaagcgtacagcggctcccgggagttctagggatctgc


 


ccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgcc


 


gtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaagg


 


tctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccc


 


cacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagtt


 


ggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatct


 


gatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttccttt


 


gaaaaacacgatgataaggatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtc


 


cccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg


 


gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcgg


 


tctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggc


 


cgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccga


 


ccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagac


 


ctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggt


 


gcatgacccgcaagcccggtgcctgaCCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGT


 


GAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTG


 


CTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTG


 


TATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGT


 


GGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACC


 


ACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAAC


 


TCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACA


 


ATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGC


 


CACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGC


 


GGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTT


 


CGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGACCA


 


AGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAG


 


GTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGA


 


TCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTAC


 


AAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCT


 


AGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCT


 


CTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC


 


GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG


 


CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC


 


GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCG


 


GTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT


 


TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG


 


AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAG


 


GAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA


 


GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA


 


GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC


 


GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC


 


TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG


 


CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG


 


TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT


 


TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT


 


ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT


 


TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT


 


TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT


 


TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT


 


TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA


 


AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGC


 


TTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT


 


GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG


 


CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC


 


AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC


 


CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTG


 


CGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG


 


GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG


 


TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC


 


GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT


 


CCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT


 


GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC


 


ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT


 


CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT


 


GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGC


 


AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC


 


TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA


 


TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAA


 


AAGTGCCACCTGACGTC






The entire transfer plasmid including the derepressible Helper (piHelper1) and TetR expression cassettes was tested for use in AAV production by transient transfection. As shown in FIG. 3, when the plasmids were co-transfected with control pRep-Cap and pAAV-GOI plasmids, the addition of Dox activated the production of AAV to the level of approximately 30% of control Helper vectors that supports constitutive helper gene expression. Therefore, the derepressible helper construct was functional for AAV production upon induction.
Example 2: Design and Validation of Derepressible Rep-Cap Genes
Challenges for producing Rep proteins are two-fold. First, the ratio of Rep78 and Rep52 needs to be maintained during induction for high titer AAV production. Second, the p19 promoter required for Rep52 expression is located inside the coding region of Rep78, which creates challenges for including a derepressible promoter. To overcome these challenges, two strategies were developed (FIGS. 4A-4C).
First, to retain the natural regulation of Rep gene expression (see FIG. 4A), the original viral promoters were retained but modified by inserting two TetO sites surround the TATA box and TSS. Two copies of TetO sites was inserted into the upstream truncated p5 promoter with core elements including TATA box, Rep Binding Element (RBE), and YY1 site. In addition, a wildtype copy of p5 promoter, serving as an enhancer, was placed downstream of cap gene to support both Rep and Cap expression (FIG. 5) (See, e.g., U.S. Pat. No. 5,622,856).
The sequences for the derepressible p5 promoters set forth in FIG. 5 are as follows







min-p5-i1


(SEQ ID NO: 3)


TATTTAATCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGAT


 


CGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACG


 


CGCAGCCGCC


 


min-p5-i2


(SEQ ID NO: 4)


TATTTAAtcTCCCTATCAGTGATAGAGAtcGCCCGAGTGAGCACGCAGGG


 


TCTCCATTTTGATCCCTATCAGTGATAGAGAAGCGGGAGGTTTGAACGCG


 


CAGCCGCC


 


min-p5-i3


(SEQ ID NO: 5)


TCCCTATCAGTGATAGAGAtcTATTTAAGCCCGAGTGAGCACGCAGTCCC


 


TATCAGTGATAGAGAGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCA


 


GCCGCC






Similarly, two copies of TetO sites were also inserted into the p19 promoter next to TSS site (FIG. 6). In order to minimize the interference of such insertion and maximize the original activity of these viral promoters, three ways of insertion of TetO sites were designed for each promoter for best performance. Thus, a total of 9 variations were examined (iRepCap 1 to iRepCap9).
The sequences for the derepressible promoters including p19 illustrated schematically in FIG. 6 are provided below:







p19-i1


(SEQ ID NO: 6)


ccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatc


 


cccaattacttgctccccaaaacccagcctgagctccagtgggcgtggac


 


taatatggaacagtatttaagcgcctgTCCCTATCAGTGATAGAGATCTC


 


CCTATCAGTGATAGAGAtttgaatctcacggag


 


p19-i2


(SEQ ID NO: 7)


ccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatc


 


cccaattacttgctccccaaaacccagcctgagTCCCTATCAGTGATAGA


 


GActccagtgggcgtggactaatatggaacagtatttaagcgcctgTCCC


 


TATCAGTGATAGAGAtttgaatctcacggag


 


p19-i3


(SEQ ID NO: 8)


ccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatc


 


cccaattacttgaccccaaaacccagcctgagctccagtgggcgtggact


 


aatatggaaTCCCTATCAGTGATAGAGAcagtatttaagcgcctgTCCCT


 


ATCAGTGATAGAGAtttgaatctcacggag






Two methods were developed for the placement of the derepressible p19 promoters. In the first method, a separate expression cassette for Rep52 was generated, driven by the derepressible p19 promoters. In the first method shown in FIG. 4B, the original p19 promoter in the Rep78 ORF was silenced by changing six nucleotides in three core regulatory elements required for p19 activity (SP1, TATA-1, and TATA-2 sites). These changes did not alter the Rep78 protein sequence. Rep78 was controlled by a derepressible p5 promoter.
In a second method, an artificial intron was created for the insertion of TetO sites in the Rep78 ORF (FIG. 4C). The chimeric intron between introns from human (3-globin and immunoglobulin heavy chain genes was adopted by replacing the non-essential internal sequence with the TetO2 sites (FIG. 7A). The new artificial intron was inserted 1 bp or 25 bp downstream of TATA-2 of p19 promoter in situ (iRepCap-10 and iRepCap 11) (FIG. 4B and FIG. 7B). The new p19 promoter with adjacent TetO-containing intron ensured the repression of Rep52 gene expression before induction, while still allowing for the expression of the Rep78 protein after the removal of the intron during mRNA splicing. The efficiency of splicing is evaluated by PCR analysis for the cDNA.
The nucleic acid sequences for the intron-based p19 promoters illustrated in FIGS. 7A-7B are provided below.







In-p19-i1


(SEQ ID NO: 9)


ccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatc


 


cccaattacttgctccccaaaacccagcctgagctccagtgggcgtggac


 


taatatggaacagtatttaaggtaagtTCCCTATCAGTGATAGAGATCTC


 


CCTATCAGTGATAGAGAtactgacatccactttgcctttctctccacagc


 


gcctgtttgaatctcacggag


 


In-p19-i2


(SEQ ID NO: 10)


ccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatc


 


cccaattacttgctccccaaaacccagcctgagctccagtgggcgtggac


 


taatatggaacagtatttaagcgcctgtttgaatctcacggaaaggtaag


 


tTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGAtactgacat


 


ccactttgcctttctctccacag






To test the activity of the 11 inducible Rep-Cap designs, HEK293 cells were transfected with one of the vectors plus standard pHelper and pAAV-GFP for AAV production. Three days after transfection, cells were harvested for Rep-Cap protein expression and AAV titer analysis. Western blot analysis revealed various expression levels of Rep and Cap proteins; many maintained similar ratios of Rep78 vs Rep52 as control RepCap vector (FIG. 8A). qPCR analysis of the AAV titer showed that the designs performed similarly or had higher titers compared to control triple transfection (FIG. 8B).
To test the performance of both inducible helpers and inducible Rep-Cap designs together, HEK293 cells were transfected with selected iRepCap vectors, iHelper1/2, and pAAV-GFP, and left untreated or treated with Doxycycline for three days. As shown in FIG. 9A, the protein expression of Rep and Cap was only induced by the addition of Dox, and the derepression of the derepressible promoters. Accordingly, the AAV titer was significantly increased over 10 to 25 fold upon derepression (FIG. 9B).
To stably integrate the iHelper and iRepCap, iHelper1/2 and iRepCap10 were chosen with AAV-GFP to assemble the functional cassettes into a single PIGGYBACTM transposon transfer vector PB007 (Transposagen, Inc., Lexington, Ky.). FIGS. 10A-10D show the plasmid constructs used for integration into mammalian cells, suitably HEK293 cells. To facilitate the future addition of specific AAV genes of interest, the AAV-GFP was not included in some of the transfer vectors (FIGS. 10A and 10B). HEK293 cells will be transfected with both the transfer vectors and transposase mRNA and the integrated cell pool will be enriched by puromycin selection. The single cell clones will be isolated and screened for AAV production with and without Dox treatment to activate derepression.
The nucleic acid sequence of the vector illustrated in FIG. 10 A is provided below:








PB007-iHelper1-iRepCap10/ PBBG7 (18,281 bp)



(SEQ ID NO: 11)



ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG



 


AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC


 


ATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCG


 


TTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATC


 


CCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC


 


AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA


 


TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG


 


GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC


 


GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG


 


GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC


 


GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT


 


TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG


 


ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGA


 


CGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGC


 


TCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTG


 


ACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTAT


 


ATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACA


 


TACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAA


 


CAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGAATTCCTGCAGCC


 


CGGGGGATCCACTAGTTCTAGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTA


 


ATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGT


 


CCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGG


 


GGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGC


 


AGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCCACAATAAGT


 


TGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATG


 


GGAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcGTTT


 


AAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGC


 


TTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAAT


 


AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT


 


AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT


 


CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT


 


GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC


 


CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC


 


AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC


 


TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA


 


CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAA


 


CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT


 


GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTG


 


ATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTC


 


AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGAC


 


CGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagagg


 


agcagcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgc


 


cgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctgg


 


tgccgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaa


 


gcccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaac


 


ccaccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcgga


 


cgcaagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtggga


 


gaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagt


 


ggaagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtga


 


cgatgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggct


 


gcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacg


 


agcgaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatc


 


tccaacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaagg


 


cgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgc


 


cactacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgc


 


ggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaacc


 


ctgtgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatgg


 


tgcgcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgt


 


gtccctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaacccgggagttctagggatctgcccctctccctcccccccc


 


cctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagg


 


gcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaag


 


gaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgc


 


ctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaaga


 


gtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtg


 


cacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa


 


ggatccaccggaggccaccatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgt


 


acagtagggatcgtctacctccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaa


 


tgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctaacgcg


 


ggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacga


 


gtcctgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcaggttttggccagctggtttaggatggtggtg


 


gatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttat


 


gaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgca


 


ctgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaagg


 


cgccttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagttt


 


attcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtagGCGGCCGCTCGAGTCT


 


AGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA


 


TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG


 


TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT


 


TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC


 


AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG


 


GGGCTCTAGGGGGTATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCC


 


GCaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttataagttccctatcagtgatag


 


agaaccggtgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccg


 


tccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattccactttg


 


gccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccggg


 


aaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccc


 


cggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctca


 


ctagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag


 


ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcg


 


gtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatctgt


 


gagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaa


 


gatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttct


 


gtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttt


 


tcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctg


 


gctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgagg


 


ataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctg


 


tctcatcattttggcaaagaattgtaatacgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaacagcg


 


cattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggc


 


atgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctg


 


gcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacag


 


aaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggg


 


gcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccatta


 


ttacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaactt


 


aaatgtgaaagtgggtccccaaaaaagaagagaaaggtcgacggcggtggtgctttgtctcctcagcactctgctgtcactcaaggaagtat


 


catcaagaacaaggagggcatggatgctaagtcactaactgcctggtcccggacactggtgaccttcaaggatgtatttgtggacttcacca


 


gggaggagtggaagctgctggacactgctcagcagatcgtgtacagaaatgtgatgctggagaactataagaacctggtttccttgggttat


 


cagcttactaagccagatgtgatcctccggttggagaagggagaagagccctggctggtggagagagaaattcaccaagagacccatcct


 


gattcagagactgcatttgaaatcaaatcatcagtttaagcgtacagcggctcccgggagttctagggatctgcccctctccctccccccccc


 


ctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg


 


cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaagg


 


aagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcct


 


ctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagt


 


caaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgc


 


acatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataag


 


gatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcacc


 


ctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaac


 


tcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag


 


cgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatgga


 


aggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg


 


ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctc


 


cccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccg


 


gtgcctgaCCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACT


 


GGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTT


 


TGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGG


 


TTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGC


 


ACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTC


 


CTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCT


 


GCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT


 


TGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCT


 


GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCC


 


CGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACG


 


AGTCGGATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGACCAAGCGACGCCCAA


 


CCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG


 


AATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGA


 


GTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAAT


 


AGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGT


 


CCAAACTCATCAATGTATCTTATCATGTCTGTAGCtGATcaATTgGCGCGCCGAATTCG


 


TTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgcttcaaaatggagaccctgcgtgctcactcgggcttaaatacccag


 


cgtgaccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaatacaggacctctagagcatggaaactagataagaaagaaa


 


tacgcagagaccaaagttcaactgaaacgaattaaacggtttattgattaacaagcaaactagtttacagattacgggtgaggtaacgggtgc


 


cgatggggcgaggctcagaataaacgccatttgtgtcaacagcaaagtccacatttgtagatttgttgtagttggaagtgtattgaatctctgg


 


gttccagcgtttgctgttttctttctgcagctcccattcaatttccacgctgacctgtccggtgctgtactgcgtgatgaacgacgcaaacttagct


 


ggactgaaggtagttggaggattcgcgggaacaggtgtattcttaatcaggatctgaggaggcgggtgtttcagtccaaagcctcccatcag


 


cggcgagggatgaaagtgtccgtccgtgtgaggaatcttggcccagataggaccctgcaggtacacgtcccggtcctgccagaccatgcc


 


aggtaaggctccttgactgttgacggtccctgtagcaggagcggtgttggccgattgcaggttagtggccaccgtgccgtactcttctgtggc


 


cactgggttggtggttttaatttcttcctcgttggttatcataacgttgtcaaggtccacgttgctatttccagctccctgtttcccaaatattaagact


 


ccgctcatcggaaaaaatttgtcttcgtcgtccttgtgggttgccatagcgggaccgggatttaccagagagtctctgccattcagatgatactt


 


ggtggcaccggtccaggcaaagttgctgttgttattttgattggttgtcttggagacgcgttgctgccggtagcagggcccgggtagccagttt


 


ttggcctgattcgccatgctactaggcccggcctgagaaaattgcaacgtccgatttcctgcggtaccactcgtggtctgagtccgagacag


 


gtagtacaggtactggtcgatgagggggttcatcagccggtccaggctttggctgtgcgcgtagctgctgtgaaaaggcacgtcctcaaac


 


gtgtagctgaactgaaagttgttgcccgttctcagcatttgagaaggaaagtattccaggcagtagaaggaggaacggcccacggcctgac


 


tgccattgttcagagtcaggtacccgtactgaggaatcatgaagacgtccgccgggaacggaggcaggcagccctggtgcgcagagccg


 


aggacgtacgggagctggtattccgagtccgtaaagacctgaaccgtgctggtaaggttattggcgatggtcgtggtgccatcattcgtcgt


 


gacctccttgacctggatgttgaagagcttgaagttgagcttcttgggccggaatccccagttgttgttgatgagtcgctgccagtcacgtggt


 


gagaagtggcagtggaatctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggtgttgtcgttggtgctgcctcccgattg


 


gctggagatttgcttgtagaggtggttgttgtaggtggggagggcccaggttcgggtgctggtggtgatgactctgtcgcccagccatgtgg


 


aatcgcaatgccaatttcctgaggcgttacccactccgtcggcgccttcgttattgtctgccattggagcgccaccgcctgcagccattgtatt


 


agatcccacaccagagggggctgcggggggttctccgagtggttgagggtcgggcactgactctgagtcgccagtctgcccaaagttga


 


gtctctttctcgcgggctgctggcctttcttgccgatgcccgaagaggagtctggttcctggggtgattgctctaccggtctcttctttccagga


 


gccgtcttagcgccttcctcaaccagaccgagaggttcgagaacccgcttcttggcctggaagactgctcgcccgaggttgcccccaaaag


 


acgtatcttcttgcagacgctcctgaaactcggcgtcggcgtggttataccgcaggtacggattgtcacccgctttgagctgctggtcgtagg


 


ccttgtcgtgctcgagggccgctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttgaagggtccgaggtacttgtagccagg


 


aagcaccagaccccggccgtcgtcctgcttttgctggttggctttgggtttcggggctccaggtttcaagtcccaccactcgcgaatgccctc


 


agagaggttgtcctcgagccaatctggaagataaccatcggcagccatacctgatttaaatcatttattgttcaaagatgcagtcatccaaatc


 


cacattgaccagatcgcaggcagtgcaagcgtctggcacctttcccatgatatgatgaatgtagcacagtttctgatacgcctttttgacgaca


 


gaaacgggttgagattctgacacgggaaagcactctaaacagtctttctgtccgtgagtgaagcagatatttgaattctgattcattctctcgca


 


ttgtctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgttttggtacctgtctgcgtagttgatcgaagcttccgcgtctg


 


acgtcgatggctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtcactgggggcgggtcttttcttggctccaccctttttg


 


acgtagaattcatgctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgacttcctgcttggtgaccttcccaaagtcatgatc


 


cagacggcgggtgagttcaaatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttgagttcccgtcaatcacggcgcaca


 


tgttggtgttggaggtgacgatcacgggagtcgggtctatctgggccgaggacttgcatttctggtccacgcgcaccttgcttcctccgagaa


 


tggctttggccgactccacgaccttggcggtcatcttcccctcctcccaccagatcaccatcttgtcgacacagtcgttgaagggaaagttctc


 


attggtccagtttacgcacccgtagaagggcacagtgtgggctatggcctccgcgatgttggtcttcccggtagttgcaggcccaaacagcc


 


agatggtgttcctcttgccgaactttttcgtggcccatcccagaaagacggaagccgcatattggggatcgtacccgtttagttccaaaatttta


 


taaatccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtcgggggcggttttagtcaggctcataatctttcccgcattgt


 


ccaaggcagccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgaggcctggtcctcctggatccactgcttctccga


 


ggtaatccccttgtccacgagccacccgaccagctccatgtacctggctgaagtttttgatctgatcaccggcgcatcagaattgggattctga


 


ttctctttgttctgctcctgcgtctgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgtgagattcaaacaggcgctgtgga


 


gagaaaggcaaagtggatgtcagtaTCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAac


 


ttaccttaaatactgttccatattagtccacgcccactggagctcaggctgggttttggggagcaagtaattggggatgtagcactcatccacc


 


accttgttcccgcctccggcgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcgatcccgcggtaaattctctgaatca


 


gtttttcgcgaatctgactcaggaaacgtcccaaaaccatggatttcaccccggtggtttccacgagcacgtgcatgtggaagtagctctctcc


 


cttctcaaattgcacaaagaaaagagcctccggggccttactcacacggcgccattccgtcagaaagtcgcgctgcagcttctcggccacg


 


gtcaggggtgcctgctcaatcagattcagatccatgtcagaatctggcggcaactcccattccttctcggccacccagttcacaaagctgtca


 


gaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcacaatctcgtaaaaccccggcatGGCGGCTGCGCGTT


 


CAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCGGGCgaTCTCTATCACT


 


GATAGGGAGATCTCTATCACTGATAGGGAgaTTAAATAgaatggCTAggATCCGGCCGGc


 


cTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAG


 


CCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGTATGGCTTTTTCCCCGTAT


 


CCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGAT


 


CCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGC


 


GGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCG


 


GGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAG


 


CGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTAGA


 


TCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGAC


 


GGTATCGATAAGCTTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACG


 


TAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCAC


 


GTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGT


 


GACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGAT


 


GTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCA


 


AGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATC


 


AGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCT


 


GGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGA


 


AAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTA


 


CCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTTCGTTC


 


AGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCA


 


GCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCC


 


GGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGG


 


GTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCG


 


GGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTC


 


ACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTA


 


ATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGG


 


AAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTT


 


TGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG


 


GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAAT


 


CAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAA


 


CCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA


 


TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT


 


ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT


 


TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA


 


CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC


 


GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC


 


AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG


 


CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACG


 


GCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG


 


GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT


 


TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT


 


TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT


 


TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA


 


AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGC


 


TTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT


 


GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG


 


CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC


 


AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC


 


CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTG


 


CGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG


 


GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTG


 


TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC


 


GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCAT


 


CCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGT


 


GTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC


 


ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT


 


CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT


 


GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGC


 


AAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence of the vector illustrated in FIG. 10B is provided below.








PB007-iHelper2-iRepCap10/ PBBG8 (17,936 bp)



(SEQ ID NO: 12)



ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG



 


AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC


 


ATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCG


 


TTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATC


 


CCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC


 


AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA


 


TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG


 


GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC


 


GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG


 


GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC


 


GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT


 


TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG


 


ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGA


 


CGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGC


 


TCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTG


 


ACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTAT


 


ATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACA


 


TACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAA


 


CAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGAATTCCTGCAGCC


 


CGGGGGATCCACTAGTTCTAGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTA


 


ATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGT


 


CCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGG


 


GGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGC


 


AGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCCACAATAAGT


 


TGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATG


 


GGAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcGTTT


 


AAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGC


 


TTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAAT


 


AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT


 


AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT


 


CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT


 


GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC


 


CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC


 


AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC


 


TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA


 


CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAA


 


CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT


 


GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTG


 


ATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTC


 


AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGAC


 


CGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagagg


 


agcagcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgc


 


cgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctgg


 


tgccgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaa


 


gcccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaac


 


ccaccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcgga


 


cgcaagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtggga


 


gaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagt


 


ggaagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtga


 


cgatgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggct


 


gcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacg


 


agcgaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatc


 


tccaacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaagg


 


cgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgc


 


cactacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgc


 


ggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaacc


 


ctgtgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatgg


 


tgcgcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgt


 


gtccctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaacccgggagttctagggatctgcccctctccctcccccccc


 


cctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagg


 


gcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaag


 


gaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgc


 


ctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaaga


 


gtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtg


 


cacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa


 


ggatccaccggaggccaccatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgt


 


acagtagggatcgtctacctccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaa


 


tgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctaacgcg


 


ggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacga


 


gtcctgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcaggttttggccagctggtttaggatggtggtg


 


gatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttat


 


gaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgca


 


ctgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaagg


 


cgccttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagttt


 


attcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtagGCGGCCGCTCGAGTCT


 


AGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA


 


TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG


 


TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT


 


TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC


 


AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG


 


GGGCTCTAGGGGGTATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCC


 


GCaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttataagttccctatcagtgatag


 


agaaccggtgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccg


 


tccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattccactttg


 


gccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccggg


 


aaacgcagcggcgccgaccctgggtctcgcacattatcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccc


 


cggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctca


 


ctagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag


 


ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcg


 


gtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatctgt


 


gagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaa


 


gatgtcctatgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattttct


 


gtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcacttttttt


 


tcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctg


 


gctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtttgagatgagg


 


ataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctg


 


tctcatcattttggcaaagaattgtaatacgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaacagcg


 


cattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggc


 


atgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctg


 


gcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacag


 


aaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggg


 


gcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccatta


 


ttacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaactt


 


aaatgtgaaagtgggtccccaaaaaagaagagaaaggtcgacggcggtggttcagtttaagcgtacagcggctcccgggagttctaggg


 


atctgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccata


 


ttgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgc


 


aaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaac


 


cccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgt


 


gagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgg


 


gatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttt


 


tcctttgaaaaacacgatgataaggatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacga


 


cgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgag


 


cgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtgg


 


cggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggct


 


ggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcc


 


cgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctgga


 


gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacct


 


ggtgcatgacccgcaagcccggtgcctgaCCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTT


 


GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGC


 


TGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCT


 


TGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAAC


 


GTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCA


 


CCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA


 


ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGA


 


CAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTT


 


GCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCA


 


GCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC


 


TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGAC


 


CAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAA


 


AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGG


 


GATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTT


 


ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT


 


CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTAGCtGATcaATTg


 


GCGCGCCGAATTCGTTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgcttcaaaatggagaccctgcgtg


 


ctcactcgggcttaaatacccagcgtgaccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaatacaggacctctagagca


 


tggaaactagataagaaagaaatacgcagagaccaaagttcaactgaaacgaattaaacggtttattgattaacaagcaaactagtttacaga


 


ttacgggtgaggtaacgggtgccgatggggcgaggctcagaataaacgccatttgtgtcaacagcaaagtccacatttgtagatttgttgtag


 


ttggaagtgtattgaatctctgggttccagcgtttgctgttttctttctgcagctcccattcaatttccacgctgacctgtccggtgctgtactgcgt


 


gatgaacgacgcaaacttagctggactgaaggtagttggaggattcgcgggaacaggtgtattcttaatcaggatctgaggaggcgggtgt


 


ttcagtccaaagcctcccatcagcggcgagggatgaaagtgtccgtccgtgtgaggaatcttggcccagataggaccctgcaggtacacgt


 


cccggtcctgccagaccatgccaggtaaggctccttgactgttgacggtccctgtagcaggagcggtgttggccgattgcaggttagtggc


 


caccgtgccgtactcttctgtggccactgggttggtggttttaatttcttcctcgttggttatcataacgttgtcaaggtccacgttgctatttccag


 


ctccctgtttcccaaatattaagactccgctcatcggaaaaaatttgtcttcgtcgtccttgtgggttgccatagcgggaccgggatttaccaga


 


gagtctctgccattcagatgatacttggtggcaccggtccaggcaaagttgctgttgttattttgattggttgtcttggagacgcgttgctgccgg


 


tagcagggcccgggtagccagtttttggcctgattcgccatgctactaggcccggcctgagaaaattgcaacgtccgatttcctgcggtacc


 


actcgtggtctgagtccgagacaggtagtacaggtactggtcgatgagggggttcatcagccggtccaggctttggctgtgcgcgtagctg


 


ctgtgaaaaggcacgtcctcaaacgtgtagctgaactgaaagttgttgcccgttctcagcatttgagaaggaaagtattccaggcagtagaa


 


ggaggaacggcccacggcctgactgccattgttcagagtcaggtacccgtactgaggaatcatgaagacgtccgccgggaacggaggc


 


aggcagccctggtgcgcagagccgaggacgtacgggagctggtattccgagtccgtaaagacctgaaccgtgctggtaaggttattggcg


 


atggtcgtggtgccatcattcgtcgtgacctccttgacctggatgttgaagagcttgaagttgagcttcttgggccggaatccccagttgttgtt


 


gatgagtcgctgccagtcacgtggtgagaagtggcagtggaatctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggt


 


gttgtcgttggtgctgcctcccgattggctggagatttgcttgtagaggtggttgttgtaggtggggagggcccaggttcgggtgctggtggt


 


gatgactctgtcgcccagccatgtggaatcgcaatgccaatttcctgaggcgttacccactccgtcggcgccttcgttattgtctgccattgga


 


gcgccaccgcctgcagccattgtattagatcccacaccagagggggctgcggggggttctccgagtggttgagggtcgggcactgactct


 


gagtcgccagtctgcccaaagttgagtctctttctcgcgggctgctggcctttcttgccgatgcccgaagaggagtctggttcctggggtgatt


 


gctctaccggtctcttctttccaggagccgtcttagcgccttcctcaaccagaccgagaggttcgagaacccgcttcttggcctggaagactg


 


ctcgcccgaggttgcccccaaaagacgtatcttcttgcagacgctcctgaaactcggcgtcggcgtggttataccgcaggtacggattgtca


 


cccgctttgagctgctggtcgtaggccttgtcgtgctcgagggccgctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttgaa


 


gggtccgaggtacttgtagccaggaagcaccagaccccggccgtcgtcctgcttttgctggttggctttgggtttcggggctccaggtttcaa


 


gtcccaccactcgcgaatgccctcagagaggttgtcctcgagccaatctggaagataaccatcggcagccatacctgatttaaatcatttattg


 


ttcaaagatgcagtcatccaaatccacattgaccagatcgcaggcagtgcaagcgtctggcacctttcccatgatatgatgaatgtagcacag


 


tttctgatacgcctttttgacgacagaaacgggttgagattctgacacgggaaagcactctaaacagtctttctgtccgtgagtgaagcagatat


 


ttgaattctgattcattctctcgcattgtctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgttttggtacctgtctgcgta


 


gttgatcgaagcttccgcgtctgacgtcgatggctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtcactgggggcgg


 


gtcttttcttggctccaccctttttgacgtagaattcatgctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgacttcctgcttg


 


gtgaccttcccaaagtcatgatccagacggcgggtgagttcaaatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttgag


 


ttcccgtcaatcacggcgcacatgttggtgttggaggtgacgatcacgggagtcgggtctatctgggccgaggacttgcatttctggtccac


 


gcgcaccttgcttcctccgagaatggctttggccgactccacgaccttggcggtcatcttcccctcctcccaccagatcaccatcttgtcgaca


 


cagtcgttgaagggaaagttctcattggtccagtttacgcacccgtagaagggcacagtgtgggctatggcctccgcgatgttggtcttcccg


 


gtagttgcaggcccaaacagccagatggtgttcctcttgccgaactttttcgtggcccatcccagaaagacggaagccgcatattggggatc


 


gtacccgtttagttccaaaattttataaatccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtcgggggcggttttagtc


 


aggctcataatctttcccgcattgtccaaggcagccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgaggcctggtc


 


ctcctggatccactgcttctccgaggtaatccccttgtccacgagccacccgaccagctccatgtacctggctgaagtttttgatctgatcacc


 


ggcgcatcagaattgggattctgattctctttgttctgctcctgcgtctgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgt


 


gagattcaaacaggcgctgtggagagaaaggcaaagtggatgtcagtaTCTCTATCACTGATAGGGAGATCTCT


 


ATCACTGATAGGGAacttaccttaaatactgttccatattagtccacgcccactggagctcaggctgggttttggggagcaagta


 


attggggatgtagcactcatccaccaccttgttcccgcctccggcgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcg


 


atcccgcggtaaattctctgaatcagtttttcgcgaatctgactcaggaaacgtcccaaaaccatggatttcaccccggtggtttccacgagca


 


cgtgcatgtggaagtagctctctcccttctcaaattgcacaaagaaaagagcctccggggccttactcacacggcgccattccgtcagaaag


 


tcgcgctgcagcttctcggccacggtcaggggtgcctgctcaatcagattcagatccatgtcagaatctggcggcaactcccattccttctcg


 


gccacccagttcacaaagctgtcagaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcacaatctcgtaaaaccccggca


 


tGGCGGCTGCGCGTTCAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCG


 


GGCgaTCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAgaTTAAATAgaatgg


 


CTAggATCCGGCCGGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGT


 


GGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGTAT


 


GGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGT


 


TCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTG


 


CCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCG


 


CTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGG


 


GGCTGTCCCTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTA


 


GTGAGGGTTAATTAGATCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCC


 


CCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATATATAT


 


ATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTT


 


AAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTT


 


ATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCC


 


AAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAG


 


AGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGG


 


TTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCC


 


CAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAG


 


GTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGA


 


GCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAA


 


CGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGAC


 


ACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACA


 


GCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATAT


 


TACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACC


 


CCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTG


 


TGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT


 


AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG


 


CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC


 


GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG


 


CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA


 


CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC


 


AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC


 


CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCC


 


GACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC


 


TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG


 


GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA


 


AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA


 


ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA


 


CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAG


 


TGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG


 


AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC


 


CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG


 


ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA


 


CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCT


 


TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTC


 


TGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGT


 


TCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA


 


CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGAT


 


TTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAAC


 


TTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTC


 


GCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG


 


CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTAC


 


ATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT


 


CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC


 


TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG


 


TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGG


 


GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT


 


TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCC


 


ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG


 


CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG


 


TTGAATACTCAT






The sequence of the vector represented in FIG. 10C is provided below.








PB007-iHelper1-iRepCap10-AAV-GFP/ PBBG9 (21,391 bp)



(SEQ ID NO: 13)



ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG



 


AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC


 


ATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCG


 


TTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATC


 


CCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC


 


AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA


 


TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG


 


GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC


 


GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG


 


GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC


 


GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT


 


TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG


 


ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGA


 


CGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGC


 


TCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTG


 


ACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTAT


 


ATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACA


 


TACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAA


 


CAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGAATTCCTGCAGCC


 


CGGGGGATCCACTAGTTCTAGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTA


 


ATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGT


 


CCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGG


 


GGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGC


 


AGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCCACAATAAGT


 


TGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATG


 


GGAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcGTTT


 


AAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGC


 


TTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAAT


 


AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT


 


AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT


 


CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT


 


GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC


 


CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC


 


AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC


 


TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA


 


CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAA


 


CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT


 


GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTG


 


ATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTC


 


AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGAC


 


CGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagagg


 


agcagcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgc


 


cgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctgg


 


tgccgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaa


 


gcccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaac


 


ccaccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcgga


 


cgcaagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtggga


 


gaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagt


 


ggaagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtga


 


cgatgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggct


 


gcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacg


 


agcgaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatc


 


tccaacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaagg


 


cgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgc


 


cactacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgc


 


ggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaacc


 


ctgtgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatgg


 


tgcgcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgt


 


gtccctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaacccgggagttctagggatctgcccctctccctcccccccc


 


cctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtatttggcaatgtgagg


 


gcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaag


 


gaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgc


 


ctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaaga


 


gtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtg


 


cacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa


 


ggatccaccggaggccaccatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgt


 


acagtagggatcgtctacctccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaa


 


tgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctaacgcg


 


ggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacga


 


gtcctgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcaggttttggccagctggtttaggatggtggtg


 


gatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttat


 


gaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgca


 


ctgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaagg


 


cgccttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagttt


 


attcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtagGCGGCCGCTCGAGTCT


 


AGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA


 


TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG


 


TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT


 


TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC


 


AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG


 


GGGCTCTAGGGGGTATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCC


 


GCaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttataagttccctatcagtgatag


 


agaaccggtgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccg


 


tccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattccactttg


 


gccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccggg


 


aaacgcagcggcgccgaccctgggtctcgcacattatcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccc


 


cggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctca


 


ctagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag


 


ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcg


 


gtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatctgt


 


gagtttggggacccttgattgttctttattttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaa


 


gatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcatt


 


ttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcact


 


tttttttcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatat


 


aaattctggctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactgtt


 


tgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggt


 


tattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaacagcg


 


cattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggc


 


atgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctg


 


gcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacag


 


aaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggg


 


gcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccatta


 


ttacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaactt


 


aaatgtgaaagtgggtccccaaaaaagaagagaaaggtcgacggcggtggtgctttgtctcctcagcactctgctgtcactcaaggaagtat


 


catcaagaacaaggagggcatggatgctaagtcactaactgcctggtcccggacactggtgaccttcaaggatgtatttgtggacttcacca


 


gggaggagtggaagctgctggacactgctcagcagatcgtgtacagaaatgtgatgctggagaactataagaacctggtttccttgggttat


 


cagcttactaagccagatgtgatcctccggttggagaagggagaagagccctggctggtggagagagaaattcaccaagagacccatcct


 


gattcagagactgcatttgaaatcaaatcatcagtttaagcgtacagcggctcccgggagttctagggatctgcccctctccctccccccccc


 


ctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg


 


cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaagg


 


aagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcct


 


ctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagt


 


caaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgc


 


acatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataag


 


gatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcacc


 


ctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaac


 


tcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag


 


cgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatgga


 


aggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctg


 


ggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctc


 


cccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccg


 


gtgcctgaCCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACT


 


GGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTT


 


TGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGG


 


TTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGC


 


ACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTC


 


CTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCT


 


GCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT


 


TGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCT


 


GCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCC


 


CGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACG


 


AGTCGGATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGACCAAGCGACGCCCAA


 


CCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGG


 


AATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGA


 


GTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAAT


 


AGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGT


 


CCAAACTCATCAATGTATCTTATCATGTCTGTAGCtGATcaATTgGCGCGCCGAATTCG


 


TTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgcttcaaaatggagaccctgcgtgctcactcgggcttaaatacccag


 


cgtgaccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaatacaggacctctagagcatggaaactagataagaaagaaa


 


tacgcagagaccaaagttcaactgaaacgaattaaacggtttattgattaacaagcaaactagtttacagattacgggtgaggtaacgggtgc


 


cgatggggcgaggctcagaataaacgccatttgtgtcaacagcaaagtccacatttgtagatttgttgtagttggaagtgtattgaatctctgg


 


gttccagcgtttgctgttttctttctgcagctcccattcaatttccacgctgacctgtccggtgctgtactgcgtgatgaacgacgcaaacttagct


 


ggactgaaggtagttggaggattcgcgggaacaggtgtattcttaatcaggatctgaggaggcgggtgtttcagtccaaagcctcccatcag


 


cggcgagggatgaaagtgtccgtccgtgtgaggaatcttggcccagataggaccctgcaggtacacgtcccggtcctgccagaccatgcc


 


aggtaaggctccttgactgttgacggtccctgtagcaggagcggtgttggccgattgcaggttagtggccaccgtgccgtactcttctgtggc


 


cactgggttggtggttttaatttcttcctcgttggttatcataacgttgtcaaggtccacgttgctatttccagctccctgtttcccaaatattaagact


 


ccgctcatcggaaaaaatttgtcttcgtcgtccttgtgggttgccatagcgggaccgggatttaccagagagtctctgccattcagatgatactt


 


ggtggcaccggtccaggcaaagttgctgttgttattttgattggttgtcttggagacgcgttgctgccggtagcagggcccgggtagccagttt


 


ttggcctgattcgccatgctactaggcccggcctgagaaaattgcaacgtccgatttcctgcggtaccactcgtggtctgagtccgagacag


 


gtagtacaggtactggtcgatgagggggttcatcagccggtccaggctttggctgtgcgcgtagctgctgtgaaaaggcacgtcctcaaac


 


gtgtagctgaactgaaagttgttgcccgttctcagcatttgagaaggaaagtattccaggcagtagaaggaggaacggcccacggcctgac


 


tgccattgttcagagtcaggtacccgtactgaggaatcatgaagacgtccgccgggaacggaggcaggcagccctggtgcgcagagccg


 


aggacgtacgggagctggtattccgagtccgtaaagacctgaaccgtgctggtaaggttattggcgatggtcgtggtgccatcattcgtcgt


 


gacctccttgacctggatgttgaagagcttgaagttgagcttcttgggccggaatccccagttgttgttgatgagtcgctgccagtcacgtggt


 


gagaagtggcagtggaatctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggtgttgtcgttggtgctgcctcccgattg


 


gctggagatttgcttgtagaggtggttgttgtaggtggggagggcccaggttcgggtgctggtggtgatgactctgtcgcccagccatgtgg


 


aatcgcaatgccaatttcctgaggcgttacccactccgtcggcgccttcgttattgtctgccattggagcgccaccgcctgcagccattgtatt


 


agatcccacaccagagggggctgcggggggttctccgagtggttgagggtcgggcactgactctgagtcgccagtctgcccaaagttga


 


gtctctttctcgcgggctgctggcctttcttgccgatgcccgaagaggagtctggttcctggggtgattgctctaccggtctcttctttccagga


 


gccgtatagcgccttcctcaaccagaccgagaggttcgagaacccgcttcttggcctggaagactgctcgcccgaggttgcccccaaaag


 


acgtatcttcttgcagacgctcctgaaactcggcgtcggcgtggttataccgcaggtacggattgtcacccgctttgagctgctggtcgtagg


 


ccttgtcgtgctcgagggccgctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttgaagggtccgaggtacttgtagccagg


 


aagcaccagaccccggccgtcgtcctgcttttgctggttggctttgggtttcggggctccaggtttcaagtcccaccactcgcgaatgccctc


 


agagaggttgtcctcgagccaatctggaagataaccatcggcagccatacctgatttaaatcatttattgttcaaagatgcagtcatccaaatc


 


cacattgaccagatcgcaggcagtgcaagcgtctggcacctttcccatgatatgatgaatgtagcacagtttctgatacgcctttttgacgaca


 


gaaacgggttgagattctgacacgggaaagcactctaaacagtctttctgtccgtgagtgaagcagatatttgaattctgattcattctctcgca


 


ttgtctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgttttggtacctgtctgcgtagttgatcgaagcttccgcgtctg


 


acgtcgatggctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtcactgggggcgggtcttttcttggctccaccctttttg


 


acgtagaattcatgctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgacttcctgcttggtgaccttcccaaagtcatgatc


 


cagacggcgggtgagttcaaatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttgagttcccgtcaatcacggcgcaca


 


tgttggtgttggaggtgacgatcacgggagtcgggtctatctgggccgaggacttgcatttctggtccacgcgcaccttgcttcctccgagaa


 


tggctttggccgactccacgaccttggcggtcatcttcccctcctcccaccagatcaccatcttgtcgacacagtcgttgaagggaaagttctc


 


attggtccagtttacgcacccgtagaagggcacagtgtgggctatggcctccgcgatgttggtcttcccggtagttgcaggcccaaacagcc


 


agatggtgttcctcttgccgaactttttcgtggcccatcccagaaagacggaagccgcatattggggatcgtacccgtttagttccaaaatttta


 


taaatccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtcgggggcggttttagtcaggctcataatctttcccgcattgt


 


ccaaggcagccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgaggcctggtcctcctggatccactgcttctccga


 


ggtaatccccttgtccacgagccacccgaccagctccatgtacctggctgaagtttttgatctgatcaccggcgcatcagaattgggattctga


 


ttctctttgttctgctcctgcgtctgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgtgagattcaaacaggcgctgtgga


 


gagaaaggcaaagtggatgtcagtaTCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAac


 


ttaccttaaatactgttccatattagtccacgcccactggagctcaggctgggttttggggagcaagtaattggggatgtagcactcatccacc


 


accttgttcccgcctccggcgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcgatcccgcggtaaattctctgaatca


 


gtttttcgcgaatctgactcaggaaacgtcccaaaaccatggatttcaccccggtggtttccacgagcacgtgcatgtggaagtagctctctcc


 


cttctcaaattgcacaaagaaaagagcctccggggccttactcacacggcgccattccgtcagaaagtcgcgctgcagcttctcggccacg


 


gtcaggggtgcctgctcaatcagattcagatccatgtcagaatctggcggcaactcccattccttctcggccacccagttcacaaagctgtca


 


gaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcacaatctcgtaaaaccccggcatGGCGGCTGCGCGTT


 


CAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCGGGCgaTCTCTATCACT


 


GATAGGGAGATCTCTATCACTGATAGGGAgaTTAAATAgaatggCTAggATCCGGCCGGc


 


cTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAG


 


CCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGTATGGCTTTTTCCCCGTAT


 


CCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGAT


 


CCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGC


 


GGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCG


 


GGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAG


 


CGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTAGA


 


TCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGAC


 


GGTATCGCCTCCAAGGCCAGCTTCCCACAATAAGTTGGGTGAATTTTGGCTCATTCC


 


TCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGT


 


CAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcAAACGCCAGCAACGCGGCCTTTTT


 


ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCAGCTGCGCGCT


 


CGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCG


 


CCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTA


 


GGGGTTCCTGCGGCCGCACGCGTGGAGCTAGTTATTAATAGTAATCAATTACGGGGT


 


CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC


 


CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTC


 


CCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGT


 


AAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG


 


ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGG


 


ACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCG


 


GTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAG


 


TCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTT


 


CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACG


 


GTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAC


 


GCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG


 


GATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAG


 


TGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTA


 


ATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCA


 


ATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAAT


 


TTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGT


 


AACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTG


 


CTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTT


 


GCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC


 


TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACATCGATTGAATTCT


 


GAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAG


 


CTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGA


 


TGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGT


 


GCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTA


 


CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT


 


CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG


 


TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC


 


AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA


 


ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATC


 


CGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC


 


CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC


 


CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG


 


TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGATCTCG


 


AGCTCAAGTAGGGATCCTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCAGCGCT


 


GCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCC


 


CTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATC


 


ATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGG


 


AGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACC


 


AAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCA


 


AGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAG


 


GCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTG


 


GTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGA


 


TTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCACGT


 


GCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGC


 


GCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT


 


GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGAT


 


GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCgtaGCtGATc


 


aATTgGCGCGCCGAATTCGTTAACAAGCTtTAATTAaCGCgtAcgATAAGCTTGATATCT


 


ATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACA


 


ATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCA


 


TTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAG


 


CACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGG


 


ATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTAC


 


GCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTT


 


TTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGC


 


CCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTG


 


CTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGT


 


GGCCATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTC


 


GTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCC


 


GAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCG


 


GTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAG


 


AAATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGG


 


TCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA


 


GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT


 


AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA


 


TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC


 


TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGC


 


TCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA


 


ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT


 


GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAG


 


TCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA


 


GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT


 


TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG


 


GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC


 


CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTA


 


TCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG


 


TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT


 


TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG


 


ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT


 


TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA


 


CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA


 


GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA


 


TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT


 


CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAAC


 


TACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC


 


CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG


 


CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG


 


GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT


 


ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC


 


AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT


 


TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA


 


TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC


 


TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC


 


TTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT


 


CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAG


 


ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC


 


ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA


 


TAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence of the vector illustrated in FIG. 10D is provided below.








PB007-iHelper2-iRepCap10-AAV-GFP/ PBB G10 (21,046 bp)



(SEQ ID NO: 14)



ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG



 


AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC


 


ATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCG


 


TTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATC


 


CCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC


 


AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA


 


TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG


 


GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC


 


GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG


 


GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC


 


GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT


 


TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG


 


ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGA


 


CGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGC


 


TCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTG


 


ACGTACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCTGTAT


 


ATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTACA


 


TACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAAA


 


CAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATCGAATTCCTGCAGCC


 


CGGGGGATCCACTAGTTCTAGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTA


 


ATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGT


 


CCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGG


 


GGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGC


 


AGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCCACAATAAGT


 


TGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATG


 


GGAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcGTTT


 


AAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCTGC


 


TTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAAT


 


AGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT


 


AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT


 


CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT


 


GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC


 


CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC


 


AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC


 


TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGA


 


CTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAA


 


CCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAAT


 


GGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTG


 


ATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTC


 


AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGAC


 


CGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCTTGCCACCatggccagtcgggaagagg


 


agcagcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgc


 


cgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctgg


 


tgccgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaa


 


gcccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaac


 


ccaccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcgga


 


cgcaagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtggga


 


gaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagt


 


ggaagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtga


 


cgatgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggct


 


gcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacg


 


agcgaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatc


 


tccaacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaagg


 


cgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgc


 


cactacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgc


 


ggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaacc


 


ctgtgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatgg


 


tgcgcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgt


 


gtccctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaacccgggagttctagggatctgcccctctccctcccccccc


 


cctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagg


 


gcccggaaacctggccctgtatcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaag


 


gaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgc


 


ctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaaga


 


gtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtg


 


cacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa


 


ggatccaccggaggccaccatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggttgtctcggcgcactccgt


 


acagtagggatcgtctacctccttttgagacagaaacccgcgctaccatactggaggatcatccgctgctgcccgaatgtaacactttgacaa


 


tgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccctgggatatggttctaacgcg


 


ggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagcatgatgatccatggttacga


 


gtcctgggctctccactgtcattgttccagtcccggttccctgcagtgtatagccggcgggcaggttttggccagctggtttaggatggtggtg


 


gatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtccagcgtgtttat


 


gaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatgagctttggatacagcgccttgca


 


ctgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaagg


 


cgccttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacggagcggcggcggcagcagttt


 


attcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtagGCGGCCGCTCGAGTCT


 


AGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA


 


TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG


 


TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT


 


TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC


 


AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG


 


GGGCTCTAGGGGGTATCCCCggggttggggttgcgccttttccaaggcATCCAGCACAGTGGCGGCC


 


GCaatatttgcatgtcgctatgtgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagacttataagttccctatcagtgatag


 


agaaccggtgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccg


 


tccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctcctttttgaattccactttg


 


gccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccggg


 


aaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccc


 


cggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctca


 


ctagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag


 


ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcg


 


gtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatctgt


 


gagtttggggacccttgattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgggaa


 


gatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtctcctcttattttcttttcattt


 


tctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcacttttgtttatttgtcagattgtaagtactttctctaatcact


 


tttttttcaaggcaatcagggtatattatattgtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcata


 


taaattctggctggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactg


 


tttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgct


 


ggttattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcgaGCCACCatggctagattagataaaagtaaagtgattaa


 


cagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggc


 


atgtaaaaaataagcgggctttgctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctg


 


gcaagattttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctacag


 


aaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcCttatatgcactcagcgcCgtggg


 


gcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaagggaaacacctactactgatagtatgccgccatta


 


ttacgacaagctatcgaattatttgatcaccaaggtgcagagccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaactt


 


aaatgtgaaagtgggtccccaaaaaagaagagaaaggtcgacggcggtggttcagtttaagcgtacagcggctcccgggagttctaggg


 


atctgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccata


 


ttgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgc


 


aaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaac


 


cccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgt


 


gagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgg


 


gatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttt


 


tcctttgaaaaacacgatgataaggatccaccggagGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacga


 


cgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgag


 


cgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtgg


 


cggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggct


 


ggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcc


 


cgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctgga


 


gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacct


 


ggtgcatgacccgcaagcccggtgcctgaCCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTT


 


GTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGC


 


TGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCT


 


TGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAAC


 


GTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCA


 


CCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGA


 


ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGA


 


CAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTT


 


GCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCA


 


GCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC


 


TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCaAAATGACCGAC


 


CAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAA


 


AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGG


 


GATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTT


 


ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT


 


CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTAGCtGATcaATTg


 


GCGCGCCGAATTCGTTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgcttcaaaatggagaccctgcgtg


 


ctcactcgggcttaaatacccagcgtgaccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaatacaggacctctagagca


 


tggaaactagataagaaagaaatacgcagagaccaaagttcaactgaaacgaattaaacggtttattgattaacaagcaaactagtttacaga


 


ttacgggtgaggtaacgggtgccgatggggcgaggctcagaataaacgccatttgtgtcaacagcaaagtccacatttgtagatttgttgtag


 


ttggaagtgtattgaatctctgggttccagcgtttgctgttttctttctgcagctcccattcaatttccacgctgacctgtccggtgctgtactgcgt


 


gatgaacgacgcaaacttagctggactgaaggtagttggaggattcgcgggaacaggtgtattcttaatcaggatctgaggaggcgggtgt


 


ttcagtccaaagcctcccatcagcggcgagggatgaaagtgtccgtccgtgtgaggaatcttggcccagataggaccctgcaggtacacgt


 


cccggtcctgccagaccatgccaggtaaggctccttgactgttgacggtccctgtagcaggagcggtgttggccgattgcaggttagtggc


 


caccgtgccgtactcttctgtggccactgggttggtggttttaatttcttcctcgttggttatcataacgttgtcaaggtccacgttgctatttccag


 


ctccctgtttcccaaatattaagactccgctcatcggaaaaaatttgtcttcgtcgtccttgtgggttgccatagcgggaccgggatttaccaga


 


gagtctctgccattcagatgatacttggtggcaccggtccaggcaaagttgctgttgttattttgattggttgtcttggagacgcgttgctgccgg


 


tagcagggcccgggtagccagtttttggcctgattcgccatgctactaggcccggcctgagaaaattgcaacgtccgatttcctgcggtacc


 


actcgtggtctgagtccgagacaggtagtacaggtactggtcgatgagggggttcatcagccggtccaggctttggctgtgcgcgtagctg


 


ctgtgaaaaggcacgtcctcaaacgtgtagctgaactgaaagttgttgcccgttctcagcatttgagaaggaaagtattccaggcagtagaa


 


ggaggaacggcccacggcctgactgccattgttcagagtcaggtacccgtactgaggaatcatgaagacgtccgccgggaacggaggc


 


aggcagccctggtgcgcagagccgaggacgtacgggagctggtattccgagtccgtaaagacctgaaccgtgctggtaaggttattggcg


 


atggtcgtggtgccatcattcgtcgtgacctccttgacctggatgttgaagagcttgaagttgagcttcttgggccggaatccccagttgttgtt


 


gatgagtcgctgccagtcacgtggtgagaagtggcagtggaatctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggt


 


gttgtcgttggtgctgcctcccgattggctggagatttgcttgtagaggtggttgttgtaggtggggagggcccaggttcgggtgctggtggt


 


gatgactctgtcgcccagccatgtggaatcgcaatgccaatttcctgaggcgttacccactccgtcggcgccttcgttattgtctgccattgga


 


gcgccaccgcctgcagccattgtattagatcccacaccagagggggctgcggggggttctccgagtggttgagggtcgggcactgactct


 


gagtcgccagtctgcccaaagttgagtctctttctcgcgggctgctggcctttcttgccgatgcccgaagaggagtctggttcctggggtgatt


 


gctctaccggtctcttctttccaggagccgtcttagcgccttcctcaaccagaccgagaggttcgagaacccgcttcttggcctggaagactg


 


ctcgcccgaggttgcccccaaaagacgtatcttcttgcagacgctcctgaaactcggcgtcggcgtggttataccgcaggtacggattgtca


 


cccgctttgagctgctggtcgtaggccttgtcgtgctcgagggccgctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttgaa


 


gggtccgaggtacttgtagccaggaagcaccagaccccggccgtcgtcctgcttttgctggttggctttgggtttcggggctccaggtttcaa


 


gtcccaccactcgcgaatgccctcagagaggttgtcctcgagccaatctggaagataaccatcggcagccatacctgatttaaatcatttattg


 


ttcaaagatgcagtcatccaaatccacattgaccagatcgcaggcagtgcaagcgtctggcacctttcccatgatatgatgaatgtagcacag


 


tttctgatacgcctttttgacgacagaaacgggttgagattctgacacgggaaagcactctaaacagtctttctgtccgtgagtgaagcagatat


 


ttgaattctgattcattctctcgcattgtctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgttttggtacctgtctgcgta


 


gttgatcgaagcttccgcgtctgacgtcgatggctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtcactgggggcgg


 


gtcttttcttggctccaccctttttgacgtagaattcatgctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgacttcctgcttg


 


gtgaccttcccaaagtcatgatccagacggcgggtgagttcaaatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttgag


 


ttcccgtcaatcacggcgcacatgttggtgttggaggtgacgatcacgggagtcgggtctatctgggccgaggacttgcatttctggtccac


 


gcgcaccttgcttcctccgagaatggctttggccgactccacgaccttggcggtcatcttcccctcctcccaccagatcaccatcttgtcgaca


 


cagtcgttgaagggaaagttctcattggtccagtttacgcacccgtagaagggcacagtgtgggctatggcctccgcgatgttggtcttcccg


 


gtagttgcaggcccaaacagccagatggtgttcctcttgccgaactttttcgtggcccatcccagaaagacggaagccgcatattggggatc


 


gtacccgtttagttccaaaattttataaatccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtcgggggcggttttagtc


 


aggctcataatctttcccgcattgtccaaggcagccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgaggcctggtc


 


ctcctggatccactgcttctccgaggtaatccccttgtccacgagccacccgaccagctccatgtacctggctgaagtttttgatctgatcacc


 


ggcgcatcagaattgggattctgattctctttgttctgctcctgcgtctgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgt


 


gagattcaaacaggcgctgtggagagaaaggcaaagtggatgtcagtaTCTCTATCACTGATAGGGAGATCTCT


 


ATCACTGATAGGGAacttaccttaaatactgttccatattagtccacgcccactggagctcaggctgggttttggggagcaagta


 


attggggatgtagcactcatccaccaccttgttcccgcctccggcgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcg


 


atcccgcggtaaattctctgaatcagtttttcgcgaatctgactcaggaaacgtcccaaaaccatggatttcaccccggtggtttccacgagca


 


cgtgcatgtggaagtagctctctcccttctcaaattgcacaaagaaaagagcctccggggccttactcacacggcgccattccgtcagaaag


 


tcgcgctgcagcttctcggccacggtcaggggtgcctgctcaatcagattcagatccatgtcagaatctggcggcaactcccattccttctcg


 


gccacccagttcacaaagctgtcagaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcacaatctcgtaaaaccccggca


 


tGGCGGCTGCGCGTTCAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCG


 


GGCgaTCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAgaTTAAATAgaatgg


 


CTAggATCCGGCCGGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGT


 


GGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGTAT


 


GGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGT


 


TCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTG


 


CCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCG


 


CTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGG


 


GGCTGTCCCTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTA


 


GTGAGGGTTAATTAGATCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCC


 


CCCCCTCGAGGTCGACGGTATCGCCTCCAAGGCCAGCTTCCCACAATAAGTTGGGTG


 


AATTTTGGCTCATTCCTCCTTTCTATAGGATTGAGGTCAGAGCTTTGTGATGGGAATT


 


CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCgcGATCgcTAGcAAACGCCAG


 


CAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGC


 


AGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCG


 


GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG


 


CCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGGAGCTAGTTATTAATAGT


 


AATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC


 


TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA


 


TAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGG


 


TGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA


 


GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT


 


ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT


 


TACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTC


 


ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGCACCAA


 


AATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGG


 


CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTC


 


AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGAC


 


CGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGA


 


TTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAA


 


AATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATC


 


TCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAG


 


AATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATT


 


TCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAA


 


TCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTC


 


CAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCT


 


GGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGA


 


ACATCGATTGAATTCTGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT


 


GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCG


 


GCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC


 


ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG


 


CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC


 


ATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA


 


CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGC


 


TGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC


 


AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAA


 


GGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC


 


ACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC


 


TACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACAT


 


GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTA


 


CAAGTACTCAGATCTCGAGCTCAAGTAGGGATCCTCTAGAGTCGACCTGCAGAAGCT


 


TGCCTCGAGCAGCGCTGCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCC


 


AGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAAT


 


AAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGG


 


AGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCG


 


GGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCT


 


CCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCA


 


GGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCAC


 


CATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCC


 


TCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATT


 


TTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTG


 


GCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC


 


CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCT


 


GCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG


 


CATACGTCgtaGCtGATcaATTgGCGCGCCGAATTCGTTAACAAGCTtTAATTAaCGCgtAc


 


gATAAGCTTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTA


 


GAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAA


 


AGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACAC


 


TTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCT


 


AAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAAT


 


GCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGAT


 


CATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCA


 


TCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAG


 


TTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATG


 


ATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCC


 


ACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCG


 


AAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGT


 


GCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATC


 


CTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGC


 


GCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT


 


TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG


 


TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGC


 


GGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG


 


GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA


 


AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA


 


AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG


 


GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG


 


GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG


 


TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC


 


CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC


 


GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG


 


CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA


 


CTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA


 


GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG


 


TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC


 


TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC


 


ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT


 


AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC


 


AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC


 


CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA


 


TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCA


 


GCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT


 


ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC


 


GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCAT


 


TCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA


 


AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT


 


TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAG


 


ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG


 


GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA


 


GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGA


 


TCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC


 


AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC


 


CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






Sequences of additional vectors for use in the practice of the present invention:








iRepCap1/pKan-Anc80-RepCap-p5i1-p19i1 (10,497 bp)



(SEQ ID NO: 15)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGATCTCCCTATC



 


AGTGATAGAGAtcGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTT


 


GAACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttct


 


gacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctga


 


ccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaag


 


ggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaa


 


actgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcggga


 


acaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaaca


 


gtaCCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaac


 


aaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggaca


 


aggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatca


 


aggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacattt


 


ccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcg


 


gcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacg


 


ggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgcca


 


aggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgact


 


cccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccgg


 


atgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggat


 


cacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcc


 


caaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttc


 


tcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaa


 


agactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatggg


 


aaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggct


 


gccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaaccca


 


aagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggg


 


ggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacct


 


gcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggc


 


caagaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcacccc


 


aggaaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcaga


 


gtcagtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctcc


 


aatggcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtca


 


tcaccaccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaac


 


gacaacacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactc


 


atcaacaacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcacc


 


acgaccatcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagg


 


gctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcct


 


ccttctactgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagca


 


gctacgcgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtg


 


gtaccgcaggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgct


 


accggcagcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggca


 


gagactctctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttggga


 


aacagggagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccaca


 


gaagagtacggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctgg


 


catggtctggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctg


 


atgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagct


 


aagtttgcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctgg


 


aacccagagattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccat


 


cggcacccgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttct


 


ttcttatctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaa


 


gcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttgggg


 


ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgg


 


gtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccct


 


aagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagc


 


gccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccg


 


ggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg


 


gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATA


 


TCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCG


 


GCATAGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGA


 


AGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTG


 


AAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTG


 


TCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTG


 


CTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACA


 


GGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTG


 


GGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGT


 


GAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCA


 


GGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGG


 


CTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT


 


GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA


 


AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT


 


GGTTTGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGA


 


AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT


 


GCGTATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatcccca


 


attacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgTCCCTATCAGTG


 


ATAGAGATCTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTT


 


TAAACgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtac


 


atggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatg


 


cggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggt


 


gggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctt


 


tctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggagg


 


ccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggt


 


gggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgca


 


agtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcga


 


acaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtc


 


aaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcc


 


cccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaacta


 


cgcagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaatt


 


caaatatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcag


 


aaactgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttga


 


acaataaACTAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGG


 


TCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcg


 


gccgctcgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaa


 


attccacagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggcca


 


acgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg


 


gtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaa


 


ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtca


 


gaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta


 


ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctcc


 


aagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacg


 


acttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa


 


ctacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaa


 


acaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta


 


cggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaa


 


aaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctc


 


tctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaact


 


gcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatg


 


gcaaggtcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgaga


 


aatcaccatgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttattccagacttgttcaacaggccagccattacgctcgtc


 


atcaaaatcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattaca


 


aacaggaatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaagg


 


ctgttttcccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtc


 


agccagttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccat


 


acaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcgg


 


acgggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattttt


 


atcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaa


 


taataaaaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacg


 


gcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatag


 


gccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaag


 


aacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtc


 


gaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaag


 


gaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctt


 


aatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcc


 


agctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtg


 


agcgcgcggcgaattgggtaccgggccccc


 


iRepCap2/ pKan-Anc80-RepCap-p5i2-p19i1 (10,495 bp)


(SEQ ID NO: 16)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGAtcGCCCGAGT



 


GAGCACGCAGGGTCTCCATTTTGATCCCTATCAGTGATAGAGAAGCGGGAGGTTTGA


 


ACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgac


 


agctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccg


 


tggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttat


 


ctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgTCCCTATCAGTGATAG


 


AGATCTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAA


 


Cgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggag


 


ctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcct


 


ccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggcc


 


agcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctggg


 


atgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatag


 


cccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggag


 


gaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcct


 


cggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacacc


 


agcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaaga


 


ctttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagt


 


gacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcaga


 


caggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatat


 


ctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgt


 


gctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataa


 


ACTAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTC


 


TGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgct


 


cgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattcca


 


cagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcg


 


cggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatca


 


gctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca


 


ggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg


 


gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat


 


acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg


 


ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatc


 


gccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacgg


 


ctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaac


 


caccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggt


 


ctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatga


 


agttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctag


 


tatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaattt


 


attcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaagg


 


tcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcacc


 


atgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaa


 


tcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacagg


 


aatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttc


 


ccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccag


 


ttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagc


 


gatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacggg


 


agcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgt


 


gcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataa


 


aaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacat


 


ttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccga


 


aatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgt


 


ggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg


 


tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaag


 


ggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatg


 


cgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct


 


ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcg


 


cgcggcgaattgggtaccgggccccc


 


iRepCap3/ pKan-Anc80-RepCap-p5i3-p19i1 (10,493 bp)


(SEQ ID NO: 17)



cctcgaggGCTAGCcattcTCCCTATCAGTGATAGAGAtcTATTTAAGCCCGAGTG



 


AGCACGCAGTCCCTATCAGTGATAGAGAGGTCTCCATTTTGAAGCGGGAGGTTTGAA


 


CGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgaca


 


gctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgt


 


ggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttat


 


ctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgTCCCTATCAGTGATAG


 


AGATCTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAA


 


Cgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggag


 


ctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcct


 


ccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggcc


 


agcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctggg


 


atgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatag


 


cccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggag


 


gaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcct


 


cggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacacc


 


agcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaaga


 


ctttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagt


 


gacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcaga


 


caggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatat


 


ctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgt


 


gctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataa


 


ACTAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTC


 


TGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgct


 


cgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattcca


 


cagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcg


 


cggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatca


 


gctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca


 


ggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg


 


gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat


 


acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg


 


ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatc


 


gccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacgg


 


ctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaac


 


caccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggt


 


ctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatga


 


agttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctag


 


tatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaattt


 


attcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaagg


 


tcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcacc


 


atgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaa


 


tcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacagg


 


aatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttc


 


ccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccag


 


ttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagc


 


gatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacggg


 


agcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgt


 


gcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataa


 


aaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacat


 


ttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccga


 


aatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgt


 


ggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg


 


tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaag


 


ggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatg


 


cgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagct


 


ggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcg


 


cgcggcgaattgggtaccgggccccc


 


iRepCap4/ pKan-Anc80-RepCap-p5i1-p19i2 (10,495 bp)


(SEQ ID NO: 18)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGATCTCCCTATC



 


AGTGATAGAGAtcGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTT


 


GAACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttct


 


gacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctga


 


ccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaag


 


ggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaa


 


actgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcggga


 


acaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaaca


 


gtaCCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaac


 


aaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggaca


 


aggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatca


 


aggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacattt


 


ccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcg


 


gcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacg


 


ggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgcca


 


aggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgact


 


cccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccgg


 


atgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggat


 


cacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcc


 


caaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttc


 


tcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaa


 


agactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatggg


 


aaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggct


 


gccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaaccca


 


aagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggg


 


ggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacct


 


gcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggc


 


caagaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcacccc


 


aggaaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcaga


 


gtcagtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctcc


 


aatggcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtca


 


tcaccaccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaac


 


gacaacacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactc


 


atcaacaacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcacc


 


acgaccatcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagg


 


gctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcct


 


ccttctactgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagca


 


gctacgcgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtg


 


gtaccgcaggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgct


 


accggcagcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggca


 


gagactctctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttggga


 


aacagggagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccaca


 


gaagagtacggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctgg


 


catggtctggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctg


 


atgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagct


 


aagtttgcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctgg


 


aacccagagattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccat


 


cggcacccgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttct


 


ttcttatctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaa


 


gcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttgggg


 


ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgg


 


gtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccct


 


aagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagc


 


gccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccg


 


ggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg


 


gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATA


 


TCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCG


 


GCATAGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGA


 


AGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTG


 


AAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTG


 


TCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTG


 


CTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACA


 


GGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTG


 


GGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGT


 


GAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCA


 


GGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGG


 


CTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT


 


GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA


 


AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT


 


GGTTTGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGA


 


AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT


 


GCGTATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatcccca


 


attacttgctccccaaaacccagcctgagTCCCTATCAGTGATAGAGActccagtgggcgtggactaatatggaacagtat


 


ttaagcgcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTA


 


AACgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatg


 


gagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcg


 


gcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtg


 


ggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttc


 


tgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggcc


 


atagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgg


 


gaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaag


 


tcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaac


 


accagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaa


 


agactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccc


 


cagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacg


 


cagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaa


 


atatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaa


 


ctgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaaca


 


ataaACTAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTC


 


TCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggcc


 


gctcgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattc


 


cacagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacg


 


cgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat


 


cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc


 


caggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagag


 


gtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg


 


gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaag


 


ctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactt


 


atcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac


 


ggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaa


 


accaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg


 


ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaa


 


tgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctc


 


tagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgca


 


atttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggca


 


aggtcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaat


 


caccatgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatc


 


aaaatcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaa


 


caggaatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggct


 


gttttcccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtca


 


gccagttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccata


 


caagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcgga


 


cgggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattttta


 


tcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaat


 


aataaaaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacgg


 


cacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatagg


 


ccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaaga


 


acgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcg


 


aggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaagg


 


aagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctta


 


atgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcca


 


gctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtga


 


gcgcgcggcgaattgggtaccgggccccc


 


iRepCap5/ pKan-Anc80-RepCap-p5i2-p19i2 (10,493 bp)


(SEQ ID NO: 19)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGAtcGCCCGAGT



 


GAGCACGCAGGGTCTCCATTTTGATCCCTATCAGTGATAGAGAAGCGGGAGGTTTGA


 


ACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgac


 


agctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccg


 


tggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgatgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttatct


 


agtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagTCCCTATCAGTGATAGAGActccagtgggcgtggactaatatggaacagtatttaagc


 


gcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAACgc


 


agacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctgg


 


tcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcctccaa


 


ctcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagca


 


gcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgg


 


gccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagccca


 


cactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggagg


 


ggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggc


 


ccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagca


 


gccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagacttttt


 


ccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgac


 


gcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacag


 


gtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgc


 


ttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgcta


 


cattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaAC


 


TAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTG


 


CGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgctcga


 


ctagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattccacag


 


cctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg


 


ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct


 


cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga


 


accgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg


 


aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc


 


tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc


 


tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc


 


actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctac


 


actagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc


 


gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctga


 


cgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttt


 


taaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctagtatat


 


aaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaatttattca


 


tatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaaggtcctg


 


gtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatga


 


gtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcac


 


tcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacaggaatc


 


gaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttccca


 


ggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccagttg


 


agacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgat


 


agattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacgggagc


 


aagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgca


 


atgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataaaaa


 


agccggattaataatctggattttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacatttcc


 


ccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatc


 


ggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggac


 


tccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgcc


 


gtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaaggga


 


agaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgc


 


cgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggc


 


gaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgc


 


ggcgaattgggtaccgggccccc


 


iRepCap6/ pKan-Anc80-RepCap-p5i3-p19i2 (10,491 bp)


(SEQ ID NO: 20)



cctcgaggGCTAGCcattcTCCCTATCAGTGATAGAGAtcTATTTAAGCCCGAGTG



 


AGCACGCAGTCCCTATCAGTGATAGAGAGGTCTCCATTTTGAAGCGGGAGGTTTGAA


 


CGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgaca


 


gctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgt


 


ggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttat


 


ctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagTCCCTATCAGTGATAGAGActccagtgggcgtggactaatatggaacagtatttaagc


 


gcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAACgc


 


agacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctgg


 


tcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcctccaa


 


ctcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagca


 


gcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgg


 


gccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagccca


 


cactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggagg


 


ggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggc


 


ccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagca


 


gccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagacttttt


 


ccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgac


 


gcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacag


 


gtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgc


 


ttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgcta


 


cattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaAC


 


TAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTG


 


CGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgctcga


 


ctagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattccacag


 


cctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg


 


ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct


 


cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga


 


accgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg


 


aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc


 


tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc


 


tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc


 


actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctac


 


actagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc


 


gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctga


 


cgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttt


 


taaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctagtatat


 


aaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaatttattca


 


tatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaaggtcctg


 


gtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatga


 


gtgacgactgaatccggtgagaatggcaagagcttgtgcatttattccagacttgttcaacaggccagccattacgctcgtcatcaaaatcac


 


tcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacaggaatc


 


gaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttccca


 


ggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccagttg


 


agacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgat


 


agattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacgggagc


 


aagacgtttcccgttgaatatggctcataacacccactgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgca


 


atgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataaaaa


 


agccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacatttcc


 


ccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatc


 


ggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggac


 


tccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgcc


 


gtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaaggga


 


agaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgc


 


cgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggc


 


gaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgc


 


ggcgaattgggtaccgggccccc


 


iRepCap7/ pKan-Anc80-RepCap-p5i1-p19i3 (10,495 bp)


(SEQ ID NO: 21)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGATCTCCCTATC



 


AGTGATAGAGAtcGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTT


 


GAACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttct


 


gacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctga


 


ccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaag


 


ggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaa


 


actgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcggga


 


acaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaaca


 


gtaCCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaac


 


aaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggaca


 


aggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatca


 


aggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacattt


 


ccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcg


 


gcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacg


 


ggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgcca


 


aggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgact


 


cccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccgg


 


atgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggat


 


cacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcc


 


caaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttc


 


tcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaa


 


agactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatggg


 


aaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggct


 


gccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaaccca


 


aagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggg


 


ggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacct


 


gcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggc


 


caagaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcacccc


 


aggaaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcaga


 


gtcagtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctcc


 


aatggcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtca


 


tcaccaccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaac


 


gacaacacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactc


 


atcaacaacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcacc


 


acgaccatcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagg


 


gctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcct


 


ccttctactgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagca


 


gctacgcgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtg


 


gtaccgcaggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgct


 


accggcagcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggca


 


gagactctctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttggga


 


aacagggagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccaca


 


gaagagtacggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctgg


 


catggtctggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctg


 


atgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagct


 


aagtttgcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctgg


 


aacccagagattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccat


 


cggcacccgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttct


 


ttcttatctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaa


 


gcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttgggg


 


ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgg


 


gtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccct


 


aagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagc


 


gccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccg


 


ggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccg


 


gagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATA


 


TCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCG


 


GCATAGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGA


 


AGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTG


 


AAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTG


 


TCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTG


 


CTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACA


 


GGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTG


 


GGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGT


 


GAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCA


 


GGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGG


 


CTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCAT


 


GCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA


 


AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT


 


GGTTTGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGA


 


AACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT


 


GCGTATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatcccca


 


attacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaaTCCCTATCAGTGATAGAGAcagtat


 


ttaagcgcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTA


 


AACgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatg


 


gagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcg


 


gcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtg


 


ggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttc


 


tgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggcc


 


atagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgg


 


gaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaag


 


tcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaac


 


accagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaa


 


agactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccc


 


cagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacg


 


cagacaggtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaa


 


atatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaa


 


ctgtgctacattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaaca


 


ataaACTAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTC


 


TCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggcc


 


gctcgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattc


 


cacagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacg


 


cgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat


 


cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc


 


caggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagag


 


gtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg


 


gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaag


 


ctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactt


 


atcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac


 


ggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaa


 


accaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg


 


ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaa


 


tgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctc


 


tagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgca


 


atttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggca


 


aggtcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaat


 


caccatgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatc


 


aaaatcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaa


 


caggaatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggct


 


gttttcccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtca


 


gccagttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccata


 


caagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcgga


 


cgggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatattttta


 


tcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaat


 


aataaaaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacgg


 


cacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatagg


 


ccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaaga


 


acgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcg


 


aggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaagg


 


aagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgctta


 


atgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcca


 


gctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtga


 


gcgcgcggcgaattgggtaccgggccccc


 


iRepCap8/ pKan-Anc80-RepCap-p5i2-p19i3 (10,493 bp)


(SEQ ID NO: 22)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGAtcGCCCGAGT



 


GAGCACGCAGGGTCTCCATTTTGATCCCTATCAGTGATAGAGAAGCGGGAGGTTTGA


 


ACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgac


 


agctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccg


 


tggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgatgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttatct


 


agtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagctccagtgggcgtggactaatatggaaTCCCTATCAGTGATAGAGAcagtatttaagc


 


gcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAACgc


 


agacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctgg


 


tcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcctccaa


 


ctcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagca


 


gcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgg


 


gccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagccca


 


cactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggagg


 


ggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggc


 


ccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagca


 


gccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagacttttt


 


ccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgac


 


gcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacag


 


gtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgc


 


ttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgcta


 


cattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaAC


 


TAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTG


 


CGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgctcga


 


ctagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattccacag


 


cctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg


 


ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct


 


cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga


 


accgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg


 


aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc


 


tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc


 


tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc


 


actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctac


 


actagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc


 


gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctga


 


cgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttt


 


taaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctagtatat


 


aaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaatttattca


 


tatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaaggtcctg


 


gtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatga


 


gtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcac


 


tcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacaggaatc


 


gaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttccca


 


ggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccagttg


 


agacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgat


 


agattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacgggagc


 


aagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgca


 


atgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataaaaa


 


agccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacatttcc


 


ccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatc


 


ggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggac


 


tccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgcc


 


gtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaaggga


 


agaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgc


 


cgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggc


 


gaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgc


 


ggcgaattgggtaccgggccccc


 


iRepCap9/ pKan-Anc80-RepCap-p5i3-p19i3 (10,491 bp)


(SEQ ID NO: 23)



cctcgaggGCTAGCcattcTCCCTATCAGTGATAGAGAtcTATTTAAGCCCGAGTG



 


AGCACGCAGTCCCTATCAGTGATAGAGAGGTCTCCATTTTGAAGCGGGAGGTTTGAA


 


CGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgaca


 


gctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgt


 


ggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaaggga


 


gagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaact


 


gattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaaca


 


aggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccaAtgggcAtggacCaaCatggaacagta


 


CCtCagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaa


 


gagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaagg


 


ggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaagg


 


ctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttcca


 


gcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggca


 


agaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggt


 


gcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaagg


 


tcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactccc


 


gtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgt


 


tcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcac


 


gtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaa


 


acgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgttctcg


 


tcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggacagaaaga


 


ctgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaa


 


ggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtatggctgcc


 


gatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaacccaaag


 


ccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaaggggga


 


gcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcg


 


gtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaa


 


gaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcaccccagg


 


aaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactcagagtca


 


gtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcgctccaatg


 


gcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacagagtcatcac


 


caccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcaccaacgaca


 


acacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaa


 


caacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggcaccacgac


 


catcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcaccagggctgcc


 


tgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgttcctccttcta


 


ctgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcacagcagctacg


 


cgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacgagtggtaccg


 


caggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccctgctaccggc


 


agcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatggcagagactc


 


tctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgggaaacagg


 


gagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggccacagaagagt


 


acggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacctggcatggtct


 


ggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgctgatgggag


 


gctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccagctaagtttgc


 


gtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgctggaacccag


 


agattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccccatcggcacc


 


cgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttat


 


ctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccga


 


gtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatcggggttggggttgcgc


 


cttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcg


 


cacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcg


 


ggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccag


 


ggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaag


 


gggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgc


 


acgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagAAGCTCCCGGGAGCTTGTATATCCA


 


TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT


 


AGTATAATACGACAAGGTGAGGAACgccaccATGGCCAAGCCTTTGTCTCAAGAAGAA


 


TCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGAC


 


TACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAAT


 


GTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCT


 


GCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGG


 


CATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGAT


 


CAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAAT


 


TGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGAC


 


TGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTC


 


GGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTG


 


GAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGC


 


AATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTT


 


TGTCCAAACTCATCAATGTATCTTAGCGCTCACTGCCCGCTTTCCAGTCGGGAAACC


 


TGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGT


 


ATTGGGCGCTCTCCTAGGccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt


 


gctccccaaaacccagcctgagctccagtgggcgtggactaatatggaaTCCCTATCAGTGATAGAGAcagtatttaagc


 


gcctgTCCCTATCAGTGATAGAGAtttgaatctcacggagcgtaaacggttggtggcgcagcatctGTTTAAACgc


 


agacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctgg


 


tcgggtggctcgtggacaaggggattacctcggagaagcagtggattcaggaggaccaggcctcatacatctccttcaatgcggcctccaa


 


ctcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagca


 


gcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgg


 


gccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagccca


 


cactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggagg


 


ggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggc


 


ccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagca


 


gccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagacttttt


 


ccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgac


 


gcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacag


 


gtaccaaaacaaatgttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgc


 


ttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgcta


 


cattcatcatatcatgggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaAC


 


TAGTTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTG


 


CGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGTATACgatatccatcacactggcggccgctcga


 


ctagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattccacag


 


cctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg


 


ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct


 


cactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga


 


accgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg


 


aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc


 


tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc


 


tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc


 


actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctac


 


actagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc


 


gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctga


 


cgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttt


 


taaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttatattctctctctagtatat


 


aaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatgaaactgcaatttattca


 


tatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaagaatggcaaggtcctg


 


gtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatga


 


gtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcac


 


tcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaattacaaacaggaatc


 


gaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaaggctgttttccca


 


ggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaactccgtcagccagttg


 


agacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgat


 


agattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaagcgcggacgggagc


 


aagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgca


 


atgtaacatcagagattttgagacacaacgtggattgttgaataaatcgaacttttgctgagttgaaggatcagctctagtaaaataataaaaa


 


agccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgacggcacatttcc


 


ccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatc


 


ggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggac


 


tccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgcc


 


gtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaaggga


 


agaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgc


 


cgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggc


 


gaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgc


 


ggcgaattgggtaccgggccccc


 


iRepCap10/ pKan-Anc80-intron-inducibleRepCap (7,567 bp)


(SEQ ID NO: 24)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGATCTCCCTATC



 


AGTGATAGAGAtcGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTT


 


GAACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttct


 


gacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctga


 


ccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaag


 


ggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaa


 


actgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcggga


 


acaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtat


 


ttaaggtaagtTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGAtactgacatccactttgc


 


ctttctctccacagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcaga


 


acaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtgga


 


caaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaat


 


caaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggaca


 


tttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttc


 


ggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctac


 


gggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgcc


 


aaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccga


 


ctcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagacc


 


ggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaag


 


gatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtga


 


gcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaat


 


gttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggaca


 


gaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcat


 


gggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtat


 


ggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaa


 


cccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaa


 


gggggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgt


 


acctgcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttcca


 


ggccaagaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcac


 


cccaggaaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactc


 


agagtcagtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcg


 


ctccaatggcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacaga


 


gtcatcaccaccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcac


 


caacgacaacacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcg


 


actcatcaacaacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggc


 


accacgaccatcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcacca


 


gggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgtt


 


cctccttctactgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcaca


 


gcagctacgcgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacga


 


gtggtaccgcaggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccct


 


gctaccggcagcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatgg


 


cagagactctctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgg


 


gaaacagggagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggcca


 


cagaagagtacggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacct


 


ggcatggtctggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgc


 


tgatgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccag


 


ctaagtttgcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgct


 


ggaacccagagattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccc


 


catcggcacccgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtat


 


ttctttcttatctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatt


 


taagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatccatcacac


 


tggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgt


 


gtgaaattccacagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatc


 


ggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggc


 


gagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccag


 


caaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca


 


agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc


 


cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt


 


cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag


 


acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg


 


cctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccg


 


gcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt


 


ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaa


 


attaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttata


 


ttctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatga


 


aactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaag


 


aatggcaaggtcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt


 


gagaaatcaccatgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttattccagacttgttcaacaggccagccattacgc


 


tcgtcatcaaaatcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaa


 


ttacaaacaggaatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctgg


 


aaggctgttttcccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaact


 


ccgtcagccagttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggctt


 


cccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaag


 


cgcggacgggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgat


 


atatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctcta


 


gtaaaataataaaaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagt


 


gtgacggcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaa


 


ccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccact


 


attaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttt


 


tggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggc


 


gagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccg


 


ccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgct


 


attacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacg


 


gccagtgagcgcgcggcgaattgggtaccgggccccc


 


iRepCap11/ pKan-Anc80-intron-inducibleRepCap-d2 (7,567 bp)


(SEQ ID NO: 25)



cctcgaggGCTAGCcattcTATTTAAtcTCCCTATCAGTGATAGAGATCTCCCTATC



 


AGTGATAGAGAtcGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTT


 


GAACGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttct


 


gacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctga


 


ccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggctcttttctttgtgcaatttgagaag


 


ggagagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaa


 


actgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcggga


 


acaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtat


 


ttaagcgcctgtttgaatctcacggaaaggtaagtTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATA


 


GAGAtactgacatccactttgcctttctctccacaggaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcaga


 


acaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtgga


 


caaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaat


 


caaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggaca


 


tttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttc


 


ggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctac


 


gggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgcc


 


aaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccga


 


ctcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagacc


 


ggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaag


 


gatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtga


 


gcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaat


 


gttctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattcaaatatctgcttcactcacggaca


 


gaaagactgtttagagtgctttcccgtgtcagaatctcaacccgtttctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcat


 


gggaaaggtgccagacgcttgcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgatttaaatcaggtat


 


ggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttgaaacctggagccccgaaa


 


cccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaagtacctcggacccttcaacggactcgacaa


 


gggggagcccgtcaacgcggcggacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaagcgggtgacaatccgt


 


acctgcggtataaccacgccgacgccgagtttcaggagcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttcca


 


ggccaagaagcgggttctcgaacctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagcaatcac


 


cccaggaaccagactcctcttcgggcatcggcaagaaaggccagcagcccgcgagaaagagactcaactttgggcagactggcgactc


 


agagtcagtgcccgaccctcaaccactcggagaaccccccgcagccccctctggtgtgggatctaatacaatggctgcaggcggtggcg


 


ctccaatggcagacaataacgaaggcgccgacggagtgggtaacgcctcaggaaattggcattgcgattccacatggctgggcgacaga


 


gtcatcaccaccagcacccgaacctgggccctccccacctacaacaaccacctctacaagcaaatctccagccaatcgggaggcagcac


 


caacgacaacacctacttcggctacagcaccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcg


 


actcatcaacaacaactggggattccggcccaagaagctcaacttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgatggc


 


accacgaccatcgccaataaccttaccagcacggttcaggtctttacggactcggaataccagctcccgtacgtcctcggctctgcgcacca


 


gggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtacctgactctgaacaatggcagtcaggccgtgggccgtt


 


cctccttctactgcctggaatactttccttctcaaatgctgagaacgggcaacaactttcagttcagctacacgtttgaggacgtgccttttcaca


 


gcagctacgcgcacagccaaagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagaccacga


 


gtggtaccgcaggaaatcggacgttgcaattttctcaggccgggcctagtagcatggcgaatcaggccaaaaactggctacccgggccct


 


gctaccggcagcaacgcgtctccaagacaaccaatcaaaataacaacagcaactttgcctggaccggtgccaccaagtatcatctgaatgg


 


cagagactctctggtaaatcccggtcccgctatggcaacccacaaggacgacgaagacaaattttttccgatgagcggagtcttaatatttgg


 


gaaacagggagctggaaatagcaacgtggaccttgacaacgttatgataaccaacgaggaagaaattaaaaccaccaacccagtggcca


 


cagaagagtacggcacggtggccactaacctgcaatcggccaacaccgctcctgctacagggaccgtcaacagtcaaggagccttacct


 


ggcatggtctggcaggaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggacactttcatccctcgccgc


 


tgatgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgaatcctccaactaccttcagtccag


 


ctaagtttgcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaaattgaatgggagctgcagaaagaaaacagcaaacgct


 


ggaacccagagattcaatacacttccaactacaacaaatctacaaatgtggactttgctgttgacacaaatggcgtttattctgagcctcgccc


 


catcggcacccgttacctcacccgtaatctgtaaactagtttgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtat


 


ttctttcttatctagtttccatgctctagaggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatt


 


taagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccaagccgaattctgcagatatccatcacac


 


tggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttcgcgcgcttggcgtaatcatggtcatagctgtttcctgt


 


gtgaaattccacagcctggggtgcctaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatc


 


ggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggc


 


gagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccag


 


caaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca


 


agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgc


 


cgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt


 


cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag


 


acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg


 


cctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccg


 


gcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt


 


ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaa


 


attaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagaaataataaaaaagccggattaataatctggctttttata


 


ttctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagtgtgactctagtattattagaaaaactcatcgagcatcaaatga


 


aactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccaaag


 


aatggcaaggtcctggtaacggtctgcgattccgacccgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagt


 


gagaaatcaccatgagtgacgactgaatccggtgagaatggcaagagcttgtgcatttctttccagacttgttcaacaggccagccattacgc


 


tcgtcatcaaaatcactcgcatcaaccaaaccgttattcatgcgtgattgcgcctgagcaagacgaaatacacgatcgctgttaaaaggacaa


 


ttacaaacaggaatcgaatgtaaccggcgcaggaacacggccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctgg


 


aaggctgttttcccaggaatcgcggtggtgagtaaccacgcatcatcaggagtacggataaaatgcttgatggtcgggagaggcataaact


 


ccgtcagccagttgagacggaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggctt


 


cccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcgtccatgttggagtttaag


 


cgcggacgggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgat


 


atatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcagctcta


 


gtaaaataataaaaaagccggattaataatctggctttttatattctctctctagtatataaacgcagaaaggcccacccgaaggtgagccagt


 


gtgacggcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaa


 


ccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccact


 


attaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttt


 


tggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggc


 


gagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccg


 


ccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgct


 


attacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacg


 


gccagtgagcgcgcggcgaattgggtaccgggccccc






The sequence for the PBBG-iHelper-Puro construct illustrated in in FIG. 11A is shown below:







PBBG-iHelper-Puro (11,801 bp)


(SEQ ID NO: 26)


ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA


 


TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT


 


CCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATT


 


TTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCA


 


ATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA


 


TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC


 


GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC


 


ACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA


 


AAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG


 


GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC


 


GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCA


 


CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC


 


AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT


 


ACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA


 


CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGC


 


GCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGG


 


TGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGC


 


TGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACG


 


TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCT


 


GTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATA


 


TTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTT


 


ATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA


 


CAAAACTTTTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAG


 


GGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCG


 


CTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCC


 


CCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAA


 


GGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCC


 


TGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCC


 


ACAATAAGTTGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAG


 


GTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGTCAGTTAGGGTGT


 


GGAAAGTCCCgcGATCgcTAGcGTTTAAACTTAAGCTTGGTACCGAGCTC


 


GGATCCACTAGTCCAGTGTGGTGGAATTCCTGCTTCGCGATGTACGGGCC


 


AGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAAT


 


TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAAC


 


TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG


 


ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA


 


TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC


 


ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT


 


AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC


 


TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC


 


GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGA


 


TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACCA


 


AAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC


 


AAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTC


 


CCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGC


 


TCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTG


 


ACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTT


 


AAACTTAAGCTTGCCACCatggccagtcgggaagaggagcagcgcgaaac


 


cacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggagg


 


acgtgtcgtccccgtccccgtcgccgccgcctccccgggcgcccccaaaa


 


aagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcaca


 


agacgcgctggtgccgcgcacacccagcccgcggccatcgacctcggcgg


 


cggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaag


 


cccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaag


 


agaagatgtggcgctacaaatggtgggtttcagcaacccaccggtgctaa


 


tcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagac


 


gacccagtggcgcgtggtatgcggacgcaagaggaagaggaagagcccag


 


cgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcg


 


tgtctgcgtgggagaagggcatggaggctgcgcgcgcgctgatggacaag


 


taccacgtggataacgatctaaaggcgaacttcaaactactgcctgacca


 


agtggaagctctggcggccgtatgcaagacctggctgaacgaggagcacc


 


gcgggttgcagctgaccttcaccagcaacaagacctttgtgacgatgatg


 


gggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaa


 


gcatcacgagcccacgggctgcgcgttgtggctgcaccgctgcgctgaga


 


tcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggag


 


cacgtgattgaaatggatgtgacgagcgaaaacgggcagcgcgcgctgaa


 


ggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatg


 


tggtgcagatctccaacaccgacgcaaggtgctgcgtgcacgacgcggcc


 


tgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctga


 


aggcgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcagg


 


cgctgtatcctaacgcccagaccgggcacggtcaccttttgatgccacta


 


cggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggca


 


gctaccaaagttgactccgttcgccctgagcaacgcggaggacctggacg


 


cggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcg


 


ctgatagtgttccagtgctgcaaccctgtgtatcgcaactcgcgcgcgca


 


gggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaa


 


acgcgttggtgatggtgcgcagcctgtggagtgaaaacttcaccgagctg


 


ccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcg


 


caacgtgtccctgccagtggcgcatagcgatgcgcggcagaacccctttg


 


atttttaacccgggagttctagggatctgcccctctccctcccccccccc


 


taacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtct


 


atatgttattttccaccatattgccgtatttggcaatgtgagggcccgga


 


aacctggccctgtatcttgacgagcattcctaggggtctttcccctctcg


 


ccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctg


 


gaagatcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcgg


 


aaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtata


 


agatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttgga


 


tagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaagggg


 


ctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcc


 


tcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctag


 


gccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa


 


ggatccaccggaggccaccatgactacgtccggcgttccatttggcatga


 


cactacgaccaacacgatctcggttgtctcggcgcactccgtacagtagg


 


gatcgtctacctccttttgagacagaaacccgcgctaccatactggagga


 


tcatccgctgctgcccgaatgtaacactttgacaatgcacaacgtgagtt


 


acgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgg


 


gttgttccctgggatatggttctaacgcgggaggagcttgtaatcctgag


 


gaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacga


 


gcatgatgatccatggttacgagtcctgggctctccactgtcattgttcc


 


agtcccggttccctgcagtgtatagccggcgggcaggttttggccagctg


 


gtttaggatggtggtggatggcgccatgtttaatcagaggtttatatggt


 


accgggaggtggtgaattacaacatgccaaaagaggtaatgtttatgtcc


 


agcgtgtttatgaggggtcgccacttaatctacctgcgcttgtggtatga


 


tggccacgtgggttctgtggtccccgccatgagctttggatacagcgcct


 


tgcactgtgggattttgaacaatattgtggtgctgtgctgcagttactgt


 


gctgatttaagtgagatcagggtgcgctgctgtgcccggaggacaaggcg


 


ccttatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgt


 


tgtattcctgcaggacggagcggcggcggcagcagtttattcgcgcgctg


 


ctgcagcaccaccgccctatcctgatgcacgattatgactctacccccat


 


gtagGCGGCCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCT


 


CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG


 


CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA


 


TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGG


 


GTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG


 


CATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAG


 


CTGGGGCTCTAGGGGGTATCCCCGGCGCGCCggggtTGAGCTATTCCAGA


 


AGTAGTGAAGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCGG


 


ATCGATtggggttgcgccttttccaaggcTTTTCCCCGTATCCCCCCAGG


 


TGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCC


 


GTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGG


 


ATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTG


 


CCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGG


 


GGCTGTCCCTCTAGGGGATCCTCTAGGGCCTCTGAGCTATTCCAGAAGTA


 


GTGAAGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCGGATCG


 


ATCGAGCGGATCCAGCACAGTGGCGGCCGCaatatttgcatgtcgctatg


 


tgttctgggaaatcaccataaacgtgaaatccctatcagtgatagagact


 


tataagttccctatcagtgatagagaaccggtgggcactatccgtggtct


 


ggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccc


 


cgtatccggccgtccgccgtgatccatgcggttaccgcccgcgtgtcgaa


 


cccaggtgtgcgacgtcagacaacgggggagtgctcattttgaattccac


 


tttggccgcggctcgagTGAGCTATTCCAGAAGTAGTGAAGAGGCTTTTT


 


TGGAGGCCTAGGCTTTTGCAAAAAGCTCCGGATCGATGCCCGGGGGATCC


 


ACTAGTTCTAGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTA


 


CGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCC


 


GGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCC


 


CGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGC


 


TGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCA


 


AGGCCAGCTTCCCACAATAAGTTGGGTGAATTTTGGCTGAGCTATTCCAG


 


AAGTAGTGAAGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCG


 


GATCGATCATATATGGCAGATATACGCGTTGACATTGATTATTGACTAGT


 


TATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGA


 


GTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA


 


ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACG


 


CCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC


 


TGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA


 


TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG


 


ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC


 


TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC


 


GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGG


 


AGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA


 


CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCT


 


ATATAAGCAGAGCTCTCTGGCTAACTATCGTCGACGAGCTCGTTTAGTGA


 


ACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGA


 


AGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAGCT


 


TGCCACCatgaccgagtacaagcccacggtgcgcctcgccacccgcgacg


 


acgtccccagggccgtacgcaccctcgccgccgcgttcgccgactacccc


 


gccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccga


 


gctgcaagaactatcctcacgcgcgtcgggctcgacatcggcaaggtgtg


 


ggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcg


 


tcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttg


 


agcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgcc


 


gcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgc


 


ccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtg


 


gaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc


 


ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacg


 


tcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggt


 


gcctgaAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTG


 


TTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT


 


CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAAC


 


TCATCAATGTATCTTATCATGTCTGTAGCtGATgtATAcCTAggATCCGG


 


CCGGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGC


 


AGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTG


 


GTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCA


 


GCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCG


 


GGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACC


 


GGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACG


 


TAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAGCGGCC


 


GCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTA


 


GATCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTC


 


GAGGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATATATAT


 


ATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGT


 


ATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGA


 


CTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAA


 


GCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACA


 


GCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCA


 


TGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCA


 


GGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGC


 


GCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTT


 


GAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGT


 


TGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGC


 


ACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCG


 


TCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAG


 


CAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGA


 


TTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTAT


 


CCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGG


 


CGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTG


 


TTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTA


 


AAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGC


 


TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG


 


AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCG


 


CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG


 


GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGA


 


TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC


 


GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC


 


GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG


 


ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC


 


CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG


 


GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGT


 


GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC


 


CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA


 


AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG


 


AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT


 


ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA


 


GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC


 


CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA


 


AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT


 


CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA


 


AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAA


 


TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC


 


AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC


 


CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTG


 


GCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGAT


 


TTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC


 


TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA


 


GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT


 


ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTC


 


CGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA


 


AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC


 


GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT


 


CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT


 


CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA


 


ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT


 


TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA


 


GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT


 


TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC


 


CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence for the PBBG-ITRGFP construct illustrated in in FIG. 11B is shown below:







PBBG-ITRGFP (7,798 bp)


(SEQ ID NO: 27)


ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA


 


TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT


 


CCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATT


 


TTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCA


 


ATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA


 


TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC


 


GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC


 


ACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA


 


AAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG


 


GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC


 


GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCA


 


CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC


 


AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT


 


ACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA


 


CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGC


 


GCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGG


 


TGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGC


 


TGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACG


 


TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCT


 


GTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATA


 


TTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTT


 


ATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA


 


CAAAACTTTTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAG


 


GGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCG


 


CTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCC


 


CCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAA


 


GGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCC


 


TGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCC


 


ACAATAAGTTGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAG


 


GTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGTCAGTTAGGGTGT


 


GGAAAGTCCCgcGATCgcTAGcAAACGCCAGCAACGCGGCCTTTTTACGG


 


TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCAGCTGCG


 


CGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGA


 


CCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTG


 


GCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGGAGCTAGTTA


 


TTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGT


 


TCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC


 


GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTC


 


AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTG


 


CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT


 


GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGAC


 


CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA


 


TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGG


 


TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG


 


TTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTC


 


CGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA


 


TAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCA


 


CGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG


 


ATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCC


 


AAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGC


 


TTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCC


 


TAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTG


 


CACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAA


 


TATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAG


 


GTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTAT


 


TTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTT


 


TGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAAC


 


GTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGA


 


ACATCGATTGAATTCTGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGG


 


GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT


 


TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC


 


CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT


 


CGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACC


 


ACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC


 


CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC


 


CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG


 


GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTAC


 


AACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGG


 


CATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC


 


AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTG


 


CTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGA


 


CCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCG


 


CCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGATCTCGAG


 


CTCAAGTAGGGATCCTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCAG


 


CGCTGCTCGAGAGATCTACGGGTGGCATCCCTGTGACCCCTCCCCAGTGC


 


CTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTA


 


ATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATAT


 


TATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAA


 


CCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCA


 


CAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCT


 


GCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCA


 


GCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGG


 


CTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAA


 


ATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGA


 


TTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGA


 


TGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG


 


CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG


 


CGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCC


 


TTACGCATCTGTGCGGTATTTCACACCGCATACGTCgtaGCtGATcaATT


 


gGCGCGCCGAATTCGTTAACAAGCTtTAATTAaCGCgtATAcCTAggATC


 


CGGCCGGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGT


 


GGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCA


 


TTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGA


 


GCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGC


 


CCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGG


 


ACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGG


 


ACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAGCG


 


GCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAA


 


TTAGATCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCC


 


CTCGAGGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATATA


 


TATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTAT


 


CGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTT


 


TGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGA


 


CAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGC


 


ACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAAT


 


GCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCA


 


TCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCT


 


GGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAG


 


GTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGA


 


CGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCA


 


TGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGT


 


TCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCAT


 


CAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGC


 


AGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGG


 


TATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACC


 


CGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA


 


TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT


 


GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG


 


CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA


 


ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT


 


CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA


 


GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGG


 


GGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA


 


ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT


 


GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC


 


AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT


 


CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT


 


TCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC


 


GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC


 


AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG


 


GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG


 


CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA


 


ACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG


 


CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC


 


CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA


 


GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC


 


GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC


 


AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT


 


CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA


 


ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGT


 


TGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT


 


CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA


 


GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGG


 


TCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG


 


CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT


 


GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAG


 


CTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCA


 


AAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG


 


GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC


 


TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA


 


AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG


 


TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT


 


CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT


 


TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA


 


TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAA


 


TGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence for the PBBG-iRC8 construct illustrated in in FIG. 11C is shown below:







PBBG-iRC8 (9,399 bp)


(SEQ ID NO: 28)


ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA


 


TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT


 


CCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATT


 


TTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCA


 


ATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA


 


TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC


 


GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC


 


ACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA


 


AAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG


 


GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC


 


GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCA


 


CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC


 


AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT


 


ACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA


 


CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGC


 


GCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGG


 


TGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGC


 


TGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACG


 


TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCT


 


GTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATA


 


TTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTT


 


ATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA


 


CAAAACTTTTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAG


 


GGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCG


 


CTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCC


 


CCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAA


 


GGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCC


 


TGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCC


 


ACAATAAGTTGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAG


 


GTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGTCAGTTAGGGTGT


 


GGAAAGTCCCgcGATCgcTAGcTTAagCGCtGATcaATTgGCGCGCCGAA


 


TTCGTTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgc


 


ttcaaaatggagaccctgcgtgctcactcgggcttaaatacccagcgtga


 


ccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaataca


 


ggacctctagagcatggaaactagataagaaagaaatacgcagagaccaa


 


agttcaactgaaacgaattaaacggtttattgattaacaagcaaactagt


 


ttacagattacgggtgaggtaacgggtgccaatggggcggggttcagagt


 


acacgccttctgtattaacagcaaagtccacacttgtagatttgtagtag


 


ttggaggtgtactggatctcggggttccagcgcttgctgttttccttctg


 


cagctcccattcaatttccacgctgacctgtccggtgctgtattgcgtga


 


tgaaagagttcagctttgactggttgaaggtggtcggaggatccgcaggt


 


acaggcgtgttcttgatcaggatctgaggcggaggatgtttcaggccaaa


 


gccgcccatcagcggagacgggtggaagttgccgtccgtgtgaggaatct


 


tggcccagatgggaccctgcaggtacacgtcccggttctgccagaccata


 


ccgggtaaggccccctggctgttgacagttccaatttgaggagccgtgtt


 


ttgctgctgcaagttatctgccacgataccgtattcctctgtagccacag


 


ggttagtggttttgatttcttcctcgctggtgagcatgacatcgctgtaa


 


tccgcattgtctctggcagcattttgtttgccaaaaatcaggatcccgtt


 


actgggaaaaaaacgctcctcgtcgtctttgtgtgttgccatagcgatgc


 


caggattagccaatgaatttcttccattcagatggtatttggtcccagca


 


gtccaggcaaagttgctattgttgttttgcccggttgtcgttgagacgcg


 


ttgttggcggtaacagggtcctggcagccagttctttgcctgattggcca


 


ttgtattaggcccaccttggctgaagcccagagtctgcgtatttgccgtg


 


cctcctgttgtttgagtccgagacaagtagtacaggtactggtcaatcag


 


aggattcatcagccggtccaagctctggctgtgggcgtagctgctgtgga


 


aaggcacgtcctcgaaggtgtaagtaaactggaagttgttgccggttctc


 


agcatctgcgaaggaaagtattccaggcagtagaaggaggagcgtcccac


 


ggcctgactaccgttgttgagtgttaggtagccgtactggggaatcatga


 


acacgtccgccgggaacggaggcaggcagccctggtgggcagagccgaga


 


acgtacggcagctggtactccgagtccgtaaacacctggatggtgctggt


 


gaggttattggcgatggtcttggtgccttcattctgcgtgacctccttga


 


cctggatgttgaagagcttgaagctgagtctcttgggccggaatccccag


 


ttgttgttgatgagtcgctgccagtcacgtggtgaaaagtggcagtggaa


 


tctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggtgt


 


tgtcgttggtggctcctcccgatgtcccgttggagatttgcttgtagagg


 


tggttgttgtaggtgggcagggcccaggttcgggtgctggtggtgatgac


 


tctgtcgcccagccatgtggaatcgcaatgccaatttcccgaggaactac


 


ccactccgtcggcgccttcgttattgtctgccattggtgcgccaccgcct


 


gcagccattgtattaggtcccacaccagagggcgctgctggaggttctcc


 


gagaggttgagggtctggaactgactctgagtcgccagtctgaccaaaat


 


tgagtctttttctggcgggctgttggcctttcttgccgatgcccgtagag


 


gagtctggagaacgctggggtgatggctctaccggtctcttctttccagg


 


agccgtcttagcgccttcctcaaccagaccgagaggttcgagaacccgct


 


tcttggcctggaagactgctcgcccgaggttgcccccaaaagacgtatct


 


tcttgcagacgctcctgaaactcggcgtcggcgtggttataccgcaggta


 


cggattgtcacccgcctgcagctgctggtcgtaggccttgtcgtgctcga


 


gggccgctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttg


 


aagggtccgaggtacttgtagccaggaagcaccagaccccggccgtcgtc


 


ctgcttttgctggttggctttgggcttcggggctccaggtttcagcgccc


 


accactcgcgaatgccctcagagaggttgtcctcgagccaatctggaaga


 


taaccatcggcagccatacctgatttaaatcatttattgttcaaagatgc


 


agtcatccaaatccacattgaccagatcgcaggcagtgcaagcgtctggc


 


acctttcccatgatatgatgaatgtagcacagtttctgatacgccttttt


 


gacgacagaaacgggttgagattctgacacgggaaagcactctaaacagt


 


ctttctgtccgtgagtgaagcagatatttgaattctgattcattctctcg


 


cattgtctgcagggaaacagcatcagattcatgcccacgtgacgagaaca


 


tttgttttggtacctgtctgcgtagttgatcgaagcttccgcgtctgacg


 


tcgatggctgcgcaactgactcgcgcacccgtttgggctcacttatatct


 


gcgtcactgggggcgggtcttttcttggctccaccctttttgacgtagaa


 


ttcatgctccacctcaaccacgtgatcctttgcccaccggaaaaagtctt


 


tgacttcctgcttggtgaccttcccaaagtcatgatccagacggcgggtg


 


agttcaaatttgaacatccggtcttgcaacggctgctggtgttcgaaggt


 


cgttgagttcccgtcaatcacggcgcacatgttggtgttggaggtgacga


 


tcacgggagtcgggtctatctgggccgaggacttgcatttctggtccacg


 


cgcaccttgcttcctccgagaatggctttggccgactccacgaccttggc


 


ggtcatcttcccctcctcccaccagatcaccatcttgtcgacacagtcgt


 


tgaagggaaagttctcattggtccagtttacgcacccgtagaagggcaca


 


gtgtgggctatggcctccgcgatgttggtcttcccggtagttgcaggccc


 


aaacagccagatggtgttcctcttgccgaactttttcgtggcccatccca


 


gaaagacggaagccgcatattggggatcgtacccgtttagttccaaaatt


 


ttataaatccgattgctggaaatgtcctccacgggctgctggcccaccag


 


gtagtcgggggcggttttagtcaggctcataatctttcccgcattgtcca


 


aggcagccttgatttgggaccgcgagttggaggccgcattgaaggagatg


 


tatgaggcctggtcctcctggatccactgcttctccgaggtaatcccctt


 


gtccacgagccacccgaccagctccatgtacctggctgaagtttttgatc


 


tgatcaccggcgcatcagaattgggattctgattctctttgttctgctcc


 


tgcgtctgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctc


 


cgtgagattcaaacaggcgctgtggagagaaaggcaaagtggatgtcagt


 


aTCTCTATCACTGATAGGGAGATCTCTATCACTGATAGGGAacttacctt


 


aaatactgttccatattagtccacgcccactggagctcaggctgggtttt


 


ggggagcaagtaattggggatgtagcactcatccaccaccttgttcccgc


 


ctccggcgccatttctggtctttgtgaccgcgaaccagtttggcaaagtc


 


ggctcgatcccgcggtaaattctctgaatcagtttttcgcgaatctgact


 


caggaaacgtcccaaaaccatggatttcaccccggtggtttccacgagca


 


cgtgcatgtggaagtagctctctcccttctcaaattgcacaaagaaaaga


 


gcctccggggccttactcacacggcgccattccgtcagaaagtcgcgctg


 


cagcttctcggccacggtcaggggtgcctgctcaatcagattcagatcca


 


tgtcagaatctggcggcaactcccattccttctcggccacccagttcaca


 


aagctgtcagaaatgccgggcagatgctcgtcaaggtcgctggggacctt


 


aatcacaatctcgtaaaaccccggcatGGCGGCTGCGCGTTCAAACCTCC


 


CGCTTCAAAATGGAGACCCTGCGTGCTCACTCGGGCgaTCTCTATCACTG


 


ATAGGGAGATCTCTATCACTGATAGGGAgaTTAAATAgaatggCTAggAT


 


CCGGCCGGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAG


 


TGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCC


 


ATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAG


 


AGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTG


 


CCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCG


 


GACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGG


 


GACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAGC


 


GGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTA


 


ATTAGATCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCC


 


CCTCGAGGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATAT


 


ATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTA


 


TCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATT


 


TTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTG


 


ACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATG


 


CACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAA


 


TGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGC


 


ATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGC


 


TGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGA


 


GGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTG


 


ACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCC


 


ATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAG


 


TTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCA


 


TCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTG


 


CAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGG


 


GTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTAC


 


CCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAA


 


ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAG


 


TGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTT


 


GCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATT


 


AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT


 


TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG


 


AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG


 


GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG


 


AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC


 


TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA


 


CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC


 


TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC


 


TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT


 


CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT


 


CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC


 


GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA


 


GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCT


 


AACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAA


 


GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA


 


CCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGC


 


AGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA


 


CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT


 


CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAA


 


TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTT


 


AATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG


 


TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA


 


TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCC


 


AGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG


 


GTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA


 


GCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT


 


TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCA


 


GCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC


 


AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTT


 


GGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA


 


CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC


 


AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC


 


GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA


 


TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTG


 


TTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGC


 


ATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA


 


ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence for the PBBG-iRC9 construct illustrated in in FIG. 11D is shown below:







PBBG-iRC9 (9,393 bp)


(SEQ ID NO: 29)


ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA


 


TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT


 


CCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATT


 


TTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCA


 


ATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA


 


TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC


 


GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC


 


ACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA


 


AAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG


 


GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC


 


GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCA


 


CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC


 


AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT


 


ACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA


 


CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGC


 


GCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGG


 


TGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGC


 


TGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACG


 


TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCT


 


GTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATA


 


TTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTT


 


ATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA


 


CAAAACTTTTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAG


 


GGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCG


 


CTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCC


 


CCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAA


 


GGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCC


 


TGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCC


 


ACAATAAGTTGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAG


 


GTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGTCAGTTAGGGTGT


 


GGAAAGTCCCgcGATCgcTAGcTTAagCGCtGATcaATTgGCGCGCCGAA


 


TTCGTTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgc


 


ttcaaaatggagaccctgcgtgctcactcgggcttaaatacccagcgtga


 


ccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaataca


 


ggacctctagagcatggaaactagataagaaagaaatacgcagagaccaa


 


agttcaactgaaacgaattaaacggtttattgattaacaagcaaactagt


 


TTACAGATTACGAGTCAGGTATCTGGTGCCAATGGGGCGGGGTTCACTAT


 


ATACACCTTCAGTATTAACAGCAAATTCAACATTATTAGACTTGTAATAG


 


TTGGAAGTGTACTGGATCTCCGGGTTCCAGCGCTTGCTGTTTTCCTTCTG


 


CAGCTCCCACTCGATCTCCACGCTGACTTGGCCAGTAGAATACTGGGTGA


 


TGAAAGAGTTCAGCTTGTCCTTGTTGAAGGCCGTTGGAGGATCCGCAGGT


 


ACAGGTGTGTTTTTGATGAGGATCTGAGGAGGCGGGTGCTTCATTCCAAA


 


CCCTCCCATCAGCGGAGAAGGGTGAAAGTTGCCGTCCGTGTGAGGAATTT


 


TGGCCCAAATGGGTCCTTGCAGGTACACATCTCTGTCCTGCCAAACCATA


 


CCCGGAAGTATTCCTTGGTTTTGAACCCAGCCGGTCTGCGCCTGTGCTTG


 


GGCACTCTGGTGGTTTGTGGCCACTTGTCCATAGGACTCCGTTGCTACCG


 


GGTTAGTAGTTTTAATTTCTTCTTCGTTGGTTATCATGACTTTGTCCGCA


 


TCCACGTTGTCTCTTCCAGTTCCTTGTTTGCCAAAAATTAAAGATCCAGA


 


CAAAGGAAAGAAACGGTCCTCTCCTTCTTTGTGGCTGGCCATAGCAGGTC


 


CAGGATTCATCAAGCTATTACGTCCATTGAGAGCCCAAGAAGAAGCTCCA


 


GGCCAAGCAAATTCGCTGTTGTTGTTTTGAGTCACAGTGGTTGAGACACG


 


TTGTTGTCGGTAGCTGGGTCCAGGTATGTAGTTTCTTCCCTGGACAGCCA


 


TGTTGCTGGGTCCGGCCACACTGAATTTTAGCGTTTGTTGATTCTGTCCA


 


GAACCGTTAATAGTCTTTGAGAGATAGTACAAGTATTGGTCGATGAGTGG


 


ATTCATTAGTCGGTCCAGGCTTTGGCTGTGAGCGTAGCTGCTATGGAAAG


 


GTACGTTCTCAAACTCGTAGCTGAACTGGAAGTTGTTACCCGTTCTTAGC


 


ATTTGCGACGGGAAATATTCCAGGCAGTAAAAGGACGAACGACCCACGGC


 


CTGGCTTCCATCATTAAGCGTCAGATACCCGTACTGAGGAATCATGAAAA


 


CGTCCGCTGGGAACGGCGGGAGGCAGCCCTCGTGAGCCGACCCGAGCACG


 


TACGGGAGCTGATAGTCTGAGTCCGTGAAGACCTGGACCGTGCTGGTAAG


 


GTTATTGGCGATGGTCTTGACTCCATTGTTGTCCGTAACCTCTTTGACCT


 


GAATGTTGAAGAGCTTGAAGTTGAGTCGCTTAGGCCGGAATCCCCAGTTG


 


TTGTTGATGAGTCGCTGCCAGTCACGTGGTGAGAAGTGGCAGTGGAATCT


 


GTTGAAGTCAAAATACCCCCAGGGGGTGCTGTAGCCGAAGTAGGCGTTGT


 


CATTTGAAGATCCTCCAGATGTGCTGTTGGAGATTTGCTTGTAGAGGTGA


 


TTGTTGTAGGTGGGCAGGGCCCAGGTTCGGGTGCTGGTGGTGATGACTCT


 


GTCCCCCAGCCATTGGGAATCGCAATGCCAATTTCCCGAGGAACTACCCA


 


CTCCATCGGCACCTTCGTTATTGTCTGCCACTGGTGCGCCACCACCTGAA


 


GCCATTGTAAGAGATCCCACACCTGAGGGGGCTGCGGGAGGTTCTCCGAT


 


TGGTTGAGGGTCTGGGACTGACTCTGTGTCGCCAGTCTGACCGAAATTGA


 


GTCTCTTTTTAGCGGGCTGTGCACCCGATTTGCCAATACCCGCGGAGGAG


 


TCCGGTTCCTGAGGAGACTGCTCTACAGGCCTCTTCTTTCCAGGAGCCGT


 


CTTAGCCGCTTCCTCAACCAGACCAAGAGGTTCAAGAAGCCTCTTTTTGG


 


CCTGGAAGACTGCTCGCCCGAGGTTGCCCCCAAAAGACGTATCTTCTTTG


 


AGCCGCTCCTGGAACTCGGCGTCGGCGTGGTTGTACTTGAGGTACGGGTT


 


GTCTCCGGCCTTGAGCTGCTGGTCGTAGGCCTTGTCGTGCTCGAGGGCCG


 


CCGCGTCTGCTGCGTTGACCGGCTCCCCCTTGTCGAGTCCGTTGCCGGGT


 


CCAAGGTATTTGTAACCCGGAAGCACAAGACCTCGAGCGTTGTCTTGATG


 


TTGTTGATTTGCCTTGGGTTGAGGGGCTCCAGGTTTCAAAGCCCACCACT


 


CGCGAATTCCTTCACTAAGGTTGTCCTCGAGCCAATCTGGAAGATAACCA


 


TCGGCAGCCATacctgatttaaatcatttattgttcaaagatgcagtcat


 


ccaaatccacattgaccagatcgcaggcagtgcaagcgtctggcaccttt


 


cccatgatatgatgaatgtagcacagtttctgatacgcctttttgacgac


 


agaaacgggttgagattctgacacgggaaagcactctaaacagtctttct


 


gtccgtgagtgaagcagatatttgaattctgattcattctctcgcattgt


 


ctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgtt


 


ttggtacctgtctgcgtagttgatcgaagcttccgcgtctgacgtcgatg


 


gctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtca


 


ctgggggcgggtcttttcttggctccaccctttttgacgtagaattcatg


 


ctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgactt


 


cctgcttggtgaccttcccaaagtcatgatccagacggcgggtgagttca


 


aatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttga


 


gttcccgtcaatcacggcgcacatgttggtgttggaggtgacgatcacgg


 


gagtcgggtctatctgggccgaggacttgcatttctggtccacgcgcacc


 


ttgcttcctccgagaatggctttggccgactccacgaccttggcggtcat


 


cttcccctcctcccaccagatcaccatcttgtcgacacagtcgttgaagg


 


gaaagttctcattggtccagtttacgcacccgtagaagggcacagtgtgg


 


gctatggcctccgcgatgttggtcttcccggtagttgcaggcccaaacag


 


ccagatggtgttcctcttgccgaactttttcgtggcccatcccagaaaga


 


cggaagccgcatattggggatcgtacccgtttagttccaaaattttataa


 


atccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtc


 


gggggcggttttagtcaggctcataatctttcccgcattgtccaaggcag


 


ccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgag


 


gcctggtcctcctggatccactgcttctccgaggtaatccccttgtccac


 


gagccacccgaccagctccatgtacctggctgaagtttttgatctgatca


 


ccggcgcatcagaattgggattctgattctctttgttctgctcctgcgtc


 


tgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgtgag


 


attcaaacaggcgctgtggagagaaaggcaaagtggatgtcagtaTCTCT


 


ATCACTGATAGGGAGATCTCTATCACTGATAGGGAacttaccttaaatac


 


tgttccatattagtccacgcccactggagctcaggctgggttttggggag


 


caagtaattggggatgtagcactcatccaccaccttgttcccgcctccgg


 


cgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcg


 


atcccgcggtaaattctctgaatcagtttttcgcgaatctgactcaggaa


 


acgtcccaaaaccatggatttcaccccggtggtttccacgagcacgtgca


 


tgtggaagtagctctctcccttctcaaattgcacaaagaaaagagcctcc


 


ggggccttactcacacggcgccattccgtcagaaagtcgcgctgcagctt


 


ctcggccacggtcaggggtgcctgctcaatcagattcagatccatgtcag


 


aatctggcggcaactcccattccttctcggccacccagttcacaaagctg


 


tcagaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcac


 


aatctcgtaaaaccccggcatGGCGGCTGCGCGTTCAAACCTCCCGCTTC


 


AAAATGGAGACCCTGCGTGCTCACTCGGGCgaTCTCTATCACTGATAGGG


 


AGATCTCTATCACTGATAGGGAgaTTAAATAgaatggCTAggATCCGGCC


 


GGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAG


 


CCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGT


 


ATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGC


 


GAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGG


 


CTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGG


 


AGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTA


 


ATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAGCGGCCGC


 


CACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTAGA


 


TCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGA


 


GGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATATATATAT


 


AATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTAT


 


GAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACT


 


CACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGC


 


ACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGC


 


GACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATG


 


CGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGG


 


ATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGC


 


TATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGA


 


AGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTG


 


AGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCAC


 


GCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTC


 


GCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCA


 


ACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATT


 


AATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCC


 


TGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCG


 


GGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTT


 


ATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAA


 


GCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC


 


ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA


 


TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT


 


TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT


 


ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA


 


ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT


 


AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA


 


GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC


 


TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT


 


GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG


 


AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT


 


AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC


 


GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG


 


ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG


 


CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC


 


GGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT


 


TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG


 


CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA


 


AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA


 


GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA


 


GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC


 


TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG


 


TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT


 


GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC


 


CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT


 


ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG


 


CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA


 


GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC


 


AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG


 


GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA


 


GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC


 


AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA


 


TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCA


 


TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAAT


 


ACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG


 


GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA


 


TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT


 


TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG


 


CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence for the PBBG-Anc80iRC construct illustrated in in FIG. 11E is shown below:







PBBG-Anc80iRC (9,393 bp)


(SEQ ID NO: 30)


ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA


 


TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTT


 


CCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATT


 


TTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCA


 


ATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA


 


TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAAC


 


GTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC


 


ACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTA


 


AAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG


 


GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC


 


GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCA


 


CACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC


 


AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT


 


ACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAA


 


CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGC


 


GCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGG


 


TGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGC


 


TGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACG


 


TACGTTAAAGATAATCATGCGTAAAATTGACGCATGTGTTTTATCGGTCT


 


GTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATA


 


TTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTT


 


ATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA


 


CAAAACTTTTATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAG


 


GGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCG


 


CTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCC


 


CCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAA


 


GGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCC


 


TGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCAAGGCCAGCTTCCC


 


ACAATAAGTTGGGTGAATTTTGGCTCATTCCTCCTTTCTATAGGATTGAG


 


GTCAGAGCTTTGTGATGGGAATTCTGTGGAATGTGTGTCAGTTAGGGTGT


 


GGAAAGTCCCgcGATCgcTAGcTTAagCGCtGATcaATTgGCGCGCCGAA


 


TTCGTTatctgcagaattcggcttggcggctgcgcgttcaaacctcccgc


 


ttcaaaatggagaccctgcgtgctcactcgggcttaaatacccagcgtga


 


ccacatggtgtcgcaaaatgtcgcaaaacactcacgtgacctctaataca


 


ggacctctagagcatggaaactagataagaaagaaatacgcagagaccaa


 


agttcaactgaaacgaattaaacggtttattgattaacaagcaaactagt


 


ttacagattacgggtgaggtaacgggtgccgatggggcgaggctcagaat


 


aaacgccatttgtgtcaacagcaaagtccacatttgtagatttgttgtag


 


ttggaagtgtattgaatctctgggttccagcgtttgctgttttctttctg


 


cagctcccattcaatttccacgctgacctgtccggtgctgtactgcgtga


 


tgaacgacgcaaacttagctggactgaaggtagttggaggattcgcggga


 


acaggtgtattcttaatcaggatctgaggaggcgggtgtttcagtccaaa


 


gcctcccatcagcggcgagggatgaaagtgtccgtccgtgtgaggaatct


 


tggcccagataggaccctgcaggtacacgtcccggtcctgccagaccatg


 


ccaggtaaggctccttgactgttgacggtccctgtagcaggagcggtgtt


 


ggccgattgcaggttagtggccaccgtgccgtactcttctgtggccactg


 


ggttggtggttttaatttcttcctcgttggttatcataacgttgtcaagg


 


tccacgttgctatttccagctccctgtttcccaaatattaagactccgct


 


catcggaaaaaatttgtcttcgtcgtccttgtgggttgccatagcgggac


 


cgggatttaccagagagtctctgccattcagatgatacttggtggcaccg


 


gtccaggcaaagttgctgttgttattttgattggttgtcttggagacgcg


 


ttgctgccggtagcagggcccgggtagccagtttttggcctgattcgcca


 


tgctactaggcccggcctgagaaaattgcaacgtccgatttcctgcggta


 


ccactcgtggtctgagtccgagacaggtagtacaggtactggtcgatgag


 


ggggttcatcagccggtccaggctttggctgtgcgcgtagctgctgtgaa


 


aaggcacgtcctcaaacgtgtagctgaactgaaagttgttgcccgttctc


 


agcatttgagaaggaaagtattccaggcagtagaaggaggaacggcccac


 


ggcctgactgccattgttcagagtcaggtacccgtactgaggaatcatga


 


agacgtccgccgggaacggaggcaggcagccctggtgcgcagagccgagg


 


acgtacgggagctggtattccgagtccgtaaagacctgaaccgtgctggt


 


aaggttattggcgatggtcgtggtgccatcattcgtcgtgacctccttga


 


cctggatgttgaagagcttgaagttgagcttcttgggccggaatccccag


 


ttgttgttgatgagtcgctgccagtcacgtggtgagaagtggcagtggaa


 


tctgttaaagtcaaaatacccccagggggtgctgtagccgaagtaggtgt


 


tgtcgttggtgctgcctcccgattggctggagatttgcttgtagaggtgg


 


ttgttgtaggtggggagggcccaggttcgggtgctggtggtgatgactct


 


gtcgcccagccatgtggaatcgcaatgccaatttcctgaggcgttaccca


 


ctccgtcggcgccttcgttattgtctgccattggagcgccaccgcctgca


 


gccattgtattagatcccacaccagagggggctgcggggggttctccgag


 


tggttgagggtcgggcactgactctgagtcgccagtctgcccaaagttga


 


gtctctttctcgcgggctgctggcctttcttgccgatgcccgaagaggag


 


tctggttcctggggtgattgctctaccggtctcttctttccaggagccgt


 


cttagcgccttcctcaaccagaccgagaggttcgagaacccgcttcttgg


 


cctggaagactgctcgcccgaggttgcccccaaaagacgtatcttcttgc


 


agacgctcctgaaactcggcgtcggcgtggttataccgcaggtacggatt


 


gtcacccgctttgagctgctggtcgtaggccttgtcgtgctcgagggccg


 


ctgcgtccgccgcgttgacgggctcccccttgtcgagtccgttgaagggt


 


ccgaggtacttgtagccaggaagcaccagaccccggccgtcgtcctgctt


 


ttgctggttggctttgggtttcggggctccaggtttcaagtcccaccact


 


cgcgaatgccctcagagaggttgtcctcgagccaatctggaagataacca


 


tcggcagccatacctgatttaaatcatttattgttcaaagatgcagtcat


 


ccaaatccacattgaccagatcgcaggcagtgcaagcgtctggcaccttt


 


cccatgatatgatgaatgtagcacagtttctgatacgcctttttgacgac


 


agaaacgggttgagattctgacacgggaaagcactctaaacagtctttct


 


gtccgtgagtgaagcagatatttgaattctgattcattctctcgcattgt


 


ctgcagggaaacagcatcagattcatgcccacgtgacgagaacatttgtt


 


ttggtacctgtctgcgtagttgatcgaagcttccgcgtctgacgtcgatg


 


gctgcgcaactgactcgcgcacccgtttgggctcacttatatctgcgtca


 


ctgggggcgggtcttttcttggctccaccctttttgacgtagaattcatg


 


ctccacctcaaccacgtgatcctttgcccaccggaaaaagtctttgactt


 


cctgcttggtgaccttcccaaagtcatgatccagacggcgggtgagttca


 


aatttgaacatccggtcttgcaacggctgctggtgttcgaaggtcgttga


 


gttcccgtcaatcacggcgcacatgttggtgttggaggtgacgatcacgg


 


gagtcgggtctatctgggccgaggacttgcatttctggtccacgcgcacc


 


ttgcttcctccgagaatggctttggccgactccacgaccttggcggtcat


 


cttcccctcctcccaccagatcaccatcttgtcgacacagtcgttgaagg


 


gaaagttctcattggtccagtttacgcacccgtagaagggcacagtgtgg


 


gctatggcctccgcgatgttggtcttcccggtagttgcaggcccaaacag


 


ccagatggtgttcctcttgccgaactttttcgtggcccatcccagaaaga


 


cggaagccgcatattggggatcgtacccgtttagttccaaaattttataa


 


atccgattgctggaaatgtcctccacgggctgctggcccaccaggtagtc


 


gggggcggttttagtcaggctcataatctttcccgcattgtccaaggcag


 


ccttgatttgggaccgcgagttggaggccgcattgaaggagatgtatgag


 


gcctggtcctcctggatccactgcttctccgaggtaatccccttgtccac


 


gagccacccgaccagctccatgtacctggctgaagtttttgatctgatca


 


ccggcgcatcagaattgggattctgattctctttgttctgctcctgcgtc


 


tgcgacacgtgcgtcagatgctgcgccaccaaccgtttacgctccgtgag


 


attcaaacaggcgctgtggagagaaaggcaaagtggatgtcagtaTCTCT


 


ATCACTGATAGGGAGATCTCTATCACTGATAGGGAacttaccttaaatac


 


tgttccatattagtccacgcccactggagctcaggctgggttttggggag


 


caagtaattggggatgtagcactcatccaccaccttgttcccgcctccgg


 


cgccatttctggtctttgtgaccgcgaaccagtttggcaaagtcggctcg


 


atcccgcggtaaattctctgaatcagtttttcgcgaatctgactcaggaa


 


acgtcccaaaaccatggatttcaccccggtggtttccacgagcacgtgca


 


tgtggaagtagctctctcccttctcaaattgcacaaagaaaagagcctcc


 


ggggccttactcacacggcgccattccgtcagaaagtcgcgctgcagctt


 


ctcggccacggtcaggggtgcctgctcaatcagattcagatccatgtcag


 


aatctggcggcaactcccattccttctcggccacccagttcacaaagctg


 


tcagaaatgccgggcagatgctcgtcaaggtcgctggggaccttaatcac


 


aatctcgtaaaaccccggcatGGCGGCTGCGCGTTCAAACCTCCCGCTTC


 


AAAATGGAGACCCTGCGTGCTCACTCGGGCgaTCTCTATCACTGATAGGG


 


AGATCTCTATCACTGATAGGGAgaTTAAATAgaatggCTAggATCCGGCC


 


GGccTGCAggTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAG


 


CCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGT


 


ATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGC


 


GAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGG


 


CTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGG


 


AGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTA


 


ATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTCTAGAGCGGCCGC


 


CACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTAGA


 


TCTTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGA


 


GGTCGACGGTATCGATAAGCTTGATATCTATAACAAGAAAATATATATAT


 


AATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTAT


 


GAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACT


 


CACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGC


 


ACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGC


 


GACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATG


 


CGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGG


 


ATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGC


 


TATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGA


 


AGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTG


 


AGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCAC


 


GCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTC


 


GCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCA


 


ACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATT


 


AATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCC


 


TGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCG


 


GGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTT


 


ATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAA


 


GCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC


 


ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA


 


TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT


 


TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT


 


ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA


 


ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT


 


AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA


 


GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC


 


TATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT


 


GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG


 


AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT


 


AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC


 


GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG


 


ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG


 


CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC


 


GGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT


 


TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG


 


CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA


 


AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA


 


GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA


 


GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC


 


TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG


 


TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT


 


GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC


 


CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT


 


ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG


 


CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA


 


GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC


 


AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG


 


GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA


 


GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC


 


AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA


 


TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCA


 


TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAAT


 


ACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG


 


GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA


 


TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT


 


TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG


 


CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT






The sequence for the pcDNA-TetR-Ins construct illustrated in in FIG. 12A is shown below:








pcDNA-TetR-Ins (7147 bp)



(SEQ ID NO: 31)



gacggatcgggagatctgagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgct



 


agggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcgggg


 


acagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacc


 


tggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaa


 


ggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccac


 


atccagcctggccttgaatgcctgcaggcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatag


 


ttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggc


 


aaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagata


 


tacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag


 


ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga


 


cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccactt


 


ggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattat


 


gcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgc


 


ggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaa


 


tgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggc


 


ggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcg


 


aaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagctttctgtgagtttggggaccctt


 


gattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgg


 


gaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtc


 


tcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcact


 


tttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtactt


 


cagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtgg


 


aaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactg


 


tttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctac


 


agctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcga


 


gccaccatggctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtt


 


taacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggcttt


 


gctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagat


 


tttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacac


 


ggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgc


 


cttatatgcactcagcgccgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaa


 


gaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcag


 


agccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccccaaa


 


aaagaagagaaaggtcgacggcggtggttcagtttaagcgtacagcgggatccactagtccagtgtggtggaattctgc


 


agatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttcta


 


gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttccta


 


ataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaag


 


ggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagct


 


ggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgac


 


cgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccc


 


cgtcaagctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatt


 


agggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaa


 


tagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgggg


 


atttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagtt


 


agggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtg


 


tggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgccc


 


ctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttattt


 


atgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttg


 


caaaaagctcccgggagcttgtatatccattttcggatctgatcagcacgtgttgacaattaatcatcggcatagtata


 


tcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcg


 


cgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggt


 


gtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgt


 


gggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggc


 


catgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtg


 


gccgaggagcaggactgacacgtgctacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcg


 


ttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttat


 


tgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagt


 


tgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcat


 


ggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa


 


agcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctg


 


tcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgc


 


tcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccac


 


agaatcaggggataacgcaggaaagagagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccc


 


tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcag


 


cgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcct


 


gcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctg


 


ggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcc


 


cagagccacatccagcctggccttgaatgcctgcaggacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaa


 


aggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg


 


gcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg


 


ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctca


 


gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccgg


 


taactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcaga


 


gcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggta


 


tctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtag


 


cggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacg


 


gggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctaga


 


tccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgctt


 


aatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataact


 


acgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttat


 


cagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaa


 


ttgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtg


 


gtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgt


 


tgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggt


 


tatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaag


 


tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagca


 


gaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccag


 


ttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaaca


 


ggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatatt


 


attgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggt


 


tccgcgcacatttccccgaaaagtgccacctgacgtc






The sequence for the pcDNA-TetR-KRAB-Ins construct illustrated in in FIG. 12B is shown below:








pcDNA-TetR-KRAB-Ins (7493 bp)



(SEQ ID NO: 32)



gacggatcgggagatctgagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgct



 


agggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcgggg


 


acagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacc


 


tggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaa


 


ggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccac


 


atccagcctggccttgaatgcctgcaggcccgatcccctatggtcgactctcagtacaatctgctctgatgccgcatag


 


ttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggc


 


aaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagata


 


tacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggag


 


ttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga


 


cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccactt


 


ggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattat


 


gcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgc


 


ggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaa


 


tgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggc


 


ggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcg


 


aaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagctttctgtgagtttggggaccctt


 


gattgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgg


 


gaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattgtc


 


tcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaattcact


 


tttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatattgtactt


 


cagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggctggcgtgg


 


aaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatgatatacactg


 


tttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttcttctttttcctac


 


agctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgactcactatagggcga


 


gccaccatggctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtt


 


taacaacccgtaaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggcttt


 


gctcgacgccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagat


 


tttttacgtaataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacac


 


ggcctacagaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgc


 


cttatatgcactcagcgccgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaa


 


gaaagggaaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcag


 


agccagccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtccccaaa


 


aaagaagagaaaggtcgacggcggtggtgctttgtctcctcagcactctgctgtcactcaaggaagtatcatcaagaac


 


aaggagggcatggatgctaagtcactaactgcctggtcccggacactggtgaccttcaaggatgtatttgtggacttca


 


ccagggaggagtggaagctgctggacactgctcagcagatcgtgtacagaaatgtgatgctggagaactataagaacct


 


ggtttccttgggttatcagcttactaagccagatgtgatcctccggttggagaagggagaagagccctggctggtggag


 


agagaaattcaccaagagacccatcctgattcagagactgcatttgaaatcaaatcatcagtttaagcgtacagcgggg


 


atccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaa


 


cccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccct


 


ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctatt


 


ctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggct


 


ctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgc


 


ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccct


 


tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgatttagtgctt


 


tacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcg


 


ccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtc


 


tattcttttgatttataagggattttggggatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacg


 


cgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaag


 


catgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcat


 


ctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctc


 


cgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtg


 


aggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcagcac


 


gtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagtt


 


gaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcc


 


cgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccagg


 


tggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtc


 


cacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgc


 


gacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctacgagatttcgattccaccgccg


 


ccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgct


 


ggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcaca


 


aataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgt


 


cgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccaca


 


caacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgc


 


tcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtt


 


tgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagc


 


tcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagagagctcacggggacagccccccc


 


ccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtcc


 


ggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcc


 


tctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctttaggctgaaagagaga


 


tttagaatgacagaatcatagaacggcctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtg


 


cagggtcatcaaccagcagcccaggctgcccagagccacatccagcctggccttgaatgcctgcaggacatgtgagcaa


 


aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagca


 


tcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagc


 


tccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgc


 


tttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc


 


cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactg


 


gcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaact


 


acggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc


 


ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga


 


tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtca


 


tgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatga


 


gtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccata


 


gttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgc


 


gagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgc


 


aactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgc


 


aacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac


 


gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaag


 


taagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgc


 


ttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgt


 


caatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaact


 


ctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttact


 


ttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgtt


 


gaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttga


 


atgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc






It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.
It is to be understood that while certain embodiments have been illustrated and described herein, the claims are not to be limited to the specific forms or arrangement of parts described and shown. In the specification, there have been disclosed illustrative embodiments and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

## Claims

1. An isolated nucleic acid molecule comprising a plasmid encoding, in operable order:
a. a derepressible p5 promoter;
b. a rep78 gene, including a silenced p19 promoter located within the rep78 gene coding region, wherein the silenced p19 promoter comprises mutations in the SP1, TATA-1, and TATA-2 sites of a p19 promoter;
c. a cap gene;
d. a p5 promoter;
e. a derepressible p19 promoter;
f. a rep52 gene; and;
h. an antibiotic resistance gene.

2. The isolated nucleic acid molecule of claim 1, wherein the cap gene is a cap gene from an AAV serotype selected from the group consisting of ANC80, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

3. The isolated nucleic acid molecule of claim 2, wherein the cap gene is an ANC80 cap gene.

4. The isolated nucleic acid molecule of claim 1, wherein the derepressible p5 promoter comprises a functional p5 promoter and two tetracycline operator sequences (TetO2).

5. The isolated nucleic acid molecule of claim 4, wherein the derepressible p5 promoter comprises SEQ ID NO:5.

6. The isolated nucleic acid molecule of claim 1, wherein the derepressible p19 promoter comprises a functional p19 promoter and two tetracycline operator sequences (TetO2).

7. The isolated nucleic acid molecule of claim 6, wherein the derepressible p19 promoter comprises SEQ ID NO: 7.

8. The isolated nucleic acid molecule of claim 1, wherein the antibiotic resistance gene is a kanamycin resistance gene.

9. The isolated nucleic acid molecule of claim 1, further comprising an inoperable antibiotic resistance gene.

10. An isolated nucleic acid molecule comprising a plasmid encoding, in operable order:
a. a derepressible p5 promoter comprising a functional p5 promoter and two tetracycline operator sequences (TetO2);
b. a rep78 gene, including a silenced p19 promoter located within the rep78 gene coding region, wherein the silenced p19 promoter comprises mutations in the SP1, TATA-1, and TATA-2 sites of a p19 promoter;
c. a viral associated (VA I) non-coding DNA;
d. a p5 promoter;
e. a derepressible p19 promoter comprising a functional p19 promoter and two tetracycline operator sequences (TetO2);
f. a rep52 gene; and;
h. an antibiotic resistance gene.

11. The isolated nucleic acid molecule of claim 10, wherein the derepressible p5 promoter comprises SEQ ID NO:5.

12. The isolated nucleic acid molecule of claim 10, wherein the derepressible p19 promoter comprises SEQ ID NO: 7.

13. The isolated nucleic acid molecule of claim 10, wherein the antibiotic resistance gene is a kanamycin resistance gene.

14. The isolated nucleic acid molecule of claim 10, further comprising an inoperable antibiotic resistance gene.