WO2016189387A1 - Genetically modified micro-organ secreting antibody and methods of use - Google Patents

Genetically modified micro-organ secreting antibody and methods of use Download PDF

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WO2016189387A1
WO2016189387A1 PCT/IB2016/000829 IB2016000829W WO2016189387A1 WO 2016189387 A1 WO2016189387 A1 WO 2016189387A1 IB 2016000829 W IB2016000829 W IB 2016000829W WO 2016189387 A1 WO2016189387 A1 WO 2016189387A1
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ac
organ
antibody
gt
genetically modified
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PCT/IB2016/000829
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French (fr)
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Reem Miari
Nir Shapir
Inbal ZAFIR-LAVIE
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Medgenics Medical Israel Ltd.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/56Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids
    • A61K31/57Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane, progesterone
    • A61K31/573Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane, progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/505Erythropoietin [EPO]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/51Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Abstract

This application relates to the field of micro-organs secreting antibodies and methods of use.

Description

GENETICALLY MODIFIED MICRO-ORGAN SECRETI NG ANTIBODY

AND METHODS OF USE

DESCRIPTION

FIELD

[001] This application relates to the field of micro-organs secreting antibodies and methods of use.

BACKGROUND

[002] Antibodies are important therapeutics for many diseases, including autoimmune and oncologic diseases. However, therapeutic antibodies are normally administered intravenously or subcutaneously on a regular basis, which can be costly and cumbersome.

[003] A system whereby a therapeutic antibody could be produced in vivo over an extended period of time would therefore be advantageous. Continuous antibody delivery in vivo could promote patient compliance and reduce the number of injections or infusions and trips to the clinics or hospitals. In addition, it has been proposed that for certain indications for therapeutic antibodies, such as those that target tumor cell elimination, mechanisms that allow for more continuous levels of antibody could be far more effective than bolus injections where spikes and dips are common (see Lindorfer MA, et al (Sep 2012) Oncoimmunology. 1(6):959-961). The ability to eliminate repeated, high-dose bolus injections of antibodies with a method that enables stable therapeutic levels over time is also proposed to be a potential means to reduce unwanted side-effects associated with monoclonal antibodies (Samaranayake H, (2009) Ann Med. 41(5):322-31).

[004] Numerous attempts have been made to ectopically generate antibodies in vivo as described in (Samaranayake 2009). Examples of models to produce long- term ectopic expression of therapeutic antibodies include electroporation of the muscle or grafting of epidermis that was reconstituted and transduced in vitro (See Noel D, et al (Feb 2002) J Invest Dermatol. 118(2):288-94 and Perez N, et al (Mar 23, 2004) Genet Vaccines Ther. 2(1):2). While these experiments indicated that antibodies could be successfully generated ectopically in vivo, they resulted in relatively low serum antibody concentrations.

[005] Monoclonal antibodies have been developed and produced

recombinantly, such as adalimumab (See Frenzel A, et al (July 29, 2013) Front Immunol. 4:217; Prescribing Information for HUMIRA ). However, the process of generating high-producer cell lines is expensive, time-consuming, and laborious (as noted in Frenzel 2013 and Samaranayake 2009). Although non-mammalian production systems have been studied, there has been no widespread use of such antibodies therapeutically because of concern regarding the risk of immunogenicity due to non-human glycosylation patterns in non-mammalian lines (Frenzel 2013). In addition, means of generating stoichiometric amounts of both the heavy chain and light chain need to be developed for production cell lines, as any imbalances in the production of the light and heavy chain can be toxic to cells or lead to

immunoglobulin chains that complicate the purification process (Samaranayake 2009). Overall, current manufacturing and purification processes limit the production capacity of monoclonal antibodies, which leads to high costs (Samaranayake 2009).

[006] Antibody fragments/domains (such as antigen binding fragments

[Fabs], single-chain variable fragments [scFvs], and domain antibodies) have been developed and produced recombinantly, as described in Singh Athwal (2009) Innov Pharmaceut Tech. While antibody fragments have been successfully approved as therapeutics (see Nelson AL (Jan-Feb 2010) MAbs. 2(l):77-83), potential

disadvantages remain. For example, antibody fragments may be associated with the potential for lower affinity, difficulties in mass production, decreased serum half-life, and aggregation (Siontorou CG, et al (2013) Int J Nanomedicine. 8:4215-27).

[007] We have previously described that human dermal micro-organs can deliver therapeutic polypeptides (see US Application 20150118187), and we herein report the successful production of active antibodies in human dermal micro-organs. In vivo production of monoclonal antibodies is a means to overcome limitations seen with production of recombinant monoclonal antibodies and also to overcome the disadvantages of bolus injections of antibodies by intravenous or subcutaneous routes. SUMMARY

[008] In accordance with the description, we have determined that genetically modified micro-organs are capable of producing active antibody in vivo. Transduction of dermal micro-organs from human tummy tuck waste tissue with viral vectors, such as HDAd, expressing a cassette containing sequences encoding the heavy chain and light chain of adalimumab or trastuzumab (ie, Transduced

Autologous Restorative Gene Therapy (TARGT)-adalimumab or TARGT- trastuzumab) led to expression and secretion of active antibody in vitro. Transplantation of TARGT-adalimumabs into SCID mice led to serum levels of adalimumab that were in the microgram per milliliter range.

[009] In one embodiment, the invention comprises a genetically modified micro-organ that produces an antibody. In some embodiments, the micro-organ comprises a vector comprising a nucleic acid sequence encoding an antibody. In some embodiments, this genetically modified micro-organ comprises a vector comprising a nucleic acid sequence encoding an antibody operably linked to an upstream MAR regulatory sequence, and comprising at least one additional regulatory sequence.

[0010] In some embodiments, the antibody is secreted from the micro-organ for a sustained period of time such as at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. In some embodiments, the antibody is secreted from the micro-organ for at least six months. The antibody secretion may be in vitro or in vivo.

[0011] The antibody produced by the transduced micro-organ is functional as determined by binding to its antigen receptor, or by its activity. In some embodiments, the antibody binds tumor necrosis factor alpha (TNFa) or human epidermal growth factor receptor 2 (HER2). In certain embodiments, the antibody comprises the light chain and heavy chain of an antibody, such as adalimumab or trastuzumab (SEQ ID NOs: 41 and 42 (adalimumab), or SEQ ID NOs: 36 or 37, and 38 (trastuzumab). Any nucleic acid sequence encoding these amino acids are encompassed. In certain embodiments, the antibody comprises a portion of the light chain and a portion of the heavy chain of an antibody, such as adalimumab or trastuzumab, wherein the antibody produced from the micro-organ binds to TNFa or HER2, respectively.

[0012] In some embodiments, the sequence of the anti-TNFa antibody comprises the nucleic acids of one or more of SEQ ID NO: 1-4, 14-15, and 18-19 or a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to any one of SEQ ID NO: 1-4, 14-15, and 18-19. In some embodiments, the nucleic acid sequence of the anti-TNFa antibody comprises any nucleic acid sequence that encodes full length or partial length, functional, adalimubab having the amino acids of SEQ ID NOs: 41 (light chain) and 42 (heavy chain). [0013] In some embodiments, the sequence of the anti-TNFa antibody comprises the nucleic acids encoding the heavy and light chains, or portions thereof, of any one or more of SEQ ID NO: 16-17, and 20-22 or a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to the heavy and light chain portions of any one of SEQ ID NO: 16-17, and 20-22.

[0014] In some embodiments, the sequence of the anti-HER2 antibody comprises the nucleic acids of one or more of SEQ ID NO: 23-24, 30-32, and 33-35 or a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to any one of SEQ ID NO: 23-24, 30-32, and 33- 35. In some embodiments, the nucleic acid sequence of the anti-HER2 antibody comprises any nucleic acid sequence that encodes full length or partial length, functional, tratuzumab having the amino acids of SEQ ID NOs: 36 or 37 (heavy chain) and 38 (light chain).

[0015] In some embodiments, the sequence of the anti-HER2 antibody comprises the nucleic acids encoding the heavy and light chains, or portions thereof, of any one or more of SEQ ID NO: 25-28, or a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to the heavy and light chain portions of any one or more of SEQ ID NO: 25-28.

[0016] In some embodiments, the sequence of the anti-TNFa antibody comprises or consists of the nucleic acids of one or more of SEQ ID NO: 1-4, 14-15, and 18-19.

[0017] In some embodiments, the sequence of the anti-TNFa antibody comprises or consists of the nucleic acids encoding the heavy and light chains, or portions thereof, of any one or more of SEQ ID NO: 16-17, and 20-22.

[0018] In some embodiments, the sequence of the anti-HER2 antibody comprises or consists of the nucleic acids of one or more of SEQ ID NO: 23-24, 30- 32, and 33-35.

[0019] In some embodiments, the sequence of the anti-HER2 antibody comprises or consists of the nucleic acids encoding the heavy and light chains, or portions thereof, of any one or more of SEQ ID NO: 25-28.

[0020] In some embodiments, the sequence of the anti-TNFa antibody comprises a light chain portion and a heavy chain portion, wherein the light chain portion is selected from an antibody-producing portion of SEQ ID NOs: 1 and 2, and the heavy chain portion is selected from an antibody-producing portion of SEQ ID NO: 3 and 4.

[0021] In some embodiments, the sequence of the anti-HER2 antibody comprises a light chain portion and a heavy chain portion, wherein the light chain portion is selected from an antibody-producing portion of SEQ ID NOs: 30 and 33, and the heavy chain portion is selected from an antibody-producing portion of SEQ ID NO: 31-32, and 34-35.

[0022] In some embodiments, the antibody is an antibody portion, fragment, region, peptide or derivative, wherein the antibody retains ability to bind antigen and/or retains functional activity as compared to a control antibody not produced by a micro-organ.

[0023] In some embodiments, the antibody can be detected in human serum after implantation of the transduced micro-organ to a human. In certain embodiments, the antibody can be detected in human serum after implantation to a human, wherein the antibody is detected in the serum for a sustained period of time of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

[0024] In some embodiments, the at least one additional regulatory sequence of the vector comprises a MAR sequence, a CAG sequence, an EFla promoter sequence, or a WPRE sequence. In certain embodiments, the nucleic acid encoding the antibody is inserted at the multiple cloning site (mcs) shown in Figure IE.

[0025] In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light and heavy chains are produced by a single cassette, and wherein a cleavable element is placed between the heavy and light chain in the cassette. In some embodiments the cleavable element is a furin, 2a, or furin 2A site.

[0026] In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light and heavy chains are produced as two separate transcripts, and wherein a stop codon is placed after the coding sequence of the first chain (heavy or light), and wherein the stop codon is followed by an IRES element, and wherein the IRES element is followed by the next chain (heavy or light). In this embodiment, the heavy and light chains are produced as separate transcripts. [0027] In certain embodiments, the regulatory sequences and/or IRES or furin 2A elements within the cassette are selected based upon secretion of antibody when the expression cassette is expressed in a heterologous cell system.

[0028] In certain embodiments, a cassette contained within a viral vector includes the optimized sequences for expression of adalimumab comprising or consisting of any one of SEQ ID NO: 1-4, or a sequence having at least 99, 98, 97, 96,

95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to any one of one of SEQ ID NO: 1-4. In certain embodiments, a cassette contained within a viral vector includes sequences for expression of the Fab fragment of adalimumab comprising or consisting of SEQ ID NO: 29, or a sequence having at least 99, 98, 97,

96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to SEQ ID NO: 29. In certain embodiments, a cassette contained within a viral vector includes sequences for expression of trastuzumab comprising or consisting of any one or more of SEQ ID NO: 23-24, and 30-35, or a sequence having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to any one of one of SEQ ID NO: 23-24, and 30-35.

[0029] In some embodiments, the viral vector is a helper dependent adenovirus (HDAd) vector or an adeno-associated virus (AAV) vector. In some embodiments, the genetically modified micro-organ is a genetically modified dermal micro-organ. In certain embodiments, the genetically modified micro-organ is a genetically modified dermal micro-organ, wherein the dermal micro-organ does not comprise epidermis.

[0030] In some embodiments, the TARGT system is employed to generate a TARGT-antibody.

[0031] In another embodiment, the invention comprises a method for treating or preventing a disease or disorder, or a symptom of a disease or disorder, in a human subject in need comprising the steps of providing at least one genetically modified micro-organ to the human subject, wherein the micro-organ provides delivery of a therapeutic antibody; determining the secretion level of said antibody by at least one genetically modified micro-organ in vitro; implanting at least one genetically modified micro-organ in said human subject; and measuring antibody levels in whole blood or serum of said subject, wherein implantation of said at least one genetically modified micro-organ increases in vivo serum antibody levels. [0032] In another embodiment, the invention comprises a method for diagnosing a disease or disorder in a human subject in need comprising the steps of providing the human subject with at least one genetically modified micro-organ, wherein the micro-organ provides delivery of a diagnostic antibody with a detection moiety; implanting the micro-organ in said human subject; and detecting the detection moiety. A disease or disorder is diagnosed by comparing to a control. The control may be the human subjects themselves, by, for example, monitoring the detection moiety over time, or a comparison to a healthy control subject.

[0033] In some embodiments, the implanting of the genetically modified micro-organ results in sustained serum levels of antibody; optionally, where these levels are sustained for at least three months.

[0034] Some embodiments further comprise administering a corticosteroid by subcutaneous injection in the vicinity of the genetically modified micro-organ, and optionally repeating administration of corticosteroid every 2 weeks post-implantation until removal of the genetically modified micro-organ. The corticosteroid may be DepoMedrol (methylprednisolone) or like compounds.

[0035] In some embodiments, the antibody produced is for therapeutic, diagnostic, or imaging purposes, or to prevent infection with an infectious agent. In some embodiments, the antibody treats an oncologic, autoimmune, or inflammatory disease, or a symptom of an oncologic, autoimmune, or inflammatory disease. In certain embodiments, the antibody prevents an oncologic, autoimmune, or inflammatory disease, or prevents a symptom of an oncologic, autoimmune, or inflammatory disease.

[0036] In some embodiments, the production of antibody by the genetically modified micro-organ produces prevention, remission, or amelioration of disease symptoms or improvement in a diagnostic marker or biomarker of disease activity. In certain embodiments, the antibody produced by the genetically modified micro-organ is used in combination with another biologic or non-biologic agent for the treatment or prevention of a disease or a symptom of a disease.

[0037] In some embodiments, the timing of removal of the genetically modified micro-organ(s) or the timing of subsequent implantations of genetically modified micro-organ(s) is determined by measurement of serum levels of the antibody. In some embodiments, the timing of removal of the genetically modified micro-organ(s) or the timing of subsequent implantations of genetically modified micro-organ(s) is determined by measurement of therapeutic benefit, such as, for example, the cessation of amelioration of one or more symptoms of the disease, such as, for example, a stalling of tumor regression, or a stalling of reduction of inflammation.

[0038] In some embodiments, the number of genetically modified micro- organs is optimized, optionally by implantation of additional micro-organs or removal of implanted micro-organs, based on serum levels of the antibody produced.

[0039] In some embodiments, the antibody produced by the genetically modified micro-organ is capable of preventing infection by an infectious agent or preventing harmful effects of exposure to a toxin.

[0040] In some embodiments, the timing of removal of the genetically micro- organ is determined based on remission or amelioration of disease symptoms or improvements in diagnostic markers, biomarkers, imaging, or tumor regression.

[0041] In some embodiments, the antibody produced by the genetically modified micro-organ is of use as a diagnostic for imaging of a tumor(s) within a subject; optionally, wherein the change in tumor size over time can be monitored by imaging with said antibody produced by the genetically modified micro-organ;

optionally where the therapeutic regimen for said subject is determined or modified based on imaging results with said antibody produced by the genetically modified micro-organ.

[0042] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0043] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0044] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figures 1A, IB, 1C, ID, IE, and IF show an exemplary

cassette/construct design for expression of light and heavy chains of adalimumab. The sequences of the adalimumab light and heavy chains were inserted on the open reading frame into a-MAR-EFl alpha cassette using different construct designs to test for optimal antibody expression and secretion. The TNFl and TNF2 constructs have a furin 2A (F2A) cleavage site between the light and heavy chains, allowing co- translational cleavage and production of equal amount of the heavy and light chains of adalimumab. As shown in Figure 1A, the TNFl cassette is organized as follows: start- signaling peptide-antibody light chain-F2A-signaling peptide-antibody heavy chain- stop. As shown in Figure IB, the TNF2 cassette is organized as follows: start- signaling peptide-heavy chain-furin cleavage sequence-2A element-signaling peptide- light chain-stop. A cassette was also developed containing an IRES element between the sequences for the optimized light chain and heavy chain of adalimumab. The IRES element is downstream of the stop codon in the sequence for the light chain to allow canonical cap-dependent translation of the light chain and cap-independent translation of the heavy chain. As shown in Figure 1C, TNF3 is organized as follows: start-signaling peptide-antibody light chain-stop-IRES-start-signaling peptide- antibody heavy chain-stop. As shown in Figure ID, TNF5 is organized as follows: start-signaling peptide-antibody light chain-furin cleavage sequence-2 A- start- signaling peptide-heavy chain variable domain (VH)-heavy chain constant domain (CH)-stop. Figure IE shows the design of the HD Ad-MAR-EF 1 alpha expression cassette used to express the TNFl, TNF2, TNF3, and TNF5 constructs. These constructs were cloned into the HD Ad-MAR-EF 1 alpha expression cassette using the multiple-cloning site. Figure IF shows levels of adalimumab secreted by fibroblast cells extracted from human skin following nucleofection with TNFl and TNF3.

Supernatant on day 1 and 2 and intracellular on day 2 levels of total adalimumab were measured for each cassette.

[0046] Figure 2 shows the in vitro secretion profile of active adalimumab from TARGT-adalimumab in vitro. Micro-organs were harvested from tummy tuck surgical waste skins, and TARGT-adalimumab was generated by transduction with 1.5X1010 viral particles of the HDAd-MAR-EFl alpha cassette comprising the TNFl construct. Levels of active adalimumab secreted into the culture supernatant per TARGT-adalimumab per day were measured by ELISA (the ELISA measuring binding to tumor necrosis factor-alpha (TNF-alpha). TNF-alpha was measured at day 6, 9, 16, 20, 27, 34, and 44 days after harvesting of the micro-organ. [0047] Figure 3 shows skin-to-skin variability in the secretion profile of TARGT-adalimumab generated from different donors (HA308 and HA305). Micro- organs from tummy tuck surgical waste skin were transduced with 1.5X1010 viral particles of the HDAd-MAR-EFl -alpha expression cassette comprising the T F1 construct to generate TARGT-adalimumab. Levels of active adalimumab secreted per TARGT, per day, were measured by ELISA (binding to TNF-alpha). Results are shown for 6, 9, 16, 20, 27, 34, and 44 days after harvesting of the micro-organ.

[0048] Figures 4A and 4B shows comparison of adalimumab secretion from fibroblasts following nucleofection with the TNF1 versus TNF2 constructs (Figure 4A) or the TNFl, T F2, or TNF5 constructs (Figure 4B).

[0049] Figure 5 shows non-reducing western blot analysis of adalimumab from TARGT-adalimumab. TARGT-adalimumab was generated via transduction of micro-organs from tummy tuck surgical waste skin with an FIDAd-MAR-EFl alpha expression cassette comprising the TNFl construct. Western blots were performed using non-reducing sample buffer. Lanes 3-5 show results from three separate TARGT-adalimumab s made from skin donor #1, while lanes 6-8 show results from three separate TARGT-adalimumab s made from skin donor #2. Commercial adalimumab was loaded in lanes 11-12 as a positive control. Lane 10 shows spent media from TARGTs expressing erythropoietin as a negative control.

[0050] Figures 6A and 6B show reducing western blot analysis of adalimumab production from TARGT-adalimumab. TARGTs were generated via transduction of micro-organs from tummy tuck surgical waste skin with an FIDAd-MAR-EFl alpha expression cassette comprising the TNFl construct. Western blots were performed with reducing sample buffer. Lanes 1, 3, and 4 show results from three separate TARGT-adalimumab s made from skin donor #1, while lanes 5-7 show results from three separate TARGT-adalimumab s made from skin donor #2. Commercial adalimumab is loaded in lanes 10-11 as a positive control. Lane 9 shows spent media from TARGTs expressing erythropoietin as a negative control. Lane 2 is the molecular weight standard. Figure 6A shows results using chemiluminescence reagent, while Figure 6B shows results using DAB reagent.

[0051] Figures 7 A and 7B show the in vivo secretion profile of TARGT- adalimumab implanted into SCID mice. TARGT-adalimumab was prepared via transduction of micro-organs from tummy tuck surgical waste skin with an FIDAd- MAR-EFl alpha expression cassette comprising the TNFl construct. TARGT- adalimumabs were implanted on day 7 after transduction into five SCID mice. Each mouse was implanted with 4 TARGT-adalimumab. Mice were treated with

DepoMedrol (methylprednisolone) every 2 weeks. Mouse blood was collected the day before TARGT-adalimumab implantation (baseline, not shown) and approximately weekly thereafter for adalimumab measurements. Figure 7A shows the serum amounts of total (measured by ELISA with anti-human Fc) and active (measured by ELISA with TNF-alpha) adalimumab over time. Figure 7B shows the active fraction of adalimumab in SCID serum over time as measured by the ratio of active to total serum adalimumab values.

[0052] Figure 8A and 8B shows rheumatoid arthritis (RA) scores from RA mice treated with TARGT-adalimumab comprising 3X105 cells (0.3M group), TARGT-adalimumab comprising 1.0X106 cells (1M group), bi-weekly IP injection with 3mg/kg adalimumab (std group), or untransduced MO (naive group) mice.

Figure 8A presents data over days from implantation, while Figure 8B presents data at Day 29 post-implantation.

[0053] Figures 9A and 9B present the designs of the trastuzumab (HER) WT and HER Mut cassettes. The HER WT cassette comprises the heavy chain of trastuzumab (Figure 9A), while the HER Mut cassette comprises a heavy chain of trastuzumab with a S254A point mutation (Figure 9B).

[0054] Figure 10 shows results for trastuzumab in vitro secretion from

TARGT-trastuzumabs transduced with p Ad-MAR-EF 1 a- HER WT (SEQ ID No: 25) or p Ad-MAR-EF 1 a- HER Mut (SEQ ID No: 26).

[0055] Figure 11 shows results from two independent experiments assessing the total and active amounts of trastuzumab secreted from TARGT-trastuzumabs transduced with p Ad-MAR-EF 1 a- HER WT (SEQ ID No: 25) or p Ad-MAR-EF 1 a- HER Mut (SEQ ID No: 26).

[0056] Figures 12A and 12B show reducing (Figure 12A) and nonreducing (Figure 12B) western-blot analysis of secreted antibody from TARGT-trastuzumabs transduced with p Ad-MAR-EF 1 a-HER WT (SEQ ID No: 25, lanes 5-6) or pAd- M AR-EF 1 a-HER Mut (SEQ ID No: 26, lanes 2-3). Secreted antibody from TARGT- adalimumabs transduced with pAd-MAR-EFla-hTNFl (SEQ ID No: 16) are shown in lanes 11-12. Results with commercially available Herceptin (trastuzumab, lane 8) or Humira (adalimumab, lane 9) are also shown. [0057] Figures 13 A - 13D show flow cytometry analysis of trastuzumab secreted from TARGT-trastuzumabs transduced with pAd-MAR-EFla-HER WT (SEQ ID No: 25) or p Ad-MAR-EF 1 a-HER Mut (SEQ ID No: 26). Left panels present data for Her2-expressing BT-474 cells, while right panels present data for non-Her2- expressing HEK cells. Figure 13 A shows results with Humira (adalimumab) that does not bind Her2. Figure 13B shows results with commercially-available Herceptin (trastuzumab). Figure 13C shows results from Herceptin (trastuzumab) secreted from TARGT-trastuzumabs transduced with p Ad-MAR-EF 1 a-HER WT (SEQ ID No: 25). Figure 13D shows results from Herceptin (trastuzumab) secreted from TARGT- trastuzumabs transduced with p Ad-MAR-EF 1 a- HER Mut (SEQ ID No: 26).

DESCRIPTION OF THE SEQUENCES

[0058] Table 1 provides a listing of certain sequences referenced herein.

Figure imgf000013_0001
AGCGGAGTCCACACTTTTCCTGCGGTCCTCCAGTCCTCCGGGCTTTACAGCCTGAG TAGTGTGGTTACCGTCCCCTCATCCTCCCTGGGTACCCAGACCTACATTTGTAATG TGAACCATAAGCCAAGCAATACAAAGGTGGATAAAAAGGTGGAGCCAAAAAGCTGC GATAAAACACATACTTGCCCTCCTTGCCCAGCGCCCGAGTTGCTCGGCGGCCCTTC CGTATTTCTTTTTCCACCGAAACCGAAGGATACACTGATGATCTCTCGGACCCCTG AGGTCACTTGTGTGGTGGTTGACGTTTCACACGAGGACCCAGAAGTGAAGTTTAAT TGGTACGTGGATGGGGTTGAGGTGCACAATGCTAAAACCAAGCCGCGCGAGGAGCA ATATAACTCTACCTATCGAGTGGTGAGCGTGCTCACCGTACTCCATCAGGACTGGC TGAACGGGAAGGAGTACAAGTGCAAGGTTTCAAACAAGGCTCTCCCTGCCCCAATA GAGAAGACCATAAGTAAAGCCAAGGGACAGCCTCGCGAGCCACAGGTCTATACTCT GCCTCCTAGTAGGGACGAGCTCACCAAGAACCAGGTAAGCCTCACCTGCTTGGTCA AGGGCTTTTATCCATCCGACATCGCCGTGGAATGGGAGAGCAACGGACAGCCTGAA AACAACTACAAAACTACCCCACCCGTTCTTGATTCAGATGGGAGCTTTTTTCTGTA CAGCAAGTTGACCGTCGATAAATCCCGATGGCAGCAGGGAAATGTTTTCTCTTGCT CAGTGATGCATGAAGCGCTGCACAACCACTATACACAGAAGAGCCTTAGCTTGTCT CCAGGAAAA

Optimized GAAGTGCAGTTGGTCGAGTCCGGTGGAGGGCTGGTCCAGCCTGGCAGAAGTCTCCG 4 heavy chain GCTGAGTTGCGCAGCCAGCGGATTCACCTTCGACGATTACGCCATGCACTGGGTGC sequence GGCAGGCCCCGGGCAAGGGCCTTGAATGGGTGTCTGCGATCACATGGAATTCCGGA from CATATTGATTACGCCGACAGCGTGGAGGGCCGATTCACCATCAGTAGGGATAATGC adalimumab TAAGAACTCCCTGTACCTGCAGATGAATAGTCTGAGGGCTGAAGACACAGCCGTGT contained ACTATTGCGCAAAAGTCAGCTACCTCTCCACTGCTTCTAGTCTGGACTACTGGGGT in TNF3 CAGGGGACGCTGGTGACGGTTTCTTCCGCATCCACTAAAGGTCCTAGCGTTTTCCC construct CCTCGCCCCCTCTTCTAAGAGCACCTCCGGAGGAACTGCAGCCCTTGGATGCTTGG

TTAAAGATTACTTTCCCGAACCCGTAACCGTAAGCTGGAACAGTGGCGCCCTGACT TCAGGGGTACACACCTTTCCGGCCGTGCTGCAGAGCAGCGGGCTCTATAGCCTTAG CTCAGTCGTGACGGTCCCATCCTCTAGTCTTGGTACTCAAACCTACATCTGCAATG TGAATCACAAGCCTTCTAACACAAAAGTTGATAAGAAAGTAGAACCCAAGAGCTGT GATAAGACACATACTTGTCCTCCCTGTCCGGCCCCCGAATTGCTTGGGGGGCCGAG TGTCTTCCTCTTCCCTCCAAAACCCAAGGACACTCTCATGATTTCAAGGACCCCTG AAGTGACTTGTGTGGTAGTTGACGTGAGCCACGAGGACCCTGAAGTGAAGTTCAAT TGGTATGTGGATGGCGTTGAGGTGCATAATGCAAAGACAAAGCCACGCGAGGAGCA GTACAATTCCACCTATAGGGTGGTATCCGTGCTGACCGTGTTGCATCAGGACTGGC TCAATGGGAAAGAGTATAAATGTAAGGTGTCCAATAAGGCCCTGCCCGCTCCCATT GAAAAAACAATTTCAAAGGCTAAGGGCCAACCCCGCGAACCACAAGTCTACACACT CCCCCCTAGTAGAGATGAGCTGACAAAAAATCAGGTGTCTCTCACATGTCTGGTAA AAGGCTTCTATCCTTCAGATATTGCTGTGGAATGGGAATCAAATGGGCAGCCAGAG AATAACTACAAAACGACACCCCCAGTCCTTGATAGTGACGGGTCCTTCTTCCTCTA CTCTAAACTCACCGTGGACAAGAGTAGATGGCAACAGGGCAATGTGTTCTCCTGTA GCGTCATGCATGAAGCACTGCACAATCATTATACTCAGAAGAGCTTGTCCCTTAGT CCAGGAAAA

Heavy chain GGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAAC 5 signal AGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCC sequence TTTCTCTCCACAGGCGCGCACTCC

containing

intron

CMV GAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCC 6 enhancer ATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAACAGGAAAGTCCC

ATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTAC ATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTG AGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGA ATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCAT TGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCAT TATTGGCACATACATAAGGTCAATAGGGGTG

EFl-alpha ACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCAC 7 promoter ATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCT

AGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCT TTTTCCCCAGGGTGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTC

CpG-free TTAATTAAAATTATCTCTAAGGCATGTGAACTGGCTGTCTTGGTTTTCATCTGTAC 8 MAR from TTCATCTGCTACCTCTGTGACCTGAAACATATTTATAATTCCATTAAGCTGTGCAT

ATGATAGATTTATCATATGTATTTTCCTTAAAGGATTTTTGTAAGAACTAATTGAA human β- TTGATACCTGTAAAGTCTTTATCACACTACCCAATAAATAATAAATCTCTTTGTTC globin gene AGCTCTCTGTTTCTATAAATATGTACCAGTTTTATTGTTTTTAGTGGTAGTGATTT

TATTCTCTTTCTATATATATACACACACATGTGTGCATTCATAAATATATACAATT TTTATGAATAAAAAATTATTAGCAATCAATATTGAAAACCACTGATTTTTGTTTAT GTGAGCAAACAGCAGATTAAAAGGCTAGCCTGCAG

MAR 5 ' AGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAA 9 region from ATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAAAATGGG human IFN- AAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGTGGGGAT beta gene AGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGATATATG

TAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGTCTTTTT CTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGAACCCAT ATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAG GCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGACTTCAGT GTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATTGTTAAT TAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAGAAAAGT CAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATTGTAATA TGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGACAAAAAT TTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTCAATCTC AGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAAAAGGCT AAGTAACATCTGTGGCTTAATTAA

SV40 CCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTG 10 polyadenyla AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTA tion signal TAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT

CAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAT GGAATTC

An CGGGCAAAACGG 11 exemplary

furin

nucleic

acid

sequence

An GCTCCCGTTAAACAGACGCTGAATTTCGATCTCCTGAAGTTGGCCGGAGACGTCGA 12 exemplary ATCAAACCCCGGCCCA

2A nucleic

acid

sequence

An ATGATAATATGGCCACAACCATG 13 exemplary

IRES

sequence

Sequence of GTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGGTCGACTCTAGACTGCCAT 14 the TNF1 GGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA construct CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC

CTTTCTCTCCACAGGCGCGCACTCCGACATCCAAATGACACAGAGTCCTTCCTCCT

Description TGTCAGCTAGTGTTGGAGACCGCGTTACTATCACATGCAGGGCGTCACAAGGCATC of the AGGAATTACTTGGCGTGGTACCAGCAGAAGCCTGGAAAAGCCCCAAAACTGCTGAT elements : ATACGCAGCCAGCACACTTCAATCAGGCGTGCCCTCTAGGTTCTCTGGCTCCGGTT PLAIN FONT CCGGAACCGACTTCACACTCACCATATCCTCACTGCAACCTGAAGACGTGGCCACA

encodes the TACTATTGTCAGCGCTATAATAGGGCACCCTACACTTTTGGCCAAGGGACGAAAGT plasmid GGAAATAAAAAGGACAGTGGCAGCTCCGTCCGTTTTTATCTTCCCTCCATCCGATG overhang; AGCAGCTTAAGTCTGGGACTGCTTCCGTAGTGTGTTTGCTGAATAATTTTTATCCC "atg" CGAGAAGCAAAGGTTCAGTGGAAGGTCGATAATGCCCTGCAGAGTGGCAATAGTCA encodes the GGAGTCCGTAACCGAGCAGGACTCTAAGGACTCCACCTATTCCCTGAGTTCCACCT start TGACCCTTTCCAAGGCCGACTATGAGAAGCACAAAGTATACGCCTGCGAGGTAACT codon; CACCAGGGATTGAGCTCCCCAGTGACAAAGTCATTTAATCGGGGCGAGTGCCTGTC UNDERLINE CAAGGCCGACTACGAAAAGCACAAAGTGTACGCCTGTGAAGTCACCCATCAGGGCC

encodes the TGTCATCTCCAGTCACGAAGTCATTCAATCGAGGGGAGTGCCGGGCAAAACGGi! l: signal

peptide ; m mmt i GGKTGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAG

GTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGA THICK CATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAAGrGCAGCrrG!TGGAG!r

UNDERLINE CTGGCGGTGGCCTCGTGCAGCCAGGCCGGAGCCTGCGGCTGAGCTGTGCAGCCAGC

encodes the GGGTTCACCTTCGATGATTATGCTATGCACTGGGTTCGCCAGGCCCCCGGAAAGGG intron; CCTGGAGTGGGTCTCAGCTATCACATGGAATTCCGGACACATCGACTACGCCGACA

BOLD GCGTGGAGGGGCGCTTTACCATTTCAAGGGACAACGCTAAAAACAGCCTGTACCTT

encodes the CAGATGAACTCCCTGCGGGCGGAAGACACAGCGGTGTACTACTGTGCCAAGGTGAG optimized CTACCTGTCCACAGCATCCTCATTGGACTATTGGGGCCAAGGCACGCTGGTTACCG light chain TTTCCAGCGCAAGCACAAAGGGACCTAGTGTGTTCCCGTTGGCCCCTTCAAGCAAA of TCCACGAGTGGAGGCACCGCTGCACTGGGCTGCCTTGTAAAGGACTACTTCCCGGA adalimumab ; GCCAGTGACTGTGTCATGGAACAGTGGCGCCCTGACAAGCGGAGTCCACACTTTTC

DASHED CTGCGGTCCTCCAGTCCTCCGGGCTTTACAGCCTGAGTAGTGTGGTTACCGTCCCC

UNDERLINE TCATCCTCCCTGGGTACCCAGACCTACATTTGTAATGTGAACCATAAGCCAAGCAA

encodes the TACAAAGGTGGATAAAAAGGTGGAGCCAAAAAGCTGCGATAAAACACATACTTGCC

Furin CTCCTTGCCCAGCGCCCGAGTTGCTCGGCGGCCCTTCCGTATTTCTTTTTCCACCG sequence ; AAACCGAAGGATACACTGATGATCTCTCGGACCCCTGAGGTCACTTGTGTGGTGGT m amm TGACGTTTCACACGAGGACCCAGAAGTGAAGTTTAATTGGTACGTGGATGGGGTTG encodes the AGGTGCACAATGCTAAAACCAAGCCGCGCGAGGAGCAATATAACTCTACCTATCGA

2A GTGGTGAGCGTGCTCACCGTACTCCATCAGGACTGGCTGAACGGGAAGGAGTACAA sequence ; GTGCAAGGTTTCAAACAAGGCTCTCCCTGCCCCAATAGAGAAGACCATAAGTAAAG

BOLD CCAAGGGACAGCCTCGCGAGCCACAGGTCTATACTCTGCCTCCTAGTAGGGACGAG

ITALICS CTCACCAAGAACCAGGTAAGCCTCACCTGCTTGGTCAAGGGCTTTTATCCATCCGA

encodes the CATCGCCGTGGAATGGGAGAGCAACGGACAGCCTGAAAACAACTACAAAACTACCC optimized CACCCGTTCTTGATTCAGATGGGAGCTTTTTTCTGTACAGCAAGTTGACCGTCGAT heavy chain AAATCCCGATGGCAGCAGGGAAATGTTTTCTCTTGCTCAGTGATGCATGAAGCGCT of GCACAACCACTATACACAGAAGAGCCTTAGCTTGTCTCCAGGAAAATGAGGNTCCC adalimumab CGGGAGATATCCTAGGCTTGG

Sequence of GTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGGTCGACTCTAGACTGCCAT 15 the TNF3 GGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA construct CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC

CTTTCTCTCCACAGGCGCGCACTCCGACATCCAGATGACGCAGTCCCCAAGCTCAC

Description TGTCCGCCTCTGTAGGTGACCGGGTAACTATCACCTGCAGAGCATCCCAGGGCATC of the CGCAATTACCTGGCCTGGTATCAGCAGAAACCTGGCAAGGCCCCAAAACTCCTCAT elements : CTACGCAGCATCCACCCTTCAGAGTGGCGTACCAAGCCGATTCTCCGGAAGCGGTA

GTGGAACCGACTTTACCCTCACAATCTCAAGTCTGCAGCCTGAAGATGTCGCTACA

PLAIN FONT TATTATTGCCAGAGATACAATAGGGCCCCATACACCTTTGGGCAGGGCACGAAAGT

encodes the GGAAATTAAGCGCACAGTTGCGGCACCAAGTGTGTTTATTTTCCCGCCCAGCGATG plasmid AACAGCTGAAATCCGGCACGGCCAGCGTTGTATGCTTGCTGAATAACTTTTACCCT overhang; AGAGAGGCCAAGGTCCAATGGAAGGTTGACAACGCACTGCAGTCCGGCAACAGTCA

"atg" AGAGAGCGTCACTGAACAAGATTCCAAGGACAGTACATACTCACTCAGCTCCACAC encodes the TGACACTCTCCAAGGCCGACTACGAGAAGCATAAGGTCTACGCTTGCGAGGTAACG start CATCAGGGCCTTTCTAGCCCAGTTACCAAAAGTTTCAATCGAGGCGAATGCCTGTC codon; AAAAGCAGACTACGAGAAACACAAGGTTTACGCCTGTGAAGTGACACACCAGGGCT

DASHED TGAGCTCCCCTGTGACAAAATCTTTTAATAGGGGAGAGTGTTGAATGATAATATfiG

UNDERLINE wmmmmmm&KTGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA encodes the GGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATG signal ACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAAGrGCAGrrGGrCGAG peptide ; TCCGGTGGAGGGCTGGTCCAGCCTGGCAGAAGTCTCCGGCTGAGTTGCGCAGCCAG

THICK CGGATTCACCTTCGACGATTACGCCATGCACTGGGTGCGGCAGGCCCCGGGCAAGG

UNDERLINE GCCTTGAATGGGTGTCTGCGATCACATGGAATTCCGGACATATTGATTACGCCGAC

encodes the AGCGTGGAGGGCCGATTCACCATCAGTAGGGATAATGCTAAGAACTCCCTGTACCT intron; GCAGATGAATAGTCTGAGGGCTGAAGACACAGCCGTGTACTATTGCGCAAAAGTCA

BOLD GCTACCTCTCCACTGCTTCTAGTCTGGACTACTGGGGTCAGGGGACGCTGGTGACG

encodes the GTTTCTTCCGCATCCACTAAAGGTCCTAGCGTTTTCCCCCTCGCCCCCTCTTCTAA optimized GAGCACCTCCGGAGGAACTGCAGCCCTTGGATGCTTGGTTAAAGATTACTTTCCCG light chain AACCCGTAACCGTAAGCTGGAACAGTGGCGCCCTGACTTCAGGGGTACACACCTTT of CCGGCCGTGCTGCAGAGCAGCGGGCTCTATAGCCTTAGCTCAGTCGTGACGGTCCC adalimumab ; ATCCTCTAGTCTTGGTACTCAAACCTACATCTGCAATGTGAATCACAAGCCTTCTA

ACACAAAAGTTGATAAGAAAGTAGAACCCAAGAGCTGTGATAAGACACATACTTGT

CCTCCCTGTCCGGCCCCCGAATTGCTTGGGGGGCCGAGTGTCTTCCTCTTCCCTCC "tga" AAAACCCAAGGACACTCTCATGATTTCAAGGACCCCTGAAGTGACTTGTGTGGTAG encodes the TTGACGTGAGCCACGAGGACCCTGAAGTGAAGTTCAATTGGTATGTGGATGGCGTT stop codon; GAGGTGCATAATGCAAAGACAAAGCCACGCGAGGAGCAGTACAATTCCACCTATAG mmmmm GGTGGTATCCGTGCTGACCGTGTTGCATCAGGACTGGCTCAATGGGAAAGAGTATA encodes the AATGTAAGGTGTCCAATAAGGCCCTGCCCGCTCCCATTGAAAAAACAATTTCAAAG IRES GCTAAGGGCCAACCCCGCGAACCACAAGTCTACACACTCCCCCCTAGTAGAGATGA

sequence ; GCTGACAAAAAATCAGGTGTCTCTCACATGTCTGGTAAAAGGCTTCTATCCTTCAG

BOLD ATATTGCTGTGGAATGGGAATCAAATGGGCAGCCAGAGAATAACTACAAAACGACA

ITALICS CCCCCAGTCCTTGATAGTGACGGGTCCTTCTTCCTCTACTCTAAACTCACCGTGGA

encodes the CAAGAGTAGATGGCAACAGGGCAATGTGTTCTCCTGTAGCGTCATGCATGAAGCAC optimized TGCACAATCATTATACTCAGAAGAGCTTGTCCCTTAGTCCAGGAAAATGAGGAT C heavy chain CCGGGAGATATCCTAGGCTTGG

of

adalimumab

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 16 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG EFla-opt TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG hTNFl; CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT

GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

Description GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG of the AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG elements : GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC BLACK GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT CAPITALS ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT

encodes the CCTCCGAGCCGCTCCGACACCGGGAG ISiSSiSiiTTAATTAAAATTATCTCTAAGGC shuttle ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT vector; GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT

TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC

mmmmm ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG encodes the TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA Ascl CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC

Restriction AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG enzyme GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA site ; TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA UNDERLINE CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT

encodes CpG TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG free MAR ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT from human CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA β globin GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG gene ; GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG ITALICS AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC

encodes CMV CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG Enhancer; CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT HEAVY GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT

UNDERLINE CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC

encodes AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT human EFl TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG promotor ; CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC DASHED TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG UNDERLINE ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC

encodes a TCCGACATCCAAATGACACAGAGTCCTTCCTCCTTGTCAGCTAGTGTTGGAGACCG multiple CGTTACTATCACATGCAGGGCGTCACAAGGCATCAGGAATTACTTGGCGTGGTACC cloning AGCAGAAGCCTGGAAAAGCCCCAAAACTGCTGATATACGCAGCCAGCACACTTCAA site ; TCAGGCGTGCCCTCTAGGTTCTCTGGCTCCGGTTCCGGAACCGACTTCACACTCAC lower case CATATCCTCACTGCAACCTGAAGACGTGGCCACATACTATTGTCAGCGCTATAATA encodes GGGCACCCTACACTTTTGGCCAAGGGACGAAAGTGGAAATAAAAAGGACAGTGGCA synthetic GCTCCGTCCGTTTTTATCTTCCCTCCATCCGATGAGCAGCTTAAGTCTGGGACTGC intron ; TTCCGTAGTGTGTTTGCTGAATAATTTTTATCCCCGAGAAGCAAAGGTTCAGTGGA

BOLD AGGTCGATAATGCCCTGCAGAGTGGCAATAGTCAGGAGTCCGTAACCGAGCAGGAC

encodes TCTAAGGACTCCACCTATTCCCTGAGTTCCACCTTGACCCTTTCCAAGGCCGACTA transgene TGAGAAGCACAAAGTATACGCCTGCGAGGTAACTCACCAGGGATTGAGCTCCCCAG (opt hTNFl) TGACAAAGTCATTTAATCGGGGCGAGTGCCTGTCCAAGGCCGACTACGAAAAGCAC from ATG to AAAGTGTACGCCTGTGAAGTCACCCATCAGGGCCTGTCATCTCCAGTCACGAAGTC stop codon; ATTCAATCGAGGGGAGTGCCGGGCAAAACGGGCTCCCGTTAAACAGACGCTGAATT

TCGATCTCCTGAAGTTGGCCGGAGACGTCGAATCAAACCCCGGCCCAGGATGGAGC

TGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGG

encodes CTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCC SV40 poly ACAGGCGCGCACTCCGAAGTGCAGCTTGTGGAGTCTGGCGGTGGCCTCGTGCAGCC Adenylation AGGCCGGAGCCTGCGGCTGAGCTGTGCAGCCAGCGGGTTCACCTTCGATGATTATG signal ; CTATGCACTGGGTTCGCCAGGCCCCCGGAAAGGGCCTGGAGTGGGTCTCAGCTATC BOLD ACATGGAATTCCGGACACATCGACTACGCCGACAGCGTGGAGGGGCGCTTTACCAT ITALICS TTCAAGGGACAACGCTAAAAACAGCCTGTACCTTCAGATGAACTCCCTGCGGGCGG

encodes MAR AAGACACAGCGGTGTACTACTGTGCCAAGGTGAGCTACCTGTCCACAGCATCCTCA 5 ' region TTGGACTATTGGGGCCAAGGCACGCTGGTTACCGTTTCCAGCGCAAGCACAAAGGG from human ACCTAGTGTGTTCCCGTTGGCCCCTTCAAGCAAATCCACGAGTGGAGGCACCGCTG IFN gene CACTGGGCTGCCTTGTAAAGGACTACTTCCCGGAGCCAGTGACTGTGTCATGGAAC

AGTGGCGCCCTGACAAGCGGAGTCCACACTTTTCCTGCGGTCCTCCAGTCCTCCGG

GCTTTACAGCCTGAGTAGTGTGGTTACCGTCCCCTCATCCTCCCTGGGTACCCAGA

CCTACATTTGTAATGTGAACCATAAGCCAAGCAATACAAAGGTGGATAAAAAGGTG

GAGCCAAAAAGCTGCGATAAAACACATACTTGCCCTCCTTGCCCAGCGCCCGAGTT

GCTCGGCGGCCCTTCCGTATTTCTTTTTCCACCGAAACCGAAGGATACACTGATGA

TCTCTCGGACCCCTGAGGTCACTTGTGTGGTGGTTGACGTTTCACACGAGGACCCA

GAAGTGAAGTTTAATTGGTACGTGGATGGGGTTGAGGTGCACAATGCTAAAACCAA

GCCGCGCGAGGAGCAATATAACTCTACCTATCGAGTGGTGAGCGTGCTCACCGTAC

TCCATCAGGACTGGCTGAACGGGAAGGAGTACAAGTGCAAGGTTTCAAACAAGGCT

CTCCCTGCCCCAATAGAGAAGACCATAAGTAAAGCCAAGGGACAGCCTCGCGAGCC

ACAGGTCTATACTCTGCCTCCTAGTAGGGACGAGCTCACCAAGAACCAGGTAAGCC

TCACCTGCTTGGTCAAGGGCTTTTATCCATCCGACATCGCCGTGGAATGGGAGAGC

AACGGACAGCCTGAAAACAACTACAAAACTACCCCACCCGTTCTTGATTCAGATGG

GAGCTTTTTTCTGTACAGCAAGTTGACCGTCGATAAATCCCGATGGCAGCAGGGAA

ATGTTTTCTCTTGCTCAGTGATGCATGAAGCGCTGCACAACCACTATACACAGAAG

AGCCTTAGCTTGTCTCCAGGAAAATGAGGATCCCCGGGAGATATC

CAGACATGA AAGA^

AAAAAATGJTT^

AAGCJTGJ^AATAAA^

AGJGJGJGJGJ^GJGJOT

GAATT AGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCAT TCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAA AATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGT GGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGA TATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGT CTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGA ACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATG GAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGAC TTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATT GTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAG AAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATT GTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGAC AAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTC AATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAA AAGGCTAAGTAACATCTGTGGCTTAATTAAGGCG GQCGGGCCCCTACGTCACCCG CCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG GCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCG AATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGA CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT GACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGA ACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG AACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTA GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCG AACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCA AACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTGCGGCCAT CATCAATAATATACCTTATTTTGGATTGAAGCCAATA

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 17 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG EFla-opt TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG hTNF3; CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT

GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

Description GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG of the AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG elements : GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC BLACK GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT CAPITALS ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT

encodes the CCTCCGAGCCGCTCCGACACCGGGAl lilSliiilTTAATTAAAATTATCTCTAAGGC shuttle ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT vector; GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT mmm TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC m m ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG encodes the TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA Ascl CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC

Restriction AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG enzyme GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA site ; TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA UNDERLINE CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT

encodes CpG TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG free MAR ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT from human CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA β globin GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG gene ; GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG

ITALICS AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC

encodes CMV CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG

Enhancer; CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT

HEAVY GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT

UNDERLINE CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC

encodes AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT human EFl TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG promotor ; CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC

DASHED TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG

UNDERLINE ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC

encodes a TCCGACATCCAGATGACGCAGTCCCCAAGCTCACTGTCCGCCTCTGTAGGTGACCG multiple GGTAACTATCACCTGCAGAGCATCCCAGGGCATCCGCAATTACCTGGCCTGGTATC cloning AGCAGAAACCTGGCAAGGCCCCAAAACTCCTCATCTACGCAGCATCCACCCTTCAG site ; AGTGGCGTACCAAGCCGATTCTCCGGAAGCGGTAGTGGAACCGACTTTACCCTCAC lower case AATCTCAAGTCTGCAGCCTGAAGATGTCGCTACATATTATTGCCAGAGATACAATA encodes GGGCCCCATACACCTTTGGGCAGGGCACGAAAGTGGAAATTAAGCGCACAGTTGCG synthetic GCACCAAGTGTGTTTATTTTCCCGCCCAGCGATGAACAGCTGAAATCCGGCACGGC intron ; CAGCGTTGTATGCTTGCTGAATAACTTTTACCCTAGAGAGGCCAAGGTCCAATGGA

BOLD AGGTTGACAACGCACTGCAGTCCGGCAACAGTCAAGAGAGCGTCACTGAACAAGAT

encodes TCCAAGGACAGTACATACTCACTCAGCTCCACACTGACACTCTCCAAGGCCGACTA transgene CGAGAAGCATAAGGTCTACGCTTGCGAGGTAACGCATCAGGGCCTTTCTAGCCCAG

(opt hTNF3) TTACCAAAAGTTTCAATCGAGGCGAATGCCTGTCAAAAGCAGACTACGAGAAACAC from ATG to AAGGTTTACGCCTGTGAAGTGACACACCAGGGCTTGAGCTCCCCTGTGACAAAATC stop codon; TTTTAATAGGGGAGAGTGTTGAATGATAATATGGCCACAACCATGATGGGATGGAG

CUJVY CTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAG

miMLINE GCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTC encodes CACAGGCGCGCACTCCGAAGTGCAGTTGGTCGAGTCCGGTGGAGGGCTGGTCCAGC

SV40 poly CTGGCAGAAGTCTCCGGCTGAGTTGCGCAGCCAGCGGATTCACCTTCGACGATTAC

Adenylation GCCATGCACTGGGTGCGGCAGGCCCCGGGCAAGGGCCTTGAATGGGTGTCTGCGAT signal ; CACATGGAATTCCGGACATATTGATTACGCCGACAGCGTGGAGGGCCGATTCACCA

BOLD TCAGTAGGGATAATGCTAAGAACTCCCTGTACCTGCAGATGAATAGTCTGAGGGCT

ITALICS GAAGACACAGCCGTGTACTATTGCGCAAAAGTCAGCTACCTCTCCACTGCTTCTAG

encodes MAR TCTGGACTACTGGGGTCAGGGGACGCTGGTGACGGTTTCTTCCGCATCCACTAAAG

5 ' region GTCCTAGCGTTTTCCCCCTCGCCCCCTCTTCTAAGAGCACCTCCGGAGGAACTGCA from human GCCCTTGGATGCTTGGTTAAAGATTACTTTCCCGAACCCGTAACCGTAAGCTGGAA

IFN gene CAGTGGCGCCCTGACTTCAGGGGTACACACCTTTCCGGCCGTGCTGCAGAGCAGCG

GGCTCTATAGCCTTAGCTCAGTCGTGACGGTCCCATCCTCTAGTCTTGGTACTCAA

ACCTACATCTGCAATGTGAATCACAAGCCTTCTAACACAAAAGTTGATAAGAAAGT

AGAACCCAAGAGCTGTGATAAGACACATACTTGTCCTCCCTGTCCGGCCCCCGAAT

TGCTTGGGGGGCCGAGTGTCTTCCTCTTCCCTCCAAAACCCAAGGACACTCTCATG

ATTTCAAGGACCCCTGAAGTGACTTGTGTGGTAGTTGACGTGAGCCACGAGGACCC

TGAAGTGAAGTTCAATTGGTATGTGGATGGCGTTGAGGTGCATAATGCAAAGACAA

AGCCACGCGAGGAGCAGTACAATTCCACCTATAGGGTGGTATCCGTGCTGACCGTG

TTGCATCAGGACTGGCTCAATGGGAAAGAGTATAAATGTAAGGTGTCCAATAAGGC

CCTGCCCGCTCCCATTGAAAAAACAATTTCAAAGGCTAAGGGCCAACCCCGCGAAC

CACAAGTCTACACACTCCCCCCTAGTAGAGATGAGCTGACAAAAAATCAGGTGTCT

CTCACATGTCTGGTAAAAGGCTTCTATCCTTCAGATATTGCTGTGGAATGGGAATC

AAATGGGCAGCCAGAGAATAACTACAAAACGACACCCCCAGTCCTTGATAGTGACG

GGTCCTTCTTCCTCTACTCTAAACTCACCGTGGACAAGAGTAGATGGCAACAGGGC

AATGTGTTCTCCTGTAGCGTCATGCATGAAGCACTGCACAATCATTATACTCAGAA

GAGCTTGTCCCTTAGTCCAGGAAAATGAGGATCCCCGGGAGATATCCTAGGCTTGG

C^ ^^GAT^^

TAAGCT^GCAATAA^

CAGJ5GJ5GAGJ5TJ^^

GGAAT AGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCA

TTCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACA

AAATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTG TGGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTG ATATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGG TCTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGG AACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAAT GGAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGA CTTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGAT TGTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAA GAAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAAT TGTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGA CAAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACT CAATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTA AAAGGCTAAGTAACATCTGTGGCTTAATTAAGGCGCGCCGGGCCCCTACGT ACCC GCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATT GGCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCTGGCGTA ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTC CCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA T T G GT T AAAAAAT GAG C T GAT T T AAC AAAAAT T T AAC G C GAAT T T T AAC AAAAT AT TAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTT GTTTATTTTTC T AAAT AC AT T C AAAT AT GTATCCGCT CAT GAGAC AAT AAC C C T GA TAAATGCTT CAAT AAT AT T GAAAAAGGAAGAGT AT GAGT AT T CAACAT TTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA CGCT GGT GAAAGTAAAAGAT GCT GAAGAT CAGTT GGGT GCACGAGT GGGTTACAT C GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTT TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTT GAGTACT CACCAGT C AC AGAAAAG CAT C T T AC G GAT G G CAT GACAGT AAGAGAAT T AT G C AGT G C T G C CAT AAC CAT GAGT GAT AAC AC T G C G G C C AAC TTACTTCT GAC AA CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCG TGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCG AACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAA GTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGAT GAAC GAAAT AGAC AGAT CGCT GAGAT AG GT G C C T C AC T GAT T AAG CAT T G GT AAC T GT C AGAC CAAGT T T AC T CAT AT AT AC T T T AGAT T GAT T TAAAAC T T CAT T T T T AAT TTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAA C GT GAGT T T T C GT T C CACT GAGC GT CAGAC C C C GT AGAAAAGAT CAAAGGAT CT T C TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTA ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTT AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCA AGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCAC AC AG C C C AG C T T G GAG C GAAC GAC C T AC AC C GAAC T GAGAT AC C T AC AG C GT GAG C TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGC GGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT GCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGA TTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC GAAC GAC C GAG C G C AG C GAGT C AGT GAG C GAG GAAG C G GAAGAG C G C C CAAT AC G C AAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTGCGGCCA T CAT CAAT AAT AT AC CTTATTTTG GAT T GAAG C CAAT A Sequence of GTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGGTCGACTCTAGACTGCCAT 18

TNF2 GGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA construct ; CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC

CTTTCTCTCCACAGGCGCGCACTCCGAGG ACAGTTGG GGAGAGCGGGGG GGAC

Description TGGTCCAGCCCGGCAGGAGTTTGAGACTGAGCTGCGCCGCCAGCGGCTTTACATTC of the GACGACTATGCTATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTCGAATGGGT elements : CAGCGCCATTACTTGGAACAGCGGTCACATAGACTATGCTGACAGCGTGGAAGGCA

PLAIN FONT GGTTCACAATTAGCCGCGACAATGCTAAGAACAGCCTCTACCTTCAGATGAATAGC

encodes the CTGAGAGCCGAGGATACAGCCGTGTATTACTGTGCAAAAGTGAGCTATCTCAGCAC plasmid GGCCTCAAGCCTGGACTACTGGGGCCAAGGAACATTGGTGACCGTCAGTAGCGCAT overhang ; CTACAAAAGGCCCTTCAGTGTTCCCCCTTGCACCCAGTAGCAAGTCAACGTCCGGC

"atg" GGTACCGCCGCCCTGGGATGTCTGGTTAAAGACTACTTCCCCGAGCCCGTGACAGT encodes the GTCTTGGAATTCCGGCGCGCTGACCAGTGGCGTGCACACATTCCCCGCCGTCCTCC start AGTCCAGTGGCCTGTACTCCCTGTCCAGCGTGGTGACAGTTCCCAGTAGCTCATTG codon ; GGCACCCAGACATATATCTGCAATGTCAATCACAAGCCATCAAATACCAAGGTAGA

UNDERLINE CAAAAAGGTTGAACCAAAGTCCTGCGACAAAACCCACACCTGTCCTCCATGTCCTG

encodes the CACCCGAGCTCCTGGGCGGTCCCAGTGTATTTCTCTTTCCTCCGAAGCCCAAGGAT signal ACGTTGATGATCTCTAGGACCCCAGAAGTTACCTGTGTAGTGGTCGACGTCAGCCA peptide ; TGAAGATCCTGAAGTTAAATTCAACTGGTATGTGGACGGAGTGGAGGTTCATAATG

THICK CGAAGACCAAGCCAAGAGAAGAGCAGTATAATAGCACTTACAGAGTCGTGTCTGTG

UNDERLINE CTTACAGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGTAAAGTCTC

encodes the TAATAAGGCATTGCCAGCCCCTATCGAAAAGACCATCTCTAAAGCAAAGGGGCAGC intron ; CAAGAGAGCCACAAGTCTATACCCTCCCACCTTCACGCGATGAGCTCACCAAGAAT

BOLD CAGGTTAGCTTGACTTGTCTGGTGAAGGGCTTCTATCCATCTGATATCGCTGTGGA

encodes the GTGGGAATCAAATGGACAGCCTGAAAATAATTACAAAACTACCCCCCCCGTGCTTG optimized ACTCTGATGGCAGCTTCTTCCTGTACAGCAAGCTCACAGTGGATAAATCCAGGTGG light chain CAGCAGGGCAACGTCTTTTCCTGCTCCGTCATGCATGAGGCTCTGCACAATCACTA of TACCCAGAAGAGTCTCAGCTTGAGCCCTGGGAAACGAGCCAAAAGG<5CTCCCGTCA adalimumab ; m mmm mm mmmm mmm m m m

DASHED Ιδϋϋ¾3!¾!§GGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGG

UNDERLINE GTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCAC

encodes the TTTGCCTTTCTCTCCACAGGCGCGCACTCCGATATACAGATGACGCAATCCCCTAG

Furin TTCCTTGAGCGCATCTGTGGGGGACAGGGTGACAATCACATGCCGCGCCAGCCAAG sequence ; GCATCAGGAATTATCTGGCTTGGTACCAGCAAAAGCCCGGCAAAGCCCCTAAGCTG mmmmm CTTATTTACGCAGCCTCAACCCTCCAGTCCGGAGTACCAAGTAGGTTTTCTGGTAG encodes the TGGCAGCGGCACAGACTTTACACTGACCATAAGCTCCCTGCAGCCCGAAGATGTGG

2A CCACATACTATTGTCAGAGATACAACCGAGCTCCATACACCTTCGGCCAAGGCACC sequence ; AAAGTCGAGATTAAGAGAACAGTGGCAGCTCCGAGTGTTTTTATCTTTCCACCAAG

BOLD CGATGAGCAGCTGAAATCTGGCACTGCATCCGTCGTCTGCCTTCTGAACAACTTCT

ITALICS ACCCACGGGAGGCTAAGGTGCAATGGAAGGTTGATAACGCCCTGCAGAGTGGCAAT

encodes the AGTCAGGAGTCTGTGACAGAACAGGACAGCAAGGATTCAACATACAGTCTGTCTAG optimized CACGCTCACTCTGAGTAAGGCAGATTACGAGAAACACAAAGTGTATGCCTGCGAGG heavy chain TGACCCACCAGGGTCTCTCATCTCCTGTCACCAAATCCTTTAACAGGGGTGAATGT of CTTAGTAAGGCTGATTATGAGAAACATAAAGTGTACGCATGTGAGGTCACACATCA adalimumab GGGGCTGTCCTCCCCCGTGACAAAGTCATTTAACAGGGGGGAATGTTAAGGATCCC

CGGGAGATATCCTAGGCTTGG

Sequence of GTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGGTCGACTCTAGACTGCCAT 19

TNF5 GGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA construct ; CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC

CTTTCTCTCCACAGGCGCGCACTCCGACATCCAGATGACCCAGAGTCCCTCAAGTT

Description TGTCTGCCAGCGTTGGAGACCGGGTGACGATTACTTGTAGGGCCTCCCAGGGAATT of the AGGAACTATCTTGCATGGTATCAGCAGAAACCAGGGAAGGCCCCCAAGTTGCTGAT elements : ATATGCCGCATCCACCCTCCAATCAGGTGTCCCTAGTCGCTTTAGTGGCTCCGGAT

PLAIN FONT CTGGCACCGACTTCACTCTGACCATTAGCAGCCTGCAGCCGGAGGATGTCGCCACA

encodes the TACTATTGCCAAAGGTACAATCGCGCGCCCTACACATTCGGGCAGGGCACCAAGGT plasmid GGAGATAAAGAGAACTGTGGCTGCCCCAAGCGTGTTCATTTTCCCACCAAGCGATG overhang ; AACAACTGAAAAGTGGCACAGCGTCTGTGGTGTGCCTGCTCAATAATTTCTATCCT

"atg" CGCGAAGCAAAAGTCCAATGGAAGGTCGATAACGCACTTCAGAGCGGTAATTCCCA encodes the GGAGTCCGTGACAGAACAGGATTCAAAAGACTCTACTTATTCACTCAGCTCAACTC start TGACCCTTAGCAAGGCCGACTATGAAAAACATAAGGTGTACGCATGCGAGGTGACC codon ; CATCAAGGCCTGAGCTCACCTGTTACAAAGAGCTTTAACAGAGGAGAGTGTCTGTC UNDERLINE TAAAGCTGATTACGAGAAGCACAAAGTTTACGCCTGCGAGGTAACTCATCAGGGCC encodes the TGTCTTCTCCTGTCACTAAATCCTTCAATCGGGGGGAGTGTAGGGCGAAGCGGiaCT signal wmmm mm wmmmmwmmm€m® m mmmmmmm® wm peptide ; «i:l :i3iil:i:lGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAG THICK GTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGA

UNDERLINE CATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAGGTACAGCrrGrGGAAa encodes the GCGGCGGAGGGTTGGTGCAGCCCGGCAGATCACTCCGGCTCTCTTGTGCAGCCTCC intron ; GGCTTCACCTTCGACGACTATGCCATGCACTGGGTGCGACAAGCTCCCGGAAAGGG BOLD ACTGGAATGGGTATCCGCCATTACCTGGAACTCCGGCCACATTGATTATGCTGACT

encodes the CAGTGGAGGGCCGCTTCACAATAAGCCGGGATAATGCTAAAAATAGCCTCTACTTG optimized CAGATGAATAGCCTCAGAGCCGAAGACACAGCTGTATATTATTGCGCCAAGGTGTC light chain CTACCTGTCTACGGCTTCCAGCCTGGACTACTGGGGCCAAGGAACTCTGGTCACCG of TTTCATCCGCCTCAACAAAGGGACCATCCGTCTTTCCTCTTGCTCCTAGCTCCAAG adalimumab ; AGCACTTCCGGGGGGACAGCCGCCCTCGGATGCCTGGTGAAGGACTACTTTCCTGA DASHED GCCAGTGACTGTAAGTTGGAACTCCGGTGCCCTGACCTCCGGTGTTCACACCTTTC UNDERLINE CCGCTGTGCTGCAGAGCAGCGGTCTCTACTCCCTTAGCTCCGTCGTTACCGTTCCC

encodes the AGCTCATCTCTGGGAACACAGACTTACATCTGTAACGTGAATCATAAACCGTCCAA Furin TACAAAGGTGGACAAAAAGGTGGAACCAAAATCTTGTGACAAGACTTAAGGNTCCC sequence ; CGGGAGATATCCTAGGCTTGG

seine

encodes the

2A

sequence ;

BOLD

ITALICS

encodes the

portions of

the

optimized

heavy chain

of

adalimumab

(heavy

chain

variable

domain and

heavy chain

constant

domain)

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 20 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG EFla-opt TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG hTNF2 ; CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT

GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

Description GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG of the AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG elements : GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC BLACK GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT CAPITALS ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT

encodes the CCTCCGAGCCGCTCCGACACCGGGAiiSiiSii!iii! TTAATTAAAATTATCTCTAAGGC shuttle ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT vector; GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT

TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC

:8ϋΙϋ:Ι1:1 ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG encodes the TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA Ascl CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC

Restriction AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG enzyme GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA site ; TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA

CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT UNDERLINE TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG encodes CpG ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT free MAR CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA from human GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG β globin GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG gene ; AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC

ITALICS CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG

encodes CMV CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT

Enhancer; GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT

HEAVY CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC

UNDERLINE AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT

encodes TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG human EFl CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC promotor ; TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG

DASHED ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC

UNDERLINE TCCGAGGTACAGTTGGTGGAGAGCGGGGGTGGACTGGTCCAGCCCGGCAGGAGTTT

encodes a GAGACTGAGCTGCGCCGCCAGCGGCTTTACATTCGACGACTATGCTATGCACTGGG multiple TGAGGCAGGCCCCTGGCAAGGGCCTCGAATGGGTCAGCGCCATTACTTGGAACAGC cloning GGTCACATAGACTATGCTGACAGCGTGGAAGGCAGGTTCACAATTAGCCGCGACAA site ; TGCTAAGAACAGCCTCTACCTTCAGATGAATAGCCTGAGAGCCGAGGATACAGCCG lower case TGTATTACTGTGCAAAAGTGAGCTATCTCAGCACGGCCTCAAGCCTGGACTACTGG encodes GGCCAAGGAACATTGGTGACCGTCAGTAGCGCATCTACAAAAGGCCCTTCAGTGTT synthetic CCCCCTTGCACCCAGTAGCAAGTCAACGTCCGGCGGTACCGCCGCCCTGGGATGTC intron ; TGGTTAAAGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAATTCCGGCGCGCTG

BOLD ACCAGTGGCGTGCACACATTCCCCGCCGTCCTCCAGTCCAGTGGCCTGTACTCCCT

encodes GTCCAGCGTGGTGACAGTTCCCAGTAGCTCATTGGGCACCCAGACATATATCTGCA transgene ATGTCAATCACAAGCCATCAAATACCAAGGTAGACAAAAAGGTTGAACCAAAGTCC

(opt hTNF2) TGCGACAAAACCCACACCTGTCCTCCATGTCCTGCACCCGAGCTCCTGGGCGGTCC from ATG to CAGTGTATTTCTCTTTCCTCCGAAGCCCAAGGATACGTTGATGATCTCTAGGACCC stop codon; CAGAAGTTACCTGTGTAGTGGTCGACGTCAGCCATGAAGATCCTGAAGTTAAATTC

AACTGGTATGTGGACGGAGTGGAGGTTCATAATGCGAAGACCAAGCCAAGAGAAGA

GCAGTATAATAGCACTTACAGAGTCGTGTCTGTGCTTACAGTGCTGCATCAGGACT

encodes GGCTGAACGGCAAGGAGTATAAGTGTAAAGTCTCTAATAAGGCATTGCCAGCCCCT

SV40 poly ATCGAAAAGACCATCTCTAAAGCAAAGGGGCAGCCAAGAGAGCCACAAGTCTATAC

Adenylation CCTCCCACCTTCACGCGATGAGCTCACCAAGAATCAGGTTAGCTTGACTTGTCTGG signal ; TGAAGGGCTTCTATCCATCTGATATCGCTGTGGAGTGGGAATCAAATGGACAGCCT

BOLD GAAAATAATTACAAAACTACCCCCCCCGTGCTTGACTCTGATGGCAGCTTCTTCCT

ITALICS GTACAGCAAGCTCACAGTGGATAAATCCAGGTGGCAGCAGGGCAACGTCTTTTCCT

encodes MAR GCTCCGTCATGCATGAGGCTCTGCACAATCACTATACCCAGAAGAGTCTCAGCTTG

5 ' region AGCCCTGGGAAACGAGCCAAAAGGGCTCCCGTCAAACAGACACTGAACTTCGACCT from human GCTGAAACTTGCAGGGGATGTTGAGTCTAACCCTGGCCCCGGATGGAGCTGTATCA

IFN gene TCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGG

TCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCG

CGCACTCCGATATACAGATGACGCAATCCCCTAGTTCCTTGAGCGCATCTGTGGGG

GACAGGGTGACAATCACATGCCGCGCCAGCCAAGGCATCAGGAATTATCTGGCTTG

GTACCAGCAAAAGCCCGGCAAAGCCCCTAAGCTGCTTATTTACGCAGCCTCAACCC

TCCAGTCCGGAGTACCAAGTAGGTTTTCTGGTAGTGGCAGCGGCACAGACTTTACA

CTGACCATAAGCTCCCTGCAGCCCGAAGATGTGGCCACATACTATTGTCAGAGATA

CAACCGAGCTCCATACACCTTCGGCCAAGGCACCAAAGTCGAGATTAAGAGAACAG

TGGCAGCTCCGAGTGTTTTTATCTTTCCACCAAGCGATGAGCAGCTGAAATCTGGC

ACTGCATCCGTCGTCTGCCTTCTGAACAACTTCTACCCACGGGAGGCTAAGGTGCA

ATGGAAGGTTGATAACGCCCTGCAGAGTGGCAATAGTCAGGAGTCTGTGACAGAAC

AGGACAGCAAGGATTCAACATACAGTCTGTCTAGCACGCTCACTCTGAGTAAGGCA

GATTACGAGAAACACAAAGTGTATGCCTGCGAGGTGACCCACCAGGGTCTCTCATC

TCCTGTCACCAAATCCTTTAACAGGGGTGAATGTCTTAGTAAGGCTGATTATGAGA

AACATAAAGTGTACGCATGTGAGGTCACACATCAGGGGCTGTCCTCCCCCGTGACA

AAGTCATTTAACAGGGGGGAATGTTAAGGATCCCCGGGAGATATCCTAGGCTTGGC

CAGACATJSATJAGATAC

AAAAAATJ^CTTT^^

5 i T AACAAG^^

AGJGJGAGJGJ^^ GAA^CAGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCAT TCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAA AATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGT GGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGA TATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGT CTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGA ACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATG GAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGAC TTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATT GTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAG AAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATT GTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGAC AAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTC AATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAA AAGGCTAAGTAACATCTGTGGCTTAATTAAGGCGCGCCGGGCCCCTACGT ACCCG CCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG GCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCG AATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT T G GT T AAAAAAT GAG C T GAT T T AAC AAAAAT T T AAC G C GAAT T T T AAC AAAAT AT T AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG TTTATTTTTC T AAAT AC AT T C AAAT AT GTATCCGCT CAT GAGAC AAT AAC C C T GAT AAATGCTT CAAT AAT AT T GAAAAAGGAAGAGT AT GAGT AT T CAACAT TTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC GCT GGT GAAAGTAAAAGAT GCT GAAGAT CAGTT GGGT GCACGAGT GGGTTACAT CG AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGA CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACT CACCAGT C AC AGAAAAG CAT C T T AC G GAT G G CAT GACAGT AAGAGAAT T A T G C AGT G C T G C CAT AAC CAT GAGT GAT AAC AC T G C G G C C AAC TTACTTCT GAC AAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT GAC AC C AC GAT GCCTGTAG CAAT G G C AAC AAC GT T G C G C AAAC TAT T AAC T G G C GA ACTACTTACTCTAGCTTCCCGG C AAC AAT T AAT AGAC T G GAT G GAG G C G GAT AAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG T AAG C C C T C C C GT AT C GT AGT TAT C T AC AC GAC G G G GAGT C AG G C AAC TAT G GAT G AAC GAAAT AGAC AGAT C G C T GAGAT AG GT G C C T C AC T GAT T AAG CAT T G GT AAC T G T C AGAC CAAGT T T AC T CAT AT AT AC T T T AGAT T GAT T TAAAAC T T CAT T T T T AAT T TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTA GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA C AG C C C AG C T T G GAG C GAAC GAC C T AC AC C GAAC T GAGAT AC C T AC AG C GT GAG C T ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCG AAC GAC C GAG C G C AG C GAGT C AGT GAG C GAG GAAG C G GAAGAG C G C C CAAT AC G C A AACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTGCGGCCAT CATCAATAATATACCTTATTTTGGATTGAAGCCAATA

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 21 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG EFla-opt TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG hTNF5; CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT

GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

Description GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG of the AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG elements : GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC BLACK GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT CAPITALS ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT

encodes the CCTCCGAGCCGCTCCGACACCGGGAGIiill :@:lTTAATTAAAATTATCTCTAAGGC shuttle ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT

GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT

TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC

Figure imgf000026_0001
ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG encodes the TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA Ascl CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC

Restriction AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG enzyme GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA site ; TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA UNDERLINE CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT

encodes CpG TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG free MAR ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT from human CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA β globin GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG gene ; GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG ITALICS AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC

encodes CMV CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG Enhancer; CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT HEAVY GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT

UNDERLINE CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC

encodes AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT human EFl TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG promotor ; CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC DASHED TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG UNDERLINE ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC

encodes a TCCGACATCCAGATGACCCAGAGTCCCTCAAGTTTGTCTGCCAGCGTTGGAGACCG multiple GGTGACGATTACTTGTAGGGCCTCCCAGGGAATTAGGAACTATCTTGCATGGTATC cloning AGCAGAAACCAGGGAAGGCCCCCAAGTTGCTGATATATGCCGCATCCACCCTCCAA site ; TCAGGTGTCCCTAGTCGCTTTAGTGGCTCCGGATCTGGCACCGACTTCACTCTGAC lower case CATTAGCAGCCTGCAGCCGGAGGATGTCGCCACATACTATTGCCAAAGGTACAATC encodes GCGCGCCCTACACATTCGGGCAGGGCACCAAGGTGGAGATAAAGAGAACTGTGGCT synthetic GCCCCAAGCGTGTTCATTTTCCCACCAAGCGATGAACAACTGAAAAGTGGCACAGC intron ; GTCTGTGGTGTGCCTGCTCAATAATTTCTATCCTCGCGAAGCAAAAGTCCAATGGA

BOLD AGGTCGATAACGCACTTCAGAGCGGTAATTCCCAGGAGTCCGTGACAGAACAGGAT

encodes TCAAAAGACTCTACTTATTCACTCAGCTCAACTCTGACCCTTAGCAAGGCCGACTA transgene TGAAAAACATAAGGTGTACGCATGCGAGGTGACCCATCAAGGCCTGAGCTCACCTG

(opt hTNF5) TTACAAAGAGCTTTAACAGAGGAGAGTGTCTGTCTAAAGCTGATTACGAGAAGCAC from ATG to AAAGTTTACGCCTGCGAGGTAACTCATCAGGGCCTGTCTTCTCCTGTCACTAAATC stop codon; CTTCAATCGGGGGGAGTGTAGGGCGAAGCGGGCTCCCGTAAAGCAGACCCTTAACT

CUJVY TCGACCTGCTGAAATTGGCCGGCGACGTCGAGAGCAACCCAGGCCCCGGATGGAGC

miMLINE TGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGG encodes CTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCC

SV40 poly ACAGGCGCGCACTCCGAGGTACAGCTTGTGGAAAGCGGCGGAGGGTTGGTGCAGCC

Adenylation CGGCAGATCACTCCGGCTCTCTTGTGCAGCCTCCGGCTTCACCTTCGACGACTATG signal ; CCATGCACTGGGTGCGACAAGCTCCCGGAAAGGGACTGGAATGGGTATCCGCCATT

BOLD ACCTGGAACTCCGGCCACATTGATTATGCTGACTCAGTGGAGGGCCGCTTCACAAT

ITALICS AAGCCGGGATAATGCTAAAAATAGCCTCTACTTGCAGATGAATAGCCTCAGAGCCG

encodes MAR AAGACACAGCTGTATATTATTGCGCCAAGGTGTCCTACCTGTCTACGGCTTCCAGC 5 ' region CTGGACTACTGGGGCCAAGGAACTCTGGTCACCGTTTCATCCGCCTCAACAAAGGG from human ACCATCCGTCTTTCCTCTTGCTCCTAGCTCCAAGAGCACTTCCGGGGGGACAGCCG IFN gene CCCTCGGATGCCTGGTGAAGGACTACTTTCCTGAGCCAGTGACTGTAAGTTGGAAC

TCCGGTGCCCTGACCTCCGGTGTTCACACCTTTCCCGCTGTGCTGCAGAGCAGCGG TCTCTACTCCCTTAGCTCCGTCGTTACCGTTCCCAGCTCATCTCTGGGAACACAGA CTTACATCTGTAACGTGAATCATAAACCGTCCAATACAAAGGTGGACAAAAAGGTG GAACCAAAATCTTGTGACAAGACTTAAGGATCCCCGGGAGATA^ CAGACATGA AAGA^

AAAAAATGJTT^

AAGCJTGJ^AATAAA^

GAATT AGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCAT TCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAA AATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGT GGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGA TATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGT CTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGA ACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATG GAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGAC TTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATT GTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAG AAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATT GTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGAC AAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTC AATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAA AAGGCTAAGTAACATCTGTGGCTTAATTAA&GQGCGCZGGGCCCCTACGTCACCCG CCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG GCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCTGGCGTAA TAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCG AATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT AAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGA CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT GACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGA ACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAG TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG AACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTG TCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAAC GTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTA GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT

GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA

GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA

CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCT

ATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG

GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT

CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG

CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT

TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT

TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCG

AACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCA

AACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTGCGGCCAT

CATCAATAATATACCTTATTTTGGATTGAAGCCAATA

Sequence of C XATGA :AA ATACCTTATTTTGGA TGAAGC 22

TNF1 in GAGTTTGTGACGTGGCGCGGGG GTGGGAA GGGGCGGGTGACGTAGTAGTGTGGC ρΔ28 GGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAA vector; GTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTT

TAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGC

Description GGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGC of the GTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAG elements : GTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATATCAA

G AY' GCTTATCGATACCGTAAACAAGTCTTTAATTCAAGCAAGACTTTAACAAGTTAAAA

CAPITALS GGAGCTTATGGGTAGGAAGTAGTGTTATGATGTATGGGCATAAAGGGTTTTAATGG

encodes the GATAGTGAAAATGTCTATAATAATACTTAAATGGCTGCCCAATCACCTACAGGATT inverted GATGTAAACATGGAAAAGGTCAAAAACTTGGGTCACTAAAATAGATGATTAATGGA terminal GAGGATGAGGTTGATAGTTAAATGTAGATAAGTGGTCTTATTCTCAATAAAAATGT repeat GAACATAAGGCGAGTTTCTACAAAGATGGACAGGACTCATTCATGAAACAGCAAAA

(ITR; ACTGGACATTTGTTCTAATCTTTGAAGAGTATGAAAAATTCCTATTTTAAAGGAAA

BLACK ACAGTAACTCACAGGAAATACCAACCCAACATAAAATCAGAAACAATAGTCTAAAG

CAPITALS TAATAAAAATCAAACGTTTGCACGATCAAATTATGAATGAAATTCACTACTAAAAT

encodes the TCACACTGATTTTGTTTCATCCACAGTGTCAATGTTGTGATGCATTTCAATTGTGT shuttle GACACAGGCAGACTGTGGATCAAAAGTGGTTTCTGGTGCGACTTACTCTCTTGAGT vector; ATACCTGCAGTCCCCTTTCTTAAGTGTGTTAAAAAAAAAGGGGGATTTCTTCAATT

!ίϋ:8ϋ:1! CGCCAATACTCTAGCTCTCCATGTGCTTTCTAGGAAACAAGTGTTAACCCACCTTA

:8ϋΙϋ:Ι1:1 TTTGTCAAACCTAGCTCCAAAGGACTTTTGACTCCCCACAAACCGATGTAGCTCAA encodes the GAGAGGGTATCTGTCACCAGTATGTATAGTGAAAAAAGTATCCCAAGTCCCAACAG

Ascl CAATTCCTAAAAGGAGTTTATTTAAAAAACCACACACACCTGTAAAATAAGTATAT

Restriction ATCCTCCAAGGTGACTAGTTTTAAAAAAACAGTATTGGCTTTGATGTAAAGTACTA enzyme GTGAATATGTTAGAAAAATCTCACTGTAACCAAGTGAAATGAAAGCAAGTATGGTT site ; TGCAGAGATTCAAAGAAAATATAAGAAAACCTACTGTTGCCACTAAAAAGAATCAT

UNDERLINE ATATTAAATATACTCACACAATAGCTCTTCAGTCTGATAAAATCTACAGTCATAGG

encodes CpG AATGGATCTATCACTATTTCTATTCAGTGCTTTGATGTAATCCAGCAGGTCAGCAA free MAR AGAATTTATAGCCCCCCTTGAGCACACAGAGGGCTACAATGTGATGGCCTCCCATC from human TCCTTCATCACATCTCGAGCAAGACGTTCAGTCCTACAGAAATAAAATCAGGAATT β globin TAATAGAAAGTTTCATACATTAAACTTTATAACAAACACCTCTTAGTCATTAAACT gene ; TCCACACCAACCTGGGCAATATAGTGAGACCCCATGCCTGCAAAAAAAAAAAAATT

ITALICS AGCCAGGCATGGTAGCATGTACCTGTAGTCCCAGCTACTTGAGAGGTGAGGTGGGA

encodes CMV AAATCACTTTAGTGCAGGATGTTGAGGCTGGAGTGAACTGTGATTGTGCCACTGCA

Enhancer; CTCCAGCCTGGACAATAGAGCAAGACCTTGTCTCAAAAAAATGCATTAAAAATTTT

HEAVY TTTTAAATCTTCCACGTAACACATCCTTTGCCCTCATGTTTCATAAGGTAAAAAAT

UNDERLINE TTGATACCTTCAAAAAAACCAAGCATACCACTATCATAATTTTTTTTAAATGCAAA

encodes TAAAAACAAGATACCATTTTCACCTATCAGACTGGCAGGTTCTGATTAAATGAAAT human EFl TTCTTGGATAATATACAATATTAAGAGAGACTGTAGAAACTGGGCCAGTGGCTCAT promotor; GCCTGTAATCCCAGCACTTTGGGAGGCTGGGTAACATGGCGAACCCTGTTTCTACA

DASHED AAATAAAAATATTAGCTGGGAGTGGTGGCGCACACCTATAGTCCCAGCTACTCAGG

UNDERLINE AGGCTGAGGTGGAAGGATCGCTTGAACCCAGGAGGTTGAGACTGCAGTGAACTGTG

encodes a ATCATTCTGCTGCACTGCACCCCAGCCTGGGCAACAGAGACCTTGTCTCAAAAAAA multiple AAAAAAAAAGAGACAAATTGTGAAGAGAAAGGTACTCTCATATAACATCAGGAGTA cloning TAAAATGATTCAACTTCTTAGAGGAAAATTTGGCAATACCAAAATATTCAATAAAC site ; TCTTTCCCCTTGACCCAGAAATTCCACTTGAATAAAGCTGAACAAGTACCAAACAT

GTAAAAGAATGTTTCTTCTAGTACAGTCGGTAAGAACAAAATAGTGTCTATCAATA lower case GTGGACTGGTTAAATCAGTTATGGTATCTCCATAAGACAGAATGCTATGCAACCTT encodes TAAAATATATTAGATAGCTCTAGACACACTAATATTAAAAGTGTCCAATAACATTT synthetic AAAACTATACTCATACGTTAAAATATAAATGTATATATGTACTTTTGCATATAGTA intron ; TACATGCATAGCCAGTGCTTGAGAAGAAATGTGTACAGAAGGCTGAAAGGAGAGAA BOLD CTTTAGTCTTCTTGTTTATGGCCTCCATAGTTAGAATATTTTATAACACAAATATT

encodes TTGATATTATAATTTTAAAATAAAAACACAGAATAGCCAGACATACAATGCAAGCA transgene TTCAATACCAGGTAAGGTTTTTCACTGTAATTGACTTAACAGAAAATTTTCAAGCT (opt hTNFl) AGATGTGCATAATAATAAAAATCTGACCTTGCCTTCATGTGATTCAGCCCCAGTCC from ATG to ATTACCCTGTTTAGGACTGAGAAATGCAAGACTCTGGCTAGAGTTCCTTCTTCCAT stop codon; CTCCCTTCAATGTTTACTTTGTTCTGGTCCCTACAGAGTCCCACTATACCACAACT

GATACTAAGTAATTAGTAAGGCCCTCCTCTTTTATTTTTAATAAAGAAGATTTTAG AAAGCATCAGTTATTTAATAAGTTGGCCTAGTTTATGTTCAAATAGCAAGTACTCA

encodes GAACAGCTGCTGATGTTTGAAATTAACACAAGAAAAAGTAAAAAACCTCATTTTAA SV40 poly GATCTTACTTACCTGTCCATAATTAGTCCATGAGGAATAAACACCCTTTCCAAATC Adenylation CTCAGCATAATGATTAGGTATGCAAAATAAATCAAGGTCATAACCTGGTTCATCAT signal ; CACTAATCTGAAAAAGAAATATAGCTGTTTCAATGAGAGCATTACAGGATACAAAC BOLD ATTTGATTGGATTAAGATGTTAAAAAATAACCTTAGTCTATCAGAGAAATTTAGGT ITALICS GTAAGATGATATTAGTAACTGTTAACTTTGTAGGTATGATAATGAATTATGTAAGA

encodes MAR AAACAACAGGCCGGGCGGGTTGGTTCACACGTGTAATCCCAGCACTTTGGGAGGCT 5 ' region GAGGCAGGCAGACTGCCTGAGCTCAGGAGTTCGAGACCAGCCTGGGCAACACGGTG from human AAATCCCGTCTCTACTAAAAATACAAAAAAATTAGCCGGGTGTGGTGACACATGCC IFN gene TGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGT

GAAGGTTGCAGTGAGCCAAGAATGCGCCACTTCACTCCAGCCTGGGAAACAGAGCA AGACTCTGTCTCAAAAAAAACAAAACAAACAAACAAAAAAACAGGCTGGGCGCGGT GGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACCTGAG GTCAGGAGTTCCAGACCAGCCTTGTCAACATGGTGAAACCTCCCCCCGCCGTCTCT ACTAAAAATACAAAAATTAGCCAGGCGTGGTGGCAGGAGCCTGTAATCCCAGCTAC TTGGGAGGCTGAGGCAGGAGAATCGCTTGTACCCAGAAGGCAGAGGTTGCACTGAG CTGAGATGGCACCATTGCACTCCAGCCTGGGGGACAAGAGCGAGATTTCGTCTTTA AAAAACAAAAAACAAAACAAAAAACCATGTAACTATATGTCTTAGTCATCTTAGTC AAGAATGTAGAAGTAAAGTGATAAGATATGGAATTTCCTTTAGGTCACAAAGAGAA AAAGAAAAATTTTAAAGAGCTAAGACAAACGCAGCAAAATCTTTATATTTAATAAT ATTCTAAACATGGGTGATGAACATACGGGTATTCATTATACTATTCTCTCCACTTT TGAGTATGTTTGAAAATTTAGTAAAACAAGTTTTAACACACTGTAGTCTAACAAGA TAAAATATCACACTGAACAGGAAAAACTGGCATGGTGTGGTGGCTCACACTTGTAA TCCCAGTGCTTTGGGAGGCTGAGACAGGAGAGTTGCTTGAGGCCAGGAGTTCAAGA CCGACATGGGGAATGTAGCAAGACCCCGTCCCTACAAAAAACTTTGTAAAAATTTG CCAGGTATGGTGGTGCATACCTGTAGTCCCAGCTACTCGGGAGGCGGAGGCAGAAG GAATCACTTGAGCCCAGGAGTTTGAGGCTGCAGTGAGCTACGATCATACCACAGCA CTCCAGCGTGGACAACAGAGTAAGACCCTATCTCAAAAACAAAACAAAACAAAACA AACAAAAAAAACCACAAGAAAAACTGCTGGCTGATGCAGCGGCTCATGCCTGTAAT CCCAGTATTTTGGGAGGCCCAGGTGGGCGTATCACCTGAGGTCAGGAGTTAGAGAC CAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAATTAGCCAGG CATGTGGCACGCGCCTGTAGTCCCAGTTACTGGGGAGGCTGAAGCAGGAGGATCAC CTGAGCCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCACACCACTGCACTCCAGC CTGGGTGACACAGCAATACCCTACCTCAAAATAAAAAAGAAAAAGAAAAGAAAAGT TGCTGTCCCCGCTACCCCAATCCCAAATCCAAACAGCCTCTCTCATCTCACAGTAA GGGGGAAAAATCACCCAAAAAAGCTAAGTGATCTTTTGAAAACCCAAACTCTTAGA AGTCTAAGATTATTATAGTCAACTCATGAAGTGTCATCATAAAAGATACTCTAATA TTATTTAAGTAGAACCACATATTGGTTGTCTTGGTATGTCTAGCCCCTGGCATACA AAATATTTAATAACACTGATATGGTACCTGTGATGTGAAAATGTACTATGAGTACA GCTTTATAAATACTATATATGTACCTATATACAGAAAAAAATACAACAAAATCATA AAAGCACTTATCTTTGAAAGAGGAGTTACAGCAATTTTATTTAGTTCTTTATTGCT TTGCTATATATTCTAAATTTTTTTCAATGAATATATATCACTTTTAAAAAAATTCA ATGGTCTTTCTTATAAATTATCTTTGGCAGCATGCGTTTTTATATATACATATAAA ATGTATGGGAAATTTTTAAAGGATACATTAAATTAAAGCAAAATATACAAACAAAA AATCAGAATACAAAAAGATAAAAAGATTGGGAAGGGAGGGAGGGAGTAAGGAGGAA GGGTGGGTGGGTATAGAGAAATATACCAAATAATGGTAAGAAGTGGGGTCTTGACA CTTTCTACACTTTTTTTAAATAAAAAAAATTTTTTTCTCTCTCTTTTTTTTTTTTA GAGACGAAGTCTCGCTATGTTGCCCAGGCTGGTCTTGAACTCCTGGGATCAAGAGA TCCTCCTGCCTCAGCCTCCCAAGGTGCTTGGATTACAGGTGTGAGCCACCACGCCT GGTCACTTTCTACACTT T AAT AT AT AT AT T T T T T CAT T T T C AAT GT CAT T T T T AT T AGT T AAT T TAT AAT AC C CAT T CAC CAT TAT AT T CAAAGT CT AT T T GAAGAAAT AAA C C AGAAAGAAT GAAAT AC T C T AG C T C AC AT G C TAT T C AAT AC T AAAT T AC C T T T C A AATCACATTCAAGAAGCTGATGATTTAAGCTTTGGCGGTTTCCAATAAATATTGGT C AAAC CAT AAT T AAAT C T C AAT AT AT C AGT T AGT AC C TAT T GAG CAT CTCCTTTTA C AAC C T AAG CAT T GT AT TAGGTGCT T AAAT AC AAG C AG C T T GAC T T T T AAT AC AT T T AAAAAT AC AT AT TTAAGACT TAAAAT C T T AT T T AT G GAAT T C AGT T AT AT T T T GA GGTTTCCAGTGCTGAGAAATTTGAGGTTTGTGCTGTCTTTCAGTCCCCAAAGCTCA GTTCTGAGTTCTCAGACTTTGGTGGAACTTCATGTATTGTCAGGTTGGCCCGTAAT ACCTGTGGGACAACTTCAGCCCCTGTGCACATGGCCAGGAGGCTGGTTGCAAACAT TTTCAGGTAGGTGGACCAGGACATGCCCCTGGTCATGGCCAGGTGGAGGCATAGTG C TAT AC AG C AG G C AGAAGT C AAT AT T GAT T T GT T T T T AAAGAAAC AT GTACTACTT TCATAAGCAGAAAAAATTTCTATTCTTGGGGGAAAAGATTATGCCAGATCCTCTAG GAT T AAAT G C T GAT G CAT C T G C T AAAC C T T CAC AT AT C AGAAC AT AT T T AC TAT AG AAAGAAT GAAAAT G G GAC AT TTGTGTGT CAC CT AT GT GAACAT T C C AAAAAT AT T T T AC AAC AAC T AAGT AT T T TAT AAAT T T T AT GAAC T GAAAT T T AGT T CAAGT T C T AG GAAAAT AC AAAC C T T G C T AGAT AT TAT AAAAAT GAT AC AAT AT AT AT T CAT T T C AG G C T CAT C AGAAT AT AT C T GT TAT CAC T T GAC AAGAAT GAAAAT G CAC CAT T T T GT A GTGCTTTAAAATCAGGAAGATCCAGAGTACTAAAAATGACTTCTTCCTTGAAGCTT ACTCACCAACTTCCTCCCAGTTACTCACTGCTTCTGCCACAAGCATAAACTAGGAC C C AG C C AGAAC T C C T T GAAAT AT AC AC T T G C AAC GAT T AC T G CAT C TAT C AAAAT G GTTCAGTGCCTGGCTACAGGTTCTGCAGATCGACTAAGAATTTGAAAAGTCTTGTT TAT T T C AAAG GAAG C C CAT GT GAAT T C T G C C C AGAGT T CAT C C C AGAT AT G C AGT C TAAGAATACAGACAGAT CAGCAGAGAT GTATT CTAAAACAGGAATT CT GGCAAT AT AACAAAT T GAT T T C CAAT CAAAACAGAT T T ACAT AC CAT ACT TAT GT CAAGAAGT T GTTTTGTTTTATTGCATCCTAGATTTTATTTTTTTGATTTATGGTTTACTTTAAGC AT AAAAAAT T T GT CAAT AC AAC T C T T C C C AAAAG G CAT AAAC AAAAAT T CAT AAAA C T T G CAT CAC T T GAGAT AC T T C AG GT AT GAAT T CAC AAC T T T GT T AC AAC T T AC T A TAT AT AT G CAC AC AT AT AT AT AT AT T T G G GT AT AT TGGGGGGGTTC T AAT T T AAGA AAT GCATAATT GGCTATAGACAGACAGTT GT CAGAACTT GGCAAT GGGTACGT GCA GGTTCATTATACCAAGTCTACTTGTAGTTGTTCAAAATGTATCATAATACAAGGCC GGGCGAGGTGGCTCACGCCTGTAATCCCAGCATTTTGGGAGGCTAAGGCAGGAGGA TTGCTTGAGGTCAGGAGTTTGTGACCAGCCTGGGCAACAGAGCAAGACCCTGTCTC CAAAAAGAAAAAAAAT AAT T T T T T ACAAAAT AAAAACAAAAT GT AT CAT CAGAC GA AAT T AAAT AAGAG G CAAT T CAT T T AAAT GAC AAC T T T T C C C AG C T T GAC AT T T AAC AAAAAGT CTAAGTCCTCT T AAT T CAT AT T T AAT GAT C AAAT AT C AAAT AC T AAT T T TTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGC AGTGGCGCGATCCTGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCC TGCCTCAGCCTCCCGAGTAGCTGGGATTACAGACATGCGCCACCACGCCCGGCTAA TTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGTTGGTCAGGCTGGTCTTGAAT TTCCCACCTCAGGTGATCTGCCTGCCTCAGCCTCACAAAGCAGTAGCTGGGACTAC AGGCACCCACCACCACACTTGGTTAATTCTTTTGTATTTTTTTTGTAAAGACGGGA TTTCACCATGTTAGCCAGGATGGTCTCGATCTCCTGATCTCATGATCCGCCCGCCT CAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCCCGCCCGGCCATCAAAT AC T AAT T C T T AAAT G GT AAG GAC C CAC TAT T C AGAAC C T GT AT C C T TAT CAC T AAT AT G C AAAT AT T TAT T GAAT AC T T AC TAT GT CAT G CAT AC T AGAGAGAGT T AGAT AA AT T T GAT AC AG C T AC C C T CAC AGAAC T T AC AGT GT AAT AGAT G G CAT GAC AT GT AC ATGAGTAACTGTGAACAGTGTTAAATTGCTATTTAAAAAAAAAGACGGCTGGGCGC TGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAAGTTGATCGCTC GAGGTCAAGAGTTCGAGACCAGCCTGGCCAACGTGGTAAAACCCCGTCTCTACTAA AAAT AC AAAAAAAAAAT T AG C C AG G CAT G GT G G CAC AG G C C T GT AAT C C C AG C T AC T AG G GAG G C T GAGAC AT G GAGAAC T G C T T GAAT C C AG GAG G C AGAG GT T AC AGT GA G C C GAGAT CAT AC CAC T AC AC T C C AG C C T GAGT GAC AGAG C GAGAC T C C GT C T AAA AAAAAAAAAAAAAAAAAAGATACAGGTTAAGTGTTATGGTAGTTGAAGAGAGAACT CAAACT CT GT CT CAGAAGCCT CACTT GCAT GT GGACCACT GAT AT GAAAT AAT ATA AAT AG GT AT AAT T CAAT AAAT AG GAAC T T C AGT T T T AAT CAT C C C AAAC AC C AAAA C T T C C TAT C AAAC AG GT C CAAT AAAC T CAAT C T C TAT AAGAG C T AGAC AGAAAT C T ACTTGGTGGCCTATAATCTTATTAGCCCTTACTTGTCCCATCTGATATTAATTAAC C C CAT C T AAT AT G GAT T AGT T AAC AAT C C AGT GGCTGCTTT GAC AG GAAC AGT T G G AGAGAGT T G G G GAT T G C AAC AT AT T CAAT TAT AC AAAAAT G CAT T C AG CAT C T AC C T T GAT T AAG G C AGT GT G C AAC AGAAT T T G C AG GAGAGT AAAAGAAT GAT TAT AAAT TTACAACCCTTAAAGAGCTTATAGCTGGGCGTGGTGGCTCATGCCTGTAAATCCCA GCACTTTGGGAGGCTGAGGCGGGTGGATCACCTGAGGCCAGAAGTTCAAGACCAGC CTAGCCAACATGGCGAAACCCTGTCTCTACAAAAAATACAAAAATTAGCCGGGTGT GGTGGCACGTGCCTGTAGTCCCAGTTACTTGGGAGGCCGAGGCAGGAGAATCGCTT GAACCTAGGAGGTGGAGGCTGCAGTGAGCCGAGATTGTGCCACTGCACTCCACTTC AGCCTGGGCGACAAGAGCAAGACTCCGTCACAAAAAAAAAAAAAAAAAAAAAGCTT AAAAT C T AGT G G GAAAG G CAT AT AT AC AT AC AAC T AAC T GT AT AG C AT AAT AAAG C T CAT AAT C T GT AAC AAAAT C T AAT T C GACAAG C C C AGAAAC T T GT GAT T T AC C AAA AACAGT T AT AT AT ACACAAAAAGT AAAC CT AGAAC C CAAAGT T AC C CAGCAC CAAT GAT TCTCTCCC T AAG C AGT AT C AAGT T T AAAG C AGT GAT T AC AT TCTACTGCCTAG AT T GT AAAC T GAGT AAAG GAGAC C AG C AC CTTTCTGCTACT GAAC TAG C AC AG C C G T GT AAAC C AAC AAG G CAAT G G C AGT G C C C AAC T T T C T GT AT GAAT AT AAGT T AC AT CTGTTTTATTATTTGTGACTTGGTGTTGCATGTGGTTATTATCAACACCTTCTGAA AGAACAACTACCT GCT CAGGCT GCCATAACAAAATACCACAGACT GAGT GACTTAA CAGAAACTTATTTCTCACAGTTTTGGAGGCTGGGAAGTCCAAAATTAAGGTACCTG CAAGGTAGGTTTCAATCTCAGGCCTCTTCTTTGGCTTGAAGGTCTTCTAACTGTGT GCTCACATGACCTCTTCTAACAAGCTCTCTGGTGTCTCTTTTTTTTTTTTTTTCTT TTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTACAGTGGCACAATCTGG GCTCACTGCAACCTCCAACTCCCGGGTTCAAGTGATTCTCATGCCTCACCCTCCCG AGTAGCTTGGATGACAGGAGCCCGCTACCACACCCAGCTAATTTTTGTATTTTTAG TAGAGATGGTGTTTCACTACATTGGCCAGGCTGGTCTCAAACTCCTGACCTCGTGA TCCACCCACCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTGCGCCC GTCCTGGTGTCTTTTCATATAAGGGCACTAATCCAATCAGACCTGGGCCCAACCCT CCCGACTTCTTCTAACTGTAATTACCTTCCAAAGGCCCTGTCTCCAAATACCATCA CACTGGGGGTTAGGACTTCAAAAAAGGTATGGGGGGGGTGTGGGAGGACATAAATG C T C AGT C CAT AAC AAG C AC C C AAC AT AAAAAT G G C T AGAAC AGAT CACAAAAAAAA GGTCCTGTATGGCTTTGGGGAAGGGCTCAACCCCAAAATATCTGAGAGCTCTGGAG GGGCCTAGAAGTGGTAAATGAATGAAAACGTGGTTACTCTCCAGATCTGCCTTTCC C AAAT AT G G C CAT TCTTGGCT GAAT C AGAAAT C AAAG GAC AG GT TAT T AAT T AC T A GCTCTAAGTTACTTACCATTTGCTGAGACAGTTCAGAAATCTGACTGCATCTCCTC AGAGAT C T AGAAC AC AGT T C T C AAAT T C T AAC TTACTTGT GAT AT AC T T GT GAAT G AT AAAAAT C G C T AC AG GT AC T T T TAT T AAT C T GAAAGAGT AT T GAGAAAT T AC C T T TCATTCTGACTTTTGTCTGGAATGAAAATCAATACTTTTGCTATAATCGATTACTG AAAT AAT T T T AC T T T C C AGT AAAAC T G G C AT T AT AAT T T T T T T T AAT T T T T AAAAC T T CAT AAT TTTTTGCCAGACTGACC CAT GT AAAC AT AC AAAT T AC T AAT AAT T AT G CACGTCACATCTGTAATAATGGCCTTCATGTAAACATTTTTGTGGTTTACACATAA AAT C T C T AAT T AC AAAG C TAT AT TAT C T AAAAT T AC AGT AAG C AAGAAAAT T AAT C C AAG C T AAGAC AAT AC T T G C AAC AT CAAT T CAT CAT C T GT GACAAG GAC T G C T T AA GTCTCTTTGTGGTTAAAAAGGAAAAAAAAAAAAAAGACATGTTGGCCAGATGCGGT GGCTCACACCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCACCCCTG GCCTGCCCAACATGGTGAAACCCCGTCTCTACTAAAAACACAAAAATTAGCTGGGC GTGGTGGCGGGCGCCTGTAATTCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGC T AGAAC C C AG GAG G C AGAGAT T G C AGT GAG C T GAGAT T G C AC CAT T G C AC T AC AGT CT GGGCAACAAAAGT GAAACT CCAT CTTAAAAAAAAAAAGACAAT GTT CGT GGGT C CAAACAAGACTTAAT GGAAGT GAGT CTAAAAAT GAGCTAT GT GGGCCAGGCGTAGT GGCTCCCACCTGTAATCCCAGCACTTTGGGAGGCCGAAGCAGGCAGATCATGAGGT C AG GAGAT G GAGAC CAT C C T G G C C AAC AC G GT GAAAT CCTGTCTC T AC AAAAAT T A GCTGGGCGTGGTGGTGCCTGCCTGTAATCCCAGCTACTCAGAAGGCTCAGGCAGGA GAATCGCTTGAACCAGGGAGTCGGTGGCTAGAGTGAGCCGAGATTGCATCACTGCA CTCCTGCCTGGT GAC AGAG C AAGAC T C CAT C T CAAAAAAAAC AAAC AAAAAT AAAA GAT AAAAAT GAG C T AT GT GAAT TAAAAGAG GT AT AAC AAT AGAT AAAC CAT AT T T T ATTTAATTCCTAGTAATGAGTAATATTTCCAAACTTCTGGAATGGGCAGAAATTGC T AGT T G G CAT AT TTTTACCTTTTATATT C AGAT AC AT T AAAAT T C T C AAAAAAAAA CACCTCAAAGCAGATGATCCGCCATCTCCTTGGATAATTTGTGTTAACTCAGGATA AC AGAAAAC C AAAAT TATGAGTTACT GAT G CAAT AT T C C T AAAT GT AAAAAT AAT T AAAG C T AAT AGT AGAT T CAT C T T C CAAT T T CAT AT C AGT C T T AC AAAT AAAC T AC A TATATAACTTGCTTGCCTTCCCTTCTGAGGGATAAAGCTGTTAGAAGAATTAAAAT C AG CAT T C T T GAC TAT T C AAC C AAG G GAG G GAT AAAT TAT TACT CAT T C T AG G GAC AT G G G C T CAT AAC T AC T AC AT GT GT AAG GAC AT GAAT T T AC C CAAT AT T AC AAT T T TTCCTTTTATTAGTGTGTACAGTGGAAGAATAGACATGTTCACTCTGGACAAAAAA AAAAT TAT AC T TAT C AGT TAT CAGAAG C AC AAT GCT GAAGAC AGT AGT T C CAT AAC AAT T T GAAGT AT GT GAT C GAACT AGT AGAT T AT CT T AGT AGT AGT GAAT TAT T GT A AAT GT T AGT AAT T T G G C AG C C AC T G G G C AGAAAAAT AAGAAT T GAG G C T C AAT AT T GAT AT T AAT G GT G GT GAT T GAC AC AT AAAT T T T AT CAAGT C T AC AC AAT AT AAAAT T AC AGAAAG GT AGAAGAGT AT AC C AGT AC AAC T T C AAC AT AT C T T C AC T AC AAG G G AGTAAAAT GAC AT GGCCTAGTTAC TAT C T AAT GAACT GCAGAAAACTAAAAGAAAA CTCCAAGGCAACTCTTCTCTGCTGATCTGGTTGGTCCTTTTCCTACCTTTTGCAAT ACCCAGATACAAACAAT GGATAGAAAACAAAGTAGACTT GTAGTAT GCAGGT CACA GTGCTAAATTCACAGAAAGAAACCCCTGAACTGAACTGCTCTATTTCCTGGTGGTC AC AAAGAGT AAT T C T G GT T T AC AC C T AC AGAT T GAT GT C AAT C T AC AC C C T GT T GA TAACAGTGTGGCCAAGGACAAAAAAAAGGTGCTCCGTTTTACCAATTCTGTAAAAA ATTATTGGCAGGGTAAGCTCGGCTAGGGCAGGATTACATTTCTAGGACTACCATCC C C GAAAT T T AGAAGAT AT TAT AT C C AC AT AAAG CAT AT C T T T C AC AT T AAT T T G C A AAAATCTAAAAGCTTTTTCTTAGCTCAAGTGTGTCCAAGTTTACCCTGGCAGTTTA AAAC GAT AGT T AC AAG C AG CAT G G GT T GT AT C AGAC AC AT T T GAG G G C C AAT T T C A TGTAAGTGATATTGGGCAAGTTACTTCAACTATCTGTGCCTCCAAGGTCATACTAG TGTTTATTTACCTAAAGGGTACCTGTTATGTAACTTTAGGGTGTTTACATTAGATA AT G C C T G C AAAAT AT T T AC T T C AAC G C C T AAAAC AT AGT T AAGT AT T C AAT AAAT A C C T AC TAT T GT C AC T AC T AAC T T AAAAGT T T AGAGAT T AAGAG C AGAAT C T G G G GT GAGACAAACTTAGGTTCAAATCCTAGTATTGTTGGGTAATCTTGGGCAAGTTACTT AAC C T C T C T GAT T T GT GT AAT T T AAAAAAT T AGT T AAT AT AC AT AAC AG G G C T T AG AAGAGT AT C T AG C AC AT AG C AC CAT T T AAG CAT T T GT TAT T G C T AAC AT G C AAAC A AT T T AAG G GAAAGAAAT T T T T T AAAAAG GAAGAG G GAT T T G C AAAC T AAAAAC AAT GAGT AT C T TAT GT T C AAAGAAAAC T AAC AAAC AG C C AG C T C T AG C AAT AAT T AAAT T C AC TAT AT AC T G G G G C AG G CAT C AC AC C C C AAAG C T AAAAG CGTCTACCTAGGCC AGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCAGAGGCGGGCAGA TCGCTTGAGCTCAGGAGTTCAAGACCAGCCTGGACAACATGGCAAAACACCATCTC T AC AAAAAAT AC AAAT AT TAGGCCGGGCG C AGT G G C T C AC G C C T GT AAT C C C AG C A CTTTGGGAGGCCAAGGCGGGTGGATCACCTGAGATCAGGAGTTCGAGAGTAGCCTG G C C AAC AT G GT GAAAC CTCGTCTC TAT TAAAAAT AC AAAAAAT T AG C C AG G CAT G G TGGCAGGCGCCTGTAATCCCAGCTACTCAGGGGGATGAGGTAGGAGAATCGCTTGA ACCCGGGAGGCAGAGGTTGCACTGAGCCGAGATCATGCCACTGTACTCCAGCCCGG GCAACAAGAGCGAAACT CCAT CT CAAAAAAT AAAT AAAT AAAT AAAT AAAAT AAAG TACAAATATTAGCCAGGGATGGTGGTGCGCACCTGTAGTCCCAGCTACTTGGGAGG CTGAAGTGGGAGAATCCCCTGAGCCTGGGGAGAATCACCCGAGCCCGGGAAGTCGA GGCTGCAGTGAGCAGTGATTGTGCCACTGCACTCCATCCTAGGTGACAGAGTGAGA CCCTGTCT C AAAAAAAAGAAAT T G G C AGAAT T AAGT AAGT T GAT GT T T AGAGAT GA AAAAT C AAC AT TTTTTCCT C AG C AAC T GAAT AAAAAC AAC AG C C AC T AC CAT T T T T T T GAGT AC C TAT TTGTAGCC TAT T T T T T AAC T G GT AT T AC T C GAGAGAGAGAGAG C TAGGTTCGAGACAGAGCTCCTTCTCTTAATAACTGTATGACCTAGGGTATGTCTGT TAGCCTCTCTGAGGCTTCAAAGGTTCCTCATCTGTAAAATGGTAATAATCATACCA T T G C T AC AG GGCTGTTTT GAAGAC T AAT TAG GAC TAT GT AAGT AAAC AT GAT GAT G GCTATTATTACTGTTCCCCGCCAGGGGCCATGCAAGGGTTGCTGATTCACATAGAC T GT C T TAT AAT C C T C T C AAT AAC T C C AAGAG GT AG C C AG C AC C T C AGAT AT AC AT A AAAT GAC T T AAG C C C AGAGAG GT GAAGT AAGT T G C C C AC AG C C AC AC AAC T AGT AA ATAGCCCAAACAAGCTGGATTCCCAGTTAGACTCCGTTAATAGCACTGCTCTTTAC CTTAAGTCATTACAATGCCTAATATGAAATAGAATCGCTTCTTTCTTAGGGTTCAA GT G GT T AAT T AT T T AAT GT AT T CAT T C AAC AAAC CAT CAT CGAGGACCTCT T AC AA GCCAAGTACTGTGCTAAGTGCTAGAGTTACGGCGGTGATTCCTGCCCTTAAAAAGT TTTAGTGG GAGAAAC AAC AG GT AAC C AG GT CAT T G C C AAAAC AAC AAAAAT AAT C A TAATAAAGCAGGCTAAAGCATATTTAACTGGCCGGGGTTTTGACTATTTTAGCAAG CAT GAT C AGAAC G GT T GAG GAG G GAG G C C AG C AG CTTGGCCGGTT C AAC AAAC AAG AAAAAAC C AGT GAG G GT G GAG C T AAGAT AC C AGAG G C T GAT T AC G GT T AAGAAT GT TCTTGAAGGTAAGGACCAGATTCTCATTTTCTATATCCTGGGGCATCGGTCAGCAT GGAATCTGGATTCTAGCACATGTGAATTTCGGCTTGAAATGACCTAATGCCTTTTC CCTAGTTCCTTCGTGTGTCAAATACGCATGGTTACCGCTACCAGAGCTGTAGTGGG G C T T C AAT GAG G C CAT GAG CAT C T C C AT AAAGAT GAAC T AC AGT GT GT G C AAAAC T AAAGGCAAAACCTGGTCCCCACACGCCCTCCCAGGTGGTCGCTTTCCGTGCCGAGG CCCCTCCAGAGGTGCCCCGAGAACCTCACCATCGCACCCCAAACTTCCAGGGAAGG GCCTCTCCCGAGAAAGCCCCCACGCCCCCACCCCGCGCCATCATTCCCGAATCTGC CCTCGGCCCCTCCCCGCAGCACGCTCGCAGGCGGCACATGTCAACCAAAACGCCAT TTCCACCTTCTCTTCCCACACGCAGTCCTCTTTTCCCAGGGCTCCCCCGAGGAGGG ACCCACCCCAAACCCCGCCATTCCGTCCTCCCTGCCGCCCTCGCGTGACGTAAAGC CGAACCCGGGAAACTGGCCGCCCCCGCCTGCGGGGTTCCCTGGGCCCGGCCGCTCT AGAACTAGTGGATCCCAATTGAAGGCCTGGTCTAAATGACTCCAAAATCACCACTT AATTCAAGAGACTGATTTCCCTGAGTCAGGCCCCTTAAAGCAGCTATTTCAATGGG ACAGGGAAACAACCCTAGGATCTGGATTAGAATCACTTGGGGGCTGCCACACCCCC AGGGCTCTGATCCTGCCCTTCTCCCACACGCACATTCACATACTGCTGCAGTGACC TTCCATTTCTAATGGGTTCCTGGGCCATCTGTCAGGTATAGGGAATGGAAAAGGGG TTGGGGAGGCTCTGCTTCAGAAAGTTTGTGTCAGGGGCTCCCAGAGCCTCCACAGA T AGAT AG C AG G G GT C C C C AC C C T AC CAT G G C AG C TAT AAAT GT GAT C AAC AT T TAT TGGCCTAGGATACAGCAGTTAGCAAAATGCCTGATGTAGTTCCCACTCCGTGGAGG T T G C AG G C T AG C C AAGAAGT CAT GAGT T C AG C AAC C C T T AC G C AC C AGT G G GAT GA GATTGGACCAGGCCGAGGGTAGTCTTGGGAACACTCAGCATTTGTCTGAGGGCCAG AAGAGGCTGCTTGCCCTCAGACAGGAGGTCAGCATCTTTATTGTAGCCCATGACAC CTCTACACCATTGCTCTTCTGGTCTTATGGAAGACATCTTTGGGCCTGATAACAGC GGAGTCTGTGTCCCACTTGTCCAGGCTGGAGTGCCACATCAGGCACACTCCAGTTG CAGGGACAGCACAGACAAGTTTCAGGAAGGCTGGTGGCCTCCAGGAGGTTAACCTT AT AAG G C C AGAT TGTAACCTAGTT GAAAAAC AT AC AC AT G C CAT GAT AAT AAAAGA AC C T AG G C AC CAT T AC AAGAGAAAAAAT CAT T T T T GT AGAT AC GAG CAT G GAT T C T TGGGTGGGTCAGACACACTGGGCTTGTGCTCTGACTGCACTGTCTCCCCTACCTGA CCTTGGGTAAACCATAAGACTGCTGCATGACTCAGTGTCCACCCCAAAAAAGTACC G GT AGAT AT T G G C C AC AGT AGAT AT CAGCTAGAGT GGACT CT CAT GACAAT GAGGG GAGATGTATTCCCCATCTTAGGCACCTGGGACTCTACCTTCCATCTTCTGCTCCGT GTCTCTCCATCCCCAGGCTCTTCAGAACTCAGGGAGTCCAGAATGTCAGCTCCCAG AT T T C AG C C T T C AGAAAG GAAAC C CAT T AC C GT T C AGT T GAAC AAAT GT T GT C T GA G C C C C AGAT C T G G G C T C AGAG G C CAT C T AG G C TAT GAGAC AAGAG G G GAAC AAAG C ACCGTCTGCACTCACTCACCACACTCACTTGCTGTCCCAGGTCACATCCATCGGGT AGAGAATCTAAGAGGCTGAGCTAGCTCCCGCCACCAGCCCAGCCCACCCCACCTGG CCCCTTCCTTCCTTCTACAAAATATGCACCACCTGTCAAAGGGTGGGCAGTGCCAG G C C T G CAT AC AGAG C AC T GAGT GT AAAAG C AGAC AT G GAC C C T GAC C T C C AG GAG C TTCCAATTTTCTTGAAGAGACAAATCAGCTGGCATTTCAGTCCAGTGTGATCTGCT CTTGGTGAGCACAGACCTAGGGAGTTGGGGCAGCTTCCCAGAAGAACTGCAGTCCA GGCTGAGGGCAGAGAAATGAGGGGAATGGCGAGGAATTGGGGAGCAGGGGGGAGCT CAGTAGAGAGCCAAGGGCGGGAGGTGAGAAGTCCGTGTTGGGCCAGGAGCTACCCT CCGGTGGCCACAGCCGAAGTCGAGGATGCCTTTGGAACTCATCCCCACTTCTCTCT TTCTGTATGTAGCCGTCCAAGAACAAGTCACCTCCAAGTGTAGCCGGATCAAGGCA AGCCCCCCATCTAGCAAGCACTTGATGCCACCCAGAACTGGGCTTCTTCAGAACAA TCTGAGTCCAGGAATGATCCCACTCACCAGGCACCAGAGCTGCGAGGGCATGGGAG TGATCTCACCAACTCTGGGGAAGCGGCAAGGAATTTTCACCTCCAGCCCCCAGTGT CCCATCCTCTCACACTCAGGCCAGACTCCCCTGGGCAGACTTGACTCTGTCTGCCA GCATATGCAGAGCCCCAAGGCCACCCCACCAGAAGTGCCCCTGCCTGGGTTCTGTC CCAGCTCCCTGGGCACCCAGTCCTTGAGTCCCCACCAGCTCAGACGGCCTAGTGTG CCAAGAATGCCCACTGCGTTCAACAATGCTGCATGGGTCACAGCGGCAGCAGCTGT GAC C AC AG C AGT T T C G G G GAAAAC AC C C C T C AG C C AAGT G GAT AAT AG C GT T C AG C AGCACTCACCTTCTGGCCAGGCCTGCCTTCAGAGGCCATCTGATTGGGAGGCACAA GTGCCCGCTGCGATGGGAACACAAGTGCCCCTGGCCAACAACCCCAGCTTCAGCCT GCTGGGCAGCCAGAGCCTCAGGCAGAGCCCGGTACAGGGCCCGGTGCCTGTAGCAA ACACCACCAAGTTCCTCCAGCAGGGTATGGCCAGCTTTAGTCCCCTGAGCCCCATA CAGGGCATCGAGCCACCAAGCTATGTGGCTGCTGCTGCCACCGCTGCTGCTGCTTC TGCCGTTGCTGCCAGCCAGTTCCCAGGTCCGTTCGACAGAACGGATATTCCCCCTG AGCTGCCACCTGCCGACTTTTTGCGCCAGCCCCAACCCCCACTAAATGATCTGATT TCGTCACCTGACTGCAATGAGGTAGATTTCATTGAAGCTCTCTTGAAAGGCTCCTG TGTGAGCCCAGATGAAGACTGGGTGTGCAACTTGAGGCTGATCGACGACATTTTGG AACAGCATGCTGCTGCTCAAAATGCCACAGCCCAGAATTCTGGGCAAGTCACCCAG GATGCTGGGGCACTTTAAATCTGAGCAGGATGCCCATAGAAACCCCCATGGTGACA TCACTCTAGGAAGTGGTGTCGATCCATACCCGCAGTTGTCTCCCGTTACAATTTGA GTGGTGTTGTCAGCCCATGCTTATCCCTCTCTCTACCTGTGACAAAATGGAAAGCT GGTGATTTTTCAAGCTACGTGTACATATTTGAAAATTTTGTAAATGGTTTTCCTAA AC AT T AAT GAC AGAAGT AT T T AT AC T T CAT TTTGTGACTTT GT AAAT AAAG C GAC G GCTTTTGTTTCAGTAGAGTTGTGTTTACTATGCATTGTTTTGTGTTTATTATACAA TGTTACAAATATGCAGACCGTGTTGTTTGCTCCAGTGATACCTTGTTAAGCTAGGT GGCTGAGTCGCTTATGGTTTTAATGCAATGAGCAATGTGGATATGACCAAGAGTTG TTGTGCAAGTTGACAAATGCCAAATAGAAAACCACTTGGCCATTTATTTCTATGTT CACTAAAAATCCTATTGCCTTGTGTGATTCTTAATCTCTTTTGCGAACCTTTCAGT CTCCGCTAGCTCTTTCCTAATGAGCTTTACAGCAGAAGCTGTTTTATCGTTAAGTG C C C C AC AGAGAC AC T T T AC C AG GAG G C T G G GAGAGT T C T C CAGAT T T G G GAGAG G C G C AGAGAC AGT GT GT GAG C C GAG CCCTGTCT C AG C AAT C C AC C T G GAG GAG C T AGA GTATCCTCCTCCCTTTACCATTCAGACCGAGAGAAAAAGCCCAGCTTGTGTGCACC CTCGTGGGGTTAAGGCGAGCTGTTCCTGGTTTAAAGCCTTTCAGTATTTGTTTTGA TGTAAGGCTCTGTGGTTTGGGGGGGAACATCTGTAAACATTATTAGTTGATTTGGG GTTTGTCTTTGATGGTTTCTATCTGCAATTATCGTCATGTATATTTAAGTGTCTGT TATAGAAAACCCACACCCACTGTCCTGTAAACTTTTCTCAGTGTCCAGACTTTCTG TAATCACATTTTAATTGCCACCTCGTATTTCACCTCTACATTTGAAATCTGGCGTC TGTTTCAAGCCAGTGTGTTTTTTCTTCGTTCTGTAATAAACAGCCAGGAGAAAAGT G C C T C TAT GT T T T TAT T T T T C AAG G GAGT AT T C AGT AC C T AC AAAC C C AAGT C AG G AAGCCTGCTAGTGGCTTTGGTTCTTTCAGAGGCTGCTCGATGCCTTGTGTGTCAGA AAGAAAGATTCAGCAGTTTTGCATCATGGCAAAGAAGCCTGTTATTTTGGGGCTCA G C C C C T CAT T T T AT AGAG GAT GAAAC AGAG G G G GAT G G GAG GT C AC AAAGAC AAC T GCCCCGGGAGCAGGTGTGGGGGAGACTTGCCCTGAGGGTCTAGACGCTCTGCACCA CCGTCCTGTCTCCCTTGCTGAAGACCACACATGCCCTTCTTTGACCAGACCCTGCC ACCTGATAGGCCAGGACCTGGTAGGCGGGTACCCAGGTTTCATGGATGGAACCACA TCTCCCCAAAAGTGGGGAGGTAGCTACTGGGATGCACGCCTCCCGCCATGTGCTAT AGGAGAGCAGCT GAAGCAACAGTT GGGAT CAGAT GTAGT CACAATT GAAT GCAT CA TCACATTTATCCCTCTAAGTGGCTGGGAGAGTTGATATCCTCATCCCTAAGGTACA AAATGTTCCAATTTGATCAGTGGCTTTCAGGAGCTGAGAAAGGCATGTGCTCTGAG GCAGAGCTGTTATGTCCCGCAGAGCCTAAAAATGCTCTAAGAACATGCTCCCTGCC AAAATT CT CAAT GGCT GT GACAAGGGACAACGAT CGACCAAT GGGGGT GGAAGCAG AC C T C C G C AGT C C AG G G G C C AGAG C T AG GAC AGAG G G GT C G GAGAAAGAGT CAT T T TCCCAACACTCCAGCTCTTGGCCAGTCCTCACACAGTCCCCTCCTGCTTCCTGCTG AGAGAGATATCCTCATAGGTCTGGGTAAAGTCCTTCAGTCAGCTTTCATTCCCTGT CACCAACTTTGTCTCTGTTCTCCCTGCCCGTCTCAGGCAGCACTCCTCAGGAAACC TCTCCAAGAGCCAGCCTCACTGCAGCGCCCACTATTGTCCCTCTGCCTCAAGTGTC CCATCCATGCCAGGCCCCAGGCAGGCTGCAGCTTTCCCTCAGGGCCACACCAAAGC ACTTGGGCTCAGCTGTGCTGTCCCCCTCCATCACTGAGCTCAGGGGCAGCAGGGGT GGGGTGCCAGGAGGCCCATTCACCCTTCTCTGGCTCTGTGTTGGACCCACCTGCCC AGCCACTGCTGCTTAGAACCTACCCGCTGGGAAAATGAAGCCCTCCCGGAGGGGCC ACCTCAACCTGAGAGCCTCACGGATCACAGTTGTCCCCACTCAGCTCTGCCAGCCC TCAGAGACCCATAGATAAAAGCTGAGCTTGGCTCGCAGAGCTGGTTCCATCTTCCA TTCCCAGAGGGTTCAACTTCCTACCCCAACCACACAGGGAACCTCAAGGCTGAGCC AGTGTGGGCTGCAGTGCAGACCAGCTTCCTGGACACGTCCTGCCACCTGACCCCAG GCTGGCCTCACTGCCCCTGGCACTCCTGACCCTATCCTCATTCCTCCTGGCAGTGC GTGTTCTGCCATTCCGCTTTCCCTTAGCTGTCCTCTCACTGTACTGTCAGCTTCTC CTTTTCCAGGTGCCCCCCAGGGGCTTTCCACATGACCCTGTCACCCCACAGCCCAT CCAGCACCAATTCCAGCTCTCTGCCACCCTTCAAAGGAGTGACAGTGCCCTGCTTC ACCTCCCACTCACCCCTCAACCCAGAGCAATCTGGCTCCAGTCTTGCCTCCTTCCC C C T AAGT AC T C T AGT C AC AGT T C C AAAT TCCTCCTGGT C AT AAAG C C AAAT GAAG C TTCCTGGTCCTCAGCGGACTTGCCACTTCAGCAGTACTGGACTCTCTCCTCCCAGA AACCTGTTTCCCCTTGGCTCCTGGAGCCCACACTCTGCTGGAATCCTTCTGCCTCT CTGGCCTGTAGCCTGGCCCTCTCTCCCAACCTGAGGTCCATTCTCTCCTGCTCCTC CACAAGATGTTGCTCCTTCCATTACTTCCTCCCTCTCAACCAAAGCTCCTTCATTA GCTCTTTATCTTCTGGTTTCTTCCCCTGGGCAGACGAATGGATTCAAGAGCCTGTG G C C C AG C AG C C C AG C AC T C C AG GAT C T C AG C AC T T C AG CAT C C C AGT AC C C T AG C A T C T CAAT AC C C C AG C AC C C C AG C AC CAT AGT AT T C C AG C AC C C CAT T GT C C AAG C A T C T C AG C AC T C C AG CAT C C C AG C AC C C C AAC AC T C C AG C AG C C C AGAAT C T C AG C A C C C T AG C AC T G C AG CAT C T C AG GAC C C C AG C AC T T C AG CAT C C C AG C AC AC T AGT A CTCCAGCATCTCGGCACCCCAGCACCTAGGCATCCCAACACCCAGCACCCCAGCAC T T AAG CAT C C C AC C AC T AC AGT AT C T C AAC AC T C C AG C AC C C C AG C AC CAT AGT GT T C C AG C AC C C C AG CAT C C C AAC AC C C C AG C AC T T AAG CAT C C C AAC AC C T C G G CAT C C C AAC AC C C C AG C AC T G C AG CAT C T C AG C AC C T T AG CAT C C C AGT G C C C T AG CAT C T CAAT G C T C C AG C AC AC C AGT AC T AC AGT AT T C C AG C AC C C C AG C AC T C C AG CAT C T C AG C AC T G C AG C AC T G C AG C AC T C C AG CAT C C C AAAAT C C C AG CAT C C C AAC AC C C C AG C AGAC C AG C AGAC C AG CAT C T C AG C AC C G C AG CAT C C AAG GAC TAT C C C AG CAT C C C AG C AAC C C AG C AC C T C AG CAT C C C AAC AC C C C AG CAT T T C AG CAT G G C AA C AC C C C AGT AC C C C AG C AC T T C AG C AC C C C AGT AT C C C AG CAT C T C AG C GAC C C AG TATCACAAAACCTCAGCATCCTAGCACCCCAGCACCCCAGCACCTTAGCACCTTAG CATCCCAGCATCTCAGCGCCTCAGCATCTTGATATTCTGGCTGAGGTCAGCGTGGT GTATCTAGTCAGGGTCCTAACTTTCACTTCGCAGGGAAATGCTGCTGGACTGGGTC TCATGTTGGGCTGAAGCTCTCTAGACCCCTTGAAGACAGCATAAAAGAGCTTGGAG ACGCTGGGTGTCCCCCATGGAAGAGTTCACTCTCATCCTGCTTTGACAACAGCCTT CTCTGGGGTCCCTCACGGGCCCCTCTTTCTTACTGCAAGTTTGTCTCTGAGAAGAC TGTGATGCAGAAGTCACTCAGCTGCCTGTGGCTCCTGAAGAGCTGAAGGTGGAGGC CTGTAGGCCTCCCTATGAGAGGCGCAGAAAAAACCATGATTGCTAGTGGGGAGGTG CTCCCTCTACAACCCACTCCATAATCTGCCCCCGCCCAGCTCTGAGGCCAGCCCCA GGGGAAAATGCCAGATCCCCAGGGAGGTGTGTGAGACCTCAGGGGCTCCCTCCTCC CTTACAGCAGGCTCAGGCCCCTGGGGGCCTCAGGGCCAAGGTCTGTGGGTAAGCTA CTATCTCTCACTTGTCCTCTAGCCACAAAAGCCAGGGAGATCTGGCAATGGACATG AGGTTCTGAAGAAGCACATATGACTGGCTTCCTAATGCGTGGTTGTTCAGTGATTC AAT AAAC AC G CAT G G G C C AG G CAT G G G GAAAT AGAC AAAC AT GAT C C C C AAC C T C T C C C AGAGT GAAC T G G GAG G GAG GAGT GT T CAT C C C T C AG GAT T AC AC C AGAGAAAC AAAC C AG C AG GAGAT AT AT AT GGTTTTGGGGGGT C AAGAAAGAG GAAAAAC C T G G C AAGGCAAGTCCAAAATCATAGGACAGGCTGTCAGGAAGGGCAGCCTGGAACCTCTC AAGCAGGAGCTGATGCTGCAGTCCACAGGCAGAATTTCTTCTTCCTCGGGGAAATC TCAGCTTTGTTCTTAAGGCCTTTCAACTGATTGGCTGAGGTCTGCCCCTTCCCCCA CAT T C T C C AG GAT AAT CTTCCTTACT T AAAGT C AAC TAT T AAT C AC AG C T AC AAAA T C C C T T C AC AG C T AC AC AT AGAT C AGT GT T T GAT T GAC GAAC AG C C C C T AC AG C C T AG C C AAGT T GAC AC AT AAAAC T AAC CAT C AC AG G G G GAC AAAT GAT GTAAACACAT C AAC AAAT AAAAC AGT AAC AAGT T AAG GT C TAT G GAAAAAAC AC AGAAG G G G C AGA GAGAAAGAAAG C AAGAAG GAGAGT C C C AGT TTGCTAGGGCTTGTGG GAAGT G G G GA GCAGTTCTCTTTAGCTAGGATATTTGGGAAAGGCATATCTGAAGGAGTGATATTTG AG C T T AGAT T AAAAGAT G G GAAG GAG C AAG C CAT G C AAAGAG C T AG GAT GT T C C AA G C AGAGAC G GAAC AG C AAGT G C AAAT GT C AG GAG GAAT AGAAG GAG GCTGGTGGGT GGGGTCCAGTGAGCAAGAGGAGGGCAGGCAGGAGAGGGGATGGGGAGGTGGGCAGG CCCAGACCACCCAGGGCCCTGGAGACTATCCTGATCCAACAAGGGAAGCCTTGAGT CACTT CAGT GT CCAT GT GGAGAAT GGACCT CAGACT GAAT GAGGGAGGCAGTAAGG AGGGCCTCTACCTCCAGGGCTTCGCCCTGTGGACTGCGCATAGACATCTCCAACTC AGAAAGTCTGAACCAAACTTTCCATAGTTCCCCCAAGTCTGGGCATCCTCCTACTC AGTGAAAGGCAGCCATCACACCTCCCTGCCCTGCTCCCGGATGCCCCAAATCCTCT TGGTCTCCAAGTCCAGAACCTGAGACTTGTCCTTGATGTTTGTCTTTCCCTCACCC TTTCTGTATTCTGGGAAGATGGGTTTTTTTCCCCCAGATGAATCTGTAAAACTTCT GT GAT C AC AAT AAAAAT T C T G G CAGT AT TAT T T T C T G GAAC AT GAC AAAGT GAT T C AAAAT TAT T TAT C T G GAAGAC T AC AAAAC AAGAAT AG C C AG GAAAT T T C T AAAAAG AAAGAAGAAG GAG GAG GAGAAAGAAG GAG GAG GAAAAG GAG GAGAAGAAGAAAAGA AAAAGAAC C AAGAAAG GGTTCTAGCTCTAC C AAAT AT T AAAAC AT AT CAT GAAG C T AT TT AAAAC AAT AT GGTT GT GGATACT GAAAAAGAT GT GAAT AAAGT GGAAGGAAA ATAAATAGAAAT GCACAT GGGGATT GAGACT GT GAAAAAG G C AG CAT C T C AC AT CA GTGAGGGATGTTCAACACCTGGTGTTGGGAAAACTGGCTAGTCATTTAAACCAAAC AAC TGGGTCCTCTACCT C AC T C C T GAC AT T AAGAT AC AT T T AGAT GAT T C AAAGAG T AAGACAGAAAAAAT AACAC GT GAAAAC AC TAT CAGAAAACAACGT GGGCCAGGT G TGGTGGGTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGACAGATCACC TGAGGTGGGGAGTTCAAGACCAGCCTGACCAACATGGTGAAATCCTGTCTCTACTA AAAATACAAAATTAGCTGAGCGTGGTGGCGCATGCCTGTAATCCCAGCTACTCAGG AGGCCGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTGTGGTGAGCCGAG AT C AC G C CAT T G C AC T C C AG C C T G G G C AAC AAGAGT GAAAAT C CAT C T AAAAAAAA AAAAAAAAG C C AAG GT G GAT AT T T T TAT AGT AT C AG G GT AGAT C AAG C T T C T C C AA T CAT GAC AT GAAAC C C AGAAAC C AT AAAAGAAAAGAAT GAT AAAAT TGCCCACGTA AAGT AAAAAG C T T G C AC AC AGAAAAAC AC CAT AC AG GT T AC AAGAT GAG C AG C AAA AT C AGAGAAAAAAC AT T G C AAT T C AG GAC AC AC AGAG G C TAT T GT T C C T AAT AT T T AAAAAT AAAAGT AGT GGAT T GT CT ACAAAAAGAT GAAGACAAGAAT T T CAGAAAAC CAAATACTGCATGTTTTCACTTACAAGTGGAAGCTAAACACTGAGTACACGTGTAC AC AAAGAAT G GAAC CAT AG G C C AG G C AC C GT G G C T C AC G C C T GT AAT C C CAGT AC T TTGCGAGGCCGAAGCGGGCGGATCACCTGAGGTGAGGAGTTCGAGACCATCCTGGC CAACATGGTGAAACCCAGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGG TGGGTGCCTGTAATCCCAGCTACTCGGGAGGCTGCGGCAGTAGAATCGCTTGAACC CTGGAGGTGGACCTTGCAGTGAGCCGAGATCGCACCACTGCACTCCAGCCTGGGCA ACAGAGT GAGACT CCAT CT CAAAAAAAAAAAAAAGGAATAGAACAATAGACACT GG GGCCTACTTGAGGGAGGAGGGTGAGGATCAAAAACCTGCCTATCAGGTACTATGCT TAT TACCTGGGTGGT GAAAT AAT C T GT AC AC C AAAC C C C AGT GAC AT G C AAT T T AC CGATGTAACAAACCTGCCCATGTACCCGCTGAACCTAAAATAAAAGTTGGAAAAAA AT AT AGAAAT T T T C T T T GT AAT AG C C AAAAAC T G C AAAC AG C C C AG GT GT C TAT T A GT AGAAT G CAT AAAC AAAC T C G G G CAT GT T CAT ACAAT GTAAAACTACT CAT CAAT AAAAAGT GATACTT CT CAGCAAT GAAAAGAAAC TAG C TACT GATACCAGCTACAAC AT G GAT G GAT T T C AAGT G C T T TAT GAT GAGAG C AAGAAG C C AGAC AC AAAAGT GT C TATATATATATACAGTATATATACGTATATATACACATATATACAGTATATATATA CATATACAT GTATATATATACT GTATATATACT GTATATATATACACAGTATATAT ATACATATATACAGT GTATATATACT GT GTATATATACAT GTATATATACT GAGTA TATATACAT GTATATATAT GTAT ACT GTATATATACT GTAT AT AT AT AT ACACATA TATACAGTATATATATACAGTATATACTGTATATATACAGTATATACGTGTATATA T ACAT AT AT ACAGT AT AT AT GT AAAT AT ACAT AT AT ACAGT AT AT AT GT AAAT AT A CATATATACAT GTAT AT AT AT ACACTAT AT AT AT ACAT AT AT AGT GTATATATACA TATATACAT GTAT AT AT T TACT AT AT GAT T C CAT T T AT AT AAAGT GC CAAAACAGT CAAAAATAATCTATGTGGAAAAAATCAACAAAGGGATCCCCCGGGCTGCAGGAATT C GATillillliTTAATTAAAATTAT CT CTAAGGCAT GT GAACT GGCT GT CTT GGT TTTCATCTGTACTTCATCTGCTACCTCTGTGACCTGAAACATATTTATAATTCCAT T AAG C T GT G CAT AT GAT AGAT T TAT CAT AT GTAT T T T C C T T AAAG GAT T T T T GT AA GAAC T AAT T GAAT T GAT AC C T GT AAAGT CTT TAT C AC AC T AC C CAAT AAAT AAT AA ATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATGTACCAGTTTTATTGTTTTTAG T G GT AGT GAT T T TAT TCTCTTTC TAT AT AT AT AC AC AC AC AT GT GT G CAT T C AT AA AT AT AT AC AAT T T T T AT GAAT AAAAAAT TAT TAG CAAT CAAT AT T GAAAAC C AC T G ATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAGGCTAGCCTGCAGGAGTCAATG GGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCCATTGGGTTT TGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAACAGGAAAGTCCCATTGGAGCC AAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCA ATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTCATTAG GGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGG AAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTG CCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCAC A TACA TAAGGTCAA TAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTG CCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGG GTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTG ATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAAGT GCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTTCTCCCTCCTGTGAGTTTGGTA AGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGGTGGGGCAGTGCAGGA AAAGTGGCACTGTGAACCCTGCAGCCCTAGACAATTGTACTAACCTTCTTCTCTTT CCTCTCCT GAC AG GT T G GT GT AC AGT AGT AG^

TCTAGACTGCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA GGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATG ACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGACATCCAAATGACACAG AGTCCTTCCTCCTTGTCAGCTAGTGTTGGAGACCGCGTTACTATCACATGCAGGGC GTCACAAGGCATCAGGAATTACTTGGCGTGGTACCAGCAGAAGCCTGGAAAAGCCC CAAAACTGCTGATATACGCAGCCAGCACACTTCAATCAGGCGTGCCCTCTAGGTTC TCTGGCTCCGGTTCCGGAACCGACTTCACACTCACCATATCCTCACTGCAACCTGA AGACGTGGCCACATACTATTGTCAGCGCTATAATAGGGCACCCTACACTTTTGGCC AAGGGACGAAAGTGGAAATAAAAAGGACAGTGGCAGCTCCGTCCGTTTTTATCTTC CCTCCATCCGATGAGCAGCTTAAGTCTGGGACTGCTTCCGTAGTGTGTTTGCTGAA TAATTTTTATCCCCGAGAAGCAAAGGTTCAGTGGAAGGTCGATAATGCCCTGCAGA GTGGCAATAGTCAGGAGTCCGTAACCGAGCAGGACTCTAAGGACTCCACCTATTCC CTGAGTTCCACCTTGACCCTTTCCAAGGCCGACTATGAGAAGCACAAAGTATACGC CTGCGAGGTAACTCACCAGGGATTGAGCTCCCCAGTGACAAAGTCATTTAATCGGG GCGAGTGCCTGTCCAAGGCCGACTACGAAAAGCACAAAGTGTACGCCTGTGAAGTC ACCCATCAGGGCCTGTCATCTCCAGTCACGAAGTCATTCAATCGAGGGGAGTGCCG GGCAAAACGGGCTCCCGTTAAACAGACGCTGAATTTCGATCTCCTGAAGTTGGCCG GAGACGTCGAATCAAACCCCGGCCCAGGATGGAGCTGTATCATCCTCTTCTTGGTA GCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATA TGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAAGTG CAGCTTGTGGAGTCTGGCGGTGGCCTCGTGCAGCCAGGCCGGAGCCTGCGGCTGAG CTGTGCAGCCAGCGGGTTCACCTTCGATGATTATGCTATGCACTGGGTTCGCCAGG CCCCCGGAAAGGGCCTGGAGTGGGTCTCAGCTATCACATGGAATTCCGGACACATC GACTACGCCGACAGCGTGGAGGGGCGCTTTACCATTTCAAGGGACAACGCTAAAAA CAGCCTGTACCTTCAGATGAACTCCCTGCGGGCGGAAGACACAGCGGTGTACTACT GTGCCAAGGTGAGCTACCTGTCCACAGCATCCTCATTGGACTATTGGGGCCAAGGC ACGCTGGTTACCGTTTCCAGCGCAAGCACAAAGGGACCTAGTGTGTTCCCGTTGGC CCCTTCAAGCAAATCCACGAGTGGAGGCACCGCTGCACTGGGCTGCCTTGTAAAGG ACTACTTCCCGGAGCCAGTGACTGTGTCATGGAACAGTGGCGCCCTGACAAGCGGA GTCCACACTTTTCCTGCGGTCCTCCAGTCCTCCGGGCTTTACAGCCTGAGTAGTGT GGTTACCGTCCCCTCATCCTCCCTGGGTACCCAGACCTACATTTGTAATGTGAACC ATAAGCCAAGCAATACAAAGGTGGATAAAAAGGTGGAGCCAAAAAGCTGCGATAAA ACACATACTTGCCCTCCTTGCCCAGCGCCCGAGTTGCTCGGCGGCCCTTCCGTATT TCTTTTTCCACCGAAACCGAAGGATACACTGATGATCTCTCGGACCCCTGAGGTCA CTTGTGTGGTGGTTGACGTTTCACACGAGGACCCAGAAGTGAAGTTTAATTGGTAC GTGGATGGGGTTGAGGTGCACAATGCTAAAACCAAGCCGCGCGAGGAGCAATATAA CTCTACCTATCGAGTGGTGAGCGTGCTCACCGTACTCCATCAGGACTGGCTGAACG GGAAGGAGTACAAGTGCAAGGTTTCAAACAAGGCTCTCCCTGCCCCAATAGAGAAG ACCATAAGTAAAGCCAAGGGACAGCCTCGCGAGCCACAGGTCTATACTCTGCCTCC TAGTAGGGACGAGCTCACCAAGAACCAGGTAAGCCTCACCTGCTTGGTCAAGGGCT TTTATCCATCCGACATCGCCGTGGAATGGGAGAGCAACGGACAGCCTGAAAACAAC TACAAAACTACCCCACCCGTTCTTGATTCAGATGGGAGCTTTTTTCTGTACAGCAA GTTGACCGTCGATAAATCCCGATGGCAGCAGGGAAATGTTTTCTCTTGCTCAGTGA TGCATGAAGCGCTGCACAACCACTATACACAGAAGAGCCTTAGCTTGTCTCCAGGA AAATGAGGATCCCCGGGAGATATCCTAGGCTTGGCCAGACATGATAAGATACATTG TTG ^GATJ^

TJ^LAAAGJ^AAGJA^

CCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAAATGTATATAGAAGCC CAAAAGACAATAACAAAAATATTCTTGTAGAACAAAATGGGAAAGAATGTTCCACT AAATATCAAGATTTAGAGCAAAGCATGAGATGTGTGGGGATAGACAGTGAGGCTGA TAAAATAGAGTAGAGCTCAGAAACAGACCCATTGATATATGTAAGTGACCTATGAA AAAAATATGGCATTTTACAATGGGAAAATGATGGTCTTTTTCTTTTTTAGAAAAAC AGGGAAATATATTTATATGTAAAAAATAAAAGGGAACCCATATGTCATACCATACA CACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAGGCAAAACTTTAAATC TTTTAGAAAATAATATAGAAGCATGCCATCAAGACTTCAGTGTAGAGAAAAATTTC TTATGACTCAAAGTCCTAACCACAAAGAAAAGATTGTTAATTAGATTGCATGAATA TTAAGACTTATTTTTAAAATTAAAAAACCATTAAGAAAAGTCAGGCCATAGAATGA CAGAAAATATTTGCAACACCCCAGTAAAGAGAATTGTAATATGCAGATTATAAAAA GAAGTCTTACAAATCAGTAAAAAATAAAACTAGACAAAAATTTGAACAGATGAAAG AGAAACTCTAAATAATCATTACACATGAGAAACTCAATCTCAGAAATCAGAGAACT ATCATTGCATATACACTAAATTAGAGAAATATTAAAAGGCTAAGTAACATCTGTGG

cTTAATTAAwmm :ACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTC

GACACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAG TATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAA AACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCA AATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAA TTCCCAACACATACAAGTTACTCCGCCCTAAAACC «CGTCACCCGC0CCG CCC

Nucleotide ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTT 23 sequence of AACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTT optimized GCCTTTCTCTCCACAGGCGCGCACTCCGATATACAGATGACACAGAGCCCCAGCAG trastuzumab CCTCAGCGCCTCCGTCGGCGACAGGGTCACGATTACCTGTCGGGCCAGCCAGGATG WT TTAATACGGCAGTGGCATGGTATCAGCAAAAGCCCGGTAAGGCTCCTAAGTTGCTC

cassette, ATTTACAGCGCTTCCTTCCTTTATTCAGGAGTTCCATCCAGGTTTAGCGGCAGCCG encoding GTCTGGTACCGATTTTACGTTGACCATCTCTAGTCTGCAGCCTGAGGACTTTGCGA heavy chain CCTACTATTGTCAGCAGCACTACACAACTCCCCCCACCTTCGGCCAGGGCACCAAG signal GTCGAGATCAAAAGGACCGTGGCAGCCCCGTCTGTATTCATTTTCCCCCCCTCAGA sequence, CGAGCAGCTTAAGAGTGGAACCGCCTCCGTTGTGTGCCTCCTGAATAACTTCTATC heavy CACGAGAAGCGAAGGTTCAATGGAAGGTAGATAATGCTCTGCAGAGCGGCAATAGT chain, CAGGAGAGCGTCACAGAGCAGGACAGCAAAGACAGTACCTATTCACTGTCCTCAAC furin, 2A, CTTGACATTGTCAAAGGCCGACTACGAAAAGCATAAGGTCTACGCATGCGAAGTTA heavy chain CCCATCAGGGACTCTCCAGCCCGGTCACCAAATCATTTAACAGAGGCGAGTGCCGG signal GCCAAGAGAGCACCAGTTAAGCAAACCCTGAACTTCGACCTGCTTAAGCTCGCCGG sequence, TGACGTAGAATCTAACCCTGGACCTGGATGGAGCTGTATCATCCTCTTCTTGGTAG and light CAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATAT chain, in GGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAGGTGC that order. AGCTTGTGGAATCCGGAGGCGGTCTTGTGCAGCCTGGGGGCTCTCTCCGCCTCTCT

TGCGCTGCTTCCGGCTTTAACATAAAAGACACCTACATTCATTGGGTCAGGCAGGC GCCGGGTAAGGGATTGGAGTGGGTCGCCCGGATCTACCCGACAAATGGGTACACTA GGTATGCAGATTCTGTAAAAGGAAGATTCACCATCTCAGCTGACACGTCAAAGAAT ACCGCATATCTTCAGATGAATTCTCTGAGGGCGGAGGATACTGCCGTGTACTACTG CTCAAGGTGGGGCGGCGATGGCTTTTACGCTATGGACTATTGGGGCCAGGGCACAC TGGTGACAGTGAGCTCCGCCTCTACGAAGGGACCCTCAGTTTTCCCACTGGCCCCT AGTTCTAAAAGCACCTCTGGGGGCACAGCTGCACTCGGATGCCTTGTTAAAGACTA CTTCCCTGAACCTGTGACTGTCTCCTGGAACAGTGGAGCATTGACCTCAGGGGTGC ATACCTTTCCTGCTGTCCTCCAGAGTTCAGGACTCTACTCACTTTCTTCCGTTGTT ACAGTACCCTCATCATCCCTCGGAACCCAGACTTATATCTGTAACGTCAATCATAA GCCCAGCAATACAAAAGTGGATAAAAAGGTGGAACCAAAGTCATGTGATAAAACCC ATACGTGCCCGCCCTGCCCAGCACCTGAGCTGCTGGGCGGGCCTTCTGTATTCCTG TTTCCACCAAAGCCAAAGGACACCTTGATGATTAGCCGAACACCAGAAGTAACCTG TGTGGTCGTAGACGTAAGTCACGAAGATCCAGAGGTCAAGTTCAACTGGTATGTCG ACGGCGTTGAAGTGCACAACGCCAAGACTAAGCCCCGGGAGGAGCAGTACAATTCA ACCTATCGGGTCGTCTCTGTGCTCACAGTCCTCCACCAAGATTGGCTTAATGGGAA GGAGTACAAATGTAAGGTGTCTAACAAAGCACTTCCCGCTCCAATCGAAAAGACTA TAAGCAAGGCAAAGGGCCAACCGCGCGAACCACAGGTTTATACACTGCCCCCAAGT AGGGACGAGCTGACGAAAAACCAGGTTTCCTTGACTTGTCTCGTAAAGGGTTTTTA TCCCTCAGACATCGCAGTTGAGTGGGAAAGCAATGGTCAGCCTGAAAACAACTATA AAACAACACCTCCTGTCCTGGATTCTGATGGCTCCTTCTTTCTGTACAGTAAACTG ACCGTAGATAAAAGCAGATGGCAGCAGGGGAATGTGTTCTCTTGCAGCGTCATGCA CGAAGCTCTGCATAACCACTACACTCAGAAGTCACTCAGCTTGTCTCCAGGGAAGT

AA

Sequence of GTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGGTCGACTCTAGACTGCCAT 24 optimized GGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA trastuzumab CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC Mut CTTTCTCTCCACAGGCGCGCACTCCGACATACAAATGACCCAGTCTCCCTCCAGTC cassette TGAGTGCTAGCGTAGGAGACCGGGTCACCATTACCTGCAGGGCTAGTCAGGACGTG encoding AATACCGCCGTCGCCTGGTACCAGCAGAAGCCAGGGAAGGCCCCAAAGCTGCTGAT heavy chain TTACTCCGCCAGTTTCCTCTACTCAGGTGTACCCAGCAGATTCTCTGGCTCACGGT signal CTGGAACCGATTTCACTCTGACGATCAGCTCTCTGCAGCCGGAAGATTTCGCCACA sequence, TACTACTGTCAGCAACACTACACCACACCCCCAACATTCGGACAGGGGACCAAAGT heavy TGAGATCAAACGCACTGTGGCTGCCCCGAGTGTATTTATATTTCCTCCCAGCGACG chain, AGCAGCTGAAAAGCGGCACTGCATCCGTGGTGTGCCTGCTGAATAACTTCTATCCA furin, 2A, CGGGAAGCAAAGGTCCAATGGAAAGTCGACAATGCTCTGCAATCCGGCAACTCACA heavy chain GGAGAGCGTCACCGAGCAGGACTCCAAAGATTCAACCTACTCACTTTCTAGCACTT signal TGACCCTGTCTAAAGCTGACTATGAGAAGCATAAAGTGTATGCCTGTGAGGTAACC sequence, CATCAGGGACTTTCCTCCCCAGTTACGAAAAGTTTTAATCGGGGCGAATGTCGGGC and light TAAGCGCGCACCTGTGAAACAGACACTCAATTTTGACCTTCTGAAGCTGGCCGGTG chain, in ATGTTGAGAGCAATCCTGGGCCTGGATGGAGCTGTATCATCCTCTTCTTGGTAGCA that order. ACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGG

GTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGAAGTCCAG TTGGTGGAGTCAGGCGGGGGTCTGGTGCAGCCAGGAGGCTCCCTTCGGCTTTCTTG TGCAGCAAGCGGTTTCAATATTAAGGACACCTATATTCACTGGGTGAGACAAGCCC CAGGGAAAGGCCTCGAGTGGGTAGCAAGGATCTACCCTACTAACGGCTACACTCGA TACGCAGACTCCGTCAAGGGGCGGTTCACTATTTCAGCAGACACATCCAAAAACAC TGCTTATTTGCAGATGAACTCCCTGAGAGCCGAAGATACAGCTGTCTATTATTGTT CTAGGTGGGGTGGGGACGGCTTCTATGCCATGGATTACTGGGGACAGGGAACACTT GTCACCGTGAGCAGTGCTTCTACCAAAGGACCTTCAGTGTTCCCACTCGCTCCTTC TTCAAAGAGTACCTCCGGAGGCACCGCCGCGCTTGGGTGTCTGGTAAAGGATTACT TTCCCGAGCCGGTGACCGTTTCCTGGAATTCCGGTGCTCTCACGTCCGGAGTCCAT ACCTTTCCCGCCGTCCTGCAGTCTAGTGGCCTTTATTCCTTGAGTAGCGTGGTGAC CGTGCCAAGCAGCTCACTGGGCACCCAAACTTACATCTGCAACGTGAACCACAAAC CATCCAACACCAAGGTGGATAAAAAGGTTGAACCTAAAAGTTGCGACAAAACACAC ACCTGCCCTCCGTGCCCTGCCCCCGAGCTCCTGGGAGGACCCTCCGTGTTCCTCTT CCCCCCAAAGCCAAAAGACACTTTGATGATAGCACGCACACCCGAAGTGACCTGCG TCGTAGTGGATGTTTCACACGAAGACCCCGAGGTCAAATTTAATTGGTACGTGGAT GGTGTCGAAGTCCATAACGCCAAAACCAAGCCTCGAGAGGAACAGTACAATAGCAC ATACCGGGTGGTCTCAGTGCTCACCGTACTGCATCAAGATTGGCTTAACGGCAAAG AATATAAATGTAAGGTGAGTAACAAAGCCCTCCCTGCGCCTATCGAAAAGACAATT TCAAAAGCTAAGGGACAGCCCCGGGAGCCCCAGGTGTATACTCTGCCCCCTAGTAG GGACGAATTGACTAAGAATCAGGTGTCACTCACCTGCCTGGTCAAGGGTTTCTACC CTTCTGATATTGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAATTACAAA ACCACGCCACCCGTGCTCGATTCTGACGGTAGCTTCTTCCTGTACAGCAAGCTCAC AGTCGACAAGAGCAGATGGCAGCAGGGAAACGTGTTTTCCTGCTCAGTCATGCACG AGGCTCTTCACAATCACTATACTCAGAAGTCCCTCTCCCTGTCCCCCGGTAAATAA

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 25 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG

EFla- TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG trastuzumab CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT WT; GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG

Description GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC

GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT

of the ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT

CCTCCGAGCCGCTCCGACACCGGGAliiilljSjijlTTAATTAAAATTATCTCTAAGGC elements : ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT

GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT

TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC

BLACK ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG CAPITALS TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA

encodes the CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC shuttle AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG vector; GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA

TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA

:8ϋΙϋ:Ι1:1 CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT encodes the TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG Ascl ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT

Restriction CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA enzyme GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG site ; GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG UNDERLINE AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC

encodes CpG CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG free MAR CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT from human GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT β globin CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC gene ; AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT ITALICS TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG

encodes CMV CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC Enhancer; TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG HEAVY ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC UNDERLINE TCCGATATACAGATGACACAGAGCCCCAGCAGCCTCAGCGCCTCCGTCGGCGACAG

encodes GGTCACGATTACCTGTCGGGCCAGCCAGGATGTTAATACGGCAGTGGCATGGTATC human EFla AGCAAAAGCCCGGTAAGGCTCCTAAGTTGCTCATTTACAGCGCTTCCTTCCTTTAT promotor ; TCAGGAGTTCCATCCAGGTTTAGCGGCAGCCGGTCTGGTACCGATTTTACGTTGAC DASHED CATCTCTAGTCTGCAGCCTGAGGACTTTGCGACCTACTATTGTCAGCAGCACTACA UNDERLINE CAACTCCCCCCACCTTCGGCCAGGGCACCAAGGTCGAGATCAAAAGGACCGTGGCA

encodes a GCCCCGTCTGTATTCATTTTCCCCCCCTCAGACGAGCAGCTTAAGAGTGGAACCGC multiple CTCCGTTGTGTGCCTCCTGAATAACTTCTATCCACGAGAAGCGAAGGTTCAATGGA cloning AGGTAGATAATGCTCTGCAGAGCGGCAATAGTCAGGAGAGCGTCACAGAGCAGGAC site ; AGCAAAGACAGTACCTATTCACTGTCCTCAACCTTGACATTGTCAAAGGCCGACTA

CGAAAAGCATAAGGTCTACGCATGCGAAGTTACCCATCAGGGACTCTCCAGCCCGG lower case TCACCAAATCATTTAACAGAGGCGAGTGCCGGGCCAAGAGAGCACCAGTTAAGCAA encodes ACCCTGAACTTCGACCTGCTTAAGCTCGCCGGTGACGTAGAATCTAACCCTGGACC synthetic TGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA intron ; CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC BOLD CTTTCTCTCCACAGGCGCGCACTCCGAGGTGCAGCTTGTGGAATCCGGAGGCGGTC

encodes TTGTGCAGCCTGGGGGCTCTCTCCGCCTCTCTTGCGCTGCTTCCGGCTTTAACATA transgene AAAGACACCTACATTCATTGGGTCAGGCAGGCGCCGGGTAAGGGATTGGAGTGGGT ( trastuzuma CGCCCGGATCTACCCGACAAATGGGTACACTAGGTATGCAGATTCTGTAAAAGGAA b WT) from GATTCACCATCTCAGCTGACACGTCAAAGAATACCGCATATCTTCAGATGAATTCT ATG to stop CTGAGGGCGGAGGATACTGCCGTGTACTACTGCTCAAGGTGGGGCGGCGATGGCTT codon ; TTACGCTATGGACTATTGGGGCCAGGGCACACTGGTGACAGTGAGCTCCGCCTCTA

CGAAGGGACCCTCAGTTTTCCCACTGGCCCCTAGTTCTAAAAGCACCTCTGGGGGC

ACAGCTGCACTCGGATGCCTTGTTAAAGACTACTTCCCTGAACCTGTGACTGTCTC

encodes CTGGAACAGTGGAGCATTGACCTCAGGGGTGCATACCTTTCCTGCTGTCCTCCAGA SV40 poly GTTCAGGACTCTACTCACTTTCTTCCGTTGTTACAGTACCCTCATCATCCCTCGGA Adenylation ACCCAGACTTATATCTGTAACGTCAATCATAAGCCCAGCAATACAAAAGTGGATAA signal ; AAAGGTGGAACCAAAGTCATGTGATAAAACCCATACGTGCCCGCCCTGCCCAGCAC BOLD CTGAGCTGCTGGGCGGGCCTTCTGTATTCCTGTTTCCACCAAAGCCAAAGGACACC ITALICS TTGATGATTAGCCGAACACCAGAAGTAACCTGTGTGGTCGTAGACGTAAGTCACGA

encodes MAR AGATCCAGAGGTCAAGTTCAACTGGTATGTCGACGGCGTTGAAGTGCACAACGCCA 5 ' region AGACTAAGCCCCGGGAGGAGCAGTACAATTCAACCTATCGGGTCGTCTCTGTGCTC from human ACAGTCCTCCACCAAGATTGGCTTAATGGGAAGGAGTACAAATGTAAGGTGTCTAA IFN gene CAAAGCACTTCCCGCTCCAATCGAAAAGACTATAAGCAAGGCAAAGGGCCAACCGC

GCGAACCACAGGTTTATACACTGCCCCCAAGTAGGGACGAGCTGACGAAAAACCAG

GTTTCCTTGACTTGTCTCGTAAAGGGTTTTTATCCCTCAGACATCGCAGTTGAGTG

GGAAAGCAATGGTCAGCCTGAAAACAACTATAAAACAACACCTCCTGTCCTGGATT

CTGATGGCTCCTTCTTTCTGTACAGTAAACTGACCGTAGATAAAAGCAGATGGCAG

CAGGGGAATGTGTTCTCTTGCAGCGTCATGCACGAAGCTCTGCATAACCACTACAC

TCAGAAGTCACTCAGCTTGTCTCCAGGGAAGTAAGGATCCCCGGGAGATATC

GCTTGGCCAGA ATOA^

GJAGJTGJAAAAAA

i^ATTATAAG I^

CAGJSTTCAGJ^^

TGGTA^GGAATTCA

ACCTCATTCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGT AGAACAAAATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGA GATGTGTGGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGAC CCATTGATATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAA TGATGGTCTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATA AAAGGGAACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGT CTAAATGGAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCA TCAAGACTTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGA AAAGATTGTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAAC CATTAAGAAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAA GAGAATTGTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAA ACTAGACAAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGA GAAACTCAATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAA ATATTAAAAGGCTAAGTAACATCTGTGGCTTAATTAAGGCGCGCCGGGCCCCTACG TCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTAT CATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCT GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTG AATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT TGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACA ACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC GGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACA AAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTT CCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGAC TTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGC TGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACT ATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTG GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATT TTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCC GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATC CCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCA ATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTG CGGCCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATA

Sequence of GGCCGATTCATTAATGCAGGGGCCGCTGCGGCCATCATCAATAATATACCTTATTT 26 pAd-MAR- TGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCG

EFla- TGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGG trastuzumab CGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGT Mut GTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTG

GGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTG

BLACK AAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGG CAPITALS GACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGC

encodes the GTTCCGGGTCAAAGTTGGCGTTTTATTATTATAGTCAGCTGACGTGTAGTGTATTT shuttle ATACCCGGTGAGTTCCTCAAGAGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCT vector; CCTCCGAGCCGCTCCGACACCGGGAG ISiSSiSiiTTAATTAAAATTATCTCTAAGGC

1:1111 ATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCT wmmmm GAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCATATGTATT encodes the TTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATC Ascl ACACTACCCAATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATG

Restriction TACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACA enzyme CACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAAATTATTAGC site ; AATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAG UNDERLINE GCTAGCCTGCAGGAGTCAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAA

encodes CpG TAGGGACTTTCCATTGGGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAA free MAR CAGGAAAGTCCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGT from human TTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTG β globin ACATGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGT gene ; CAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATA

GGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATG GGTTTTTCCCATTATTGGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAG ITALICS AGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTC encodes CMV CCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG Enhancer; CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGT HEAVY GGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTT

UNDERLINE CTCCCTCCTGTGAGTTTGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGC

encodes AGGAGGTGGGGCAGTGCAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAAT human EFl TGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAG promotor ; CTTGCATGCCTGCAGGTCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTC

DASHED TTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGG

UNDERLINE ACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCAC

encodes a TCCGACATACAAATGACCCAGTCTCCCTCCAGTCTGAGTGCTAGCGTAGGAGACCG multiple GGTCACCATTACCTGCAGGGCTAGTCAGGACGTGAATACCGCCGTCGCCTGGTACC cloning AGCAGAAGCCAGGGAAGGCCCCAAAGCTGCTGATTTACTCCGCCAGTTTCCTCTAC site ; TCAGGTGTACCCAGCAGATTCTCTGGCTCACGGTCTGGAACCGATTTCACTCTGAC lower case GATCAGCTCTCTGCAGCCGGAAGATTTCGCCACATACTACTGTCAGCAACACTACA encodes CCACACCCCCAACATTCGGACAGGGGACCAAAGTTGAGATCAAACGCACTGTGGCT synthetic GCCCCGAGTGTATTTATATTTCCTCCCAGCGACGAGCAGCTGAAAAGCGGCACTGC intron ; ATCCGTGGTGTGCCTGCTGAATAACTTCTATCCACGGGAAGCAAAGGTCCAATGGA

BOLD AAGTCGACAATGCTCTGCAATCCGGCAACTCACAGGAGAGCGTCACCGAGCAGGAC

encodes TCCAAAGATTCAACCTACTCACTTTCTAGCACTTTGACCCTGTCTAAAGCTGACTA transgene TGAGAAGCATAAAGTGTATGCCTGTGAGGTAACCCATCAGGGACTTTCCTCCCCAG

(HER Mut) TTACGAAAAGTTTTAATCGGGGCGAATGTCGGGCTAAGCGCGCACCTGTGAAACAG from ATG to ACACTCAATTTTGACCTTCTGAAGCTGGCCGGTGATGTTGAGAGCAATCCTGGGCC stop codon; TGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAA

CAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGC

CTTTCTCTCCACAGGCGCGCACTCCGAAGTCCAGTTGGTGGAGTCAGGCGGGGGTC

encodes TGGTGCAGCCAGGAGGCTCCCTTCGGCTTTCTTGTGCAGCAAGCGGTTTCAATATT

SV40 poly AAGGACACCTATATTCACTGGGTGAGACAAGCCCCAGGGAAAGGCCTCGAGTGGGT

Adenylation AGCAAGGATCTACCCTACTAACGGCTACACTCGATACGCAGACTCCGTCAAGGGGC signal ; GGTTCACTATTTCAGCAGACACATCCAAAAACACTGCTTATTTGCAGATGAACTCC

BOLD CTGAGAGCCGAAGATACAGCTGTCTATTATTGTTCTAGGTGGGGTGGGGACGGCTT

ITALICS CTATGCCATGGATTACTGGGGACAGGGAACACTTGTCACCGTGAGCAGTGCTTCTA

encodes MAR CCAAAGGACCTTCAGTGTTCCCACTCGCTCCTTCTTCAAAGAGTACCTCCGGAGGC

5 ' region ACCGCCGCGCTTGGGTGTCTGGTAAAGGATTACTTTCCCGAGCCGGTGACCGTTTC from human CTGGAATTCCGGTGCTCTCACGTCCGGAGTCCATACCTTTCCCGCCGTCCTGCAGT

IFN gene CTAGTGGCCTTTATTCCTTGAGTAGCGTGGTGACCGTGCCAAGCAGCTCACTGGGC

ACCCAAACTTACATCTGCAACGTGAACCACAAACCATCCAACACCAAGGTGGATAA

AAAGGTTGAACCTAAAAGTTGCGACAAAACACACACCTGCCCTCCGTGCCCTGCCC

CCGAGCTCCTGGGAGGACCCTCCGTGTTCCTCTTCCCCCCAAAGCCAAAAGACACT

TTGATGATAGCACGCACACCCGAAGTGACCTGCGTCGTAGTGGATGTTTCACACGA

AGACCCCGAGGTCAAATTTAATTGGTACGTGGATGGTGTCGAAGTCCATAACGCCA

AAACCAAGCCTCGAGAGGAACAGTACAATAGCACATACCGGGTGGTCTCAGTGCTC

ACCGTACTGCATCAAGATTGGCTTAACGGCAAAGAATATAAATGTAAGGTGAGTAA

CAAAGCCCTCCCTGCGCCTATCGAAAAGACAATTTCAAAAGCTAAGGGACAGCCCC

GGGAGCCCCAGGTGTATACTCTGCCCCCTAGTAGGGACGAATTGACTAAGAATCAG

GTGTCACTCACCTGCCTGGTCAAGGGTTTCTACCCTTCTGATATTGCCGTGGAGTG

GGAGTCCAACGGCCAGCCTGAGAACAATTACAAAACCACGCCACCCGTGCTCGATT

CTGACGGTAGCTTCTTCCTGTACAGCAAGCTCACAGTCGACAAGAGCAGATGGCAG

CAGGGAAACGTGTTTTCCTGCTCAGTCATGCACGAGGCTCTTCACAATCACTATAC

TCAGAAGTCCCTCTCCCTGTCCCCCGGTAAATAAGGATCCCCGGGAGATATCCTAG

GCTTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAAT

GJJAGJTJGAAAAAAA^

^ATTAT^AAG^

CAGJ5TTCAGJ5G^^

TJGJATJGJAATJ^

ACCTCATTCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGT

AGAACAAAATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGA

GATGTGTGGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGAC

CCATTGATATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAA

TGATGGTCTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATA

AAAGGGAACCCATATGTCATACCATACACACAAAAAAATTCCAGTGAATTATAAGT CTAAATGGAGAAGGCAAAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCA TCAAGACTTCAGTGTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGA AAAGATTGTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAAC CATTAAGAAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAA GAGAATTGTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAA ACTAGACAAAAATTTGAACAGATGAAAGAGAAACTCTAAATAATCATTACACATGA GAAACTCAATCTCAGAAATCAGAGAACTATCATTGCATATACACTAAATTAGAGAA ATATTAAAAGGCTAAGTAACATCTGTGGCTTAATTAAGGCGCGCCGGGCCCCTACG TCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTAT CATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGGCCGCAGCGGCCCT GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTG AATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT TGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCC CTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACA ACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC GGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACA AAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTT CCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGAC TTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGC TGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACT ATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTG GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATT TTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCC GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGC TAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATC CCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCA ATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGGGCCGCTG CGGCCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATA

Sequence of 27 trastuzumab

WT in ρΔ28 GCGGAGTAACTTGTATGTGTTGGGAATTGTAGTTTTCTTAAAATGGGAAGTGACGT plasmid; AACGTGGGAAAACGGAAGTGACGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGT

TTCTGGGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGTGGACTTTAACCGT Description TACGTCATTTTTTAGTCCTATATATACTCGCTCTGCACTTGGCCCTTTTTTACACT of the GTGACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTTAATAGGTTTTCTTTT elements : TTACTGGTAGGCGCGCCTTAATTAAAATTATCTCTAAGGCATGTGAACTGGCTGTC

GPAY TTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCTGAAACATATTTATAAT

C 1TAPS TCCATTAAGCTGTGCATATGATAGATTTATCATATGTATTTTCCTTAAAGGATTTT encodes the TGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATCACACTACCCAATAAAT inverted AATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATGTACCAGTTTTATTGTT terminal TTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACACACACATGTGTGCATT repeat CATAAATATATACAATTTTTATGAATAAAAAATTATTAGCAATCAATATTGAAAAC

(ITR; CACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAGGCTAGCCTGCAGGAGT

BLACK CAATGGGAAAAACCCATTGGAGCCAAGTACACTGACTCAATAGGGACTTTCCATTG

CAPITALS GGTTTTGCCCAGTACATAAGGTCAATAGGGGGTGAGTCAACAGGAAAGTCCCATTG

encodes the GAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAA shuttle GGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGACATGTATACTGAGTC vector; ATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCA

ACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGG

m m m TTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATT encodes the GGCACATACATAAGGTCAATAGGGGTGACTAGTGGAGAAGAGCATGCTTGAGGGCT

Ascl GAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGG

Restriction GAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGA enzyme AAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATA site ; TAAGTGCAGTAGTCTCTGTGAACATTCAAGCATCTGCCTTCTCCCTCCTGTGAGTT

UNDERLINE TGGTAAGTCACTGACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGGTGGGGCAGTG

encodes CpG CAGGAAAAGTGGCACTGTGAACCCTGCAGCCCTAGACAATTGTACTAACCTTCTTC free MAR TCTTTCCTCTCCTGACAGGTTGGTGTACAGTAGTAGCAAGCTTGCATGCCTGCAGG from human TCGACTCTAGACTGCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAG β globin CTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGA gene ; CAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGATATACAGATGA

ITALICS CACAGAGCCCCAGCAGCCTCAGCGCCTCCGTCGGCGACAGGGTCACGATTACCTGT

encodes CMV CGGGCCAGCCAGGATGTTAATACGGCAGTGGCATGGTATCAGCAAAAGCCCGGTAA

Enhancer; GGCTCCTAAGTTGCTCATTTACAGCGCTTCCTTCCTTTATTCAGGAGTTCCATCCA

HEAVY GGTTTAGCGGCAGCCGGTCTGGTACCGATTTTACGTTGACCATCTCTAGTCTGCAG

UNDERLINE CCTGAGGACTTTGCGACCTACTATTGTCAGCAGCACTACACAACTCCCCCCACCTT

encodes CGGCCAGGGCACCAAGGTCGAGATCAAAAGGACCGTGGCAGCCCCGTCTGTATTCA human EFl TTTTCCCCCCCTCAGACGAGCAGCTTAAGAGTGGAACCGCCTCCGTTGTGTGCCTC promotor ; CTGAATAACTTCTATCCACGAGAAGCGAAGGTTCAATGGAAGGTAGATAATGCTCT

DASHED GCAGAGCGGCAATAGTCAGGAGAGCGTCACAGAGCAGGACAGCAAAGACAGTACCT

UNDERLINE ATTCACTGTCCTCAACCTTGACATTGTCAAAGGCCGACTACGAAAAGCATAAGGTC

encodes a TACGCATGCGAAGTTACCCATCAGGGACTCTCCAGCCCGGTCACCAAATCATTTAA multiple CAGAGGCGAGTGCCGGGCCAAGAGAGCACCAGTTAAGCAAACCCTGAACTTCGACC cloning TGCTTAAGCTCGCCGGTGACGTAGAATCTAACCCTGGACCTGGATGGAGCTGTATC site ; ATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAG lower case GTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGC encodes GCGCACTCCGAGGTGCAGCTTGTGGAATCCGGAGGCGGTCTTGTGCAGCCTGGGGG synthetic CTCTCTCCGCCTCTCTTGCGCTGCTTCCGGCTTTAACATAAAAGACACCTACATTC intron ; ATTGGGTCAGGCAGGCGCCGGGTAAGGGATTGGAGTGGGTCGCCCGGATCTACCCG

BOLD ACAAATGGGTACACTAGGTATGCAGATTCTGTAAAAGGAAGATTCACCATCTCAGC

encodes TGACACGTCAAAGAATACCGCATATCTTCAGATGAATTCTCTGAGGGCGGAGGATA transgene CTGCCGTGTACTACTGCTCAAGGTGGGGCGGCGATGGCTTTTACGCTATGGACTAT

(HER WT) TGGGGCCAGGGCACACTGGTGACAGTGAGCTCCGCCTCTACGAAGGGACCCTCAGT from ATG to TTTCCCACTGGCCCCTAGTTCTAAAAGCACCTCTGGGGGCACAGCTGCACTCGGAT stop codon; GCCTTGTTAAAGACTACTTCCCTGAACCTGTGACTGTCTCCTGGAACAGTGGAGCA

CUJVY TTGACCTCAGGGGTGCATACCTTTCCTGCTGTCCTCCAGAGTTCAGGACTCTACTC

UN ERLINE ACTTTCTTCCGTTGTTACAGTACCCTCATCATCCCTCGGAACCCAGACTTATATCT

encodes GTAACGTCAATCATAAGCCCAGCAATACAAAAGTGGATAAAAAGGTGGAACCAAAG

SV40 poly TCATGTGATAAAACCCATACGTGCCCGCCCTGCCCAGCACCTGAGCTGCTGGGCGG

Adenylation GCCTTCTGTATTCCTGTTTCCACCAAAGCCAAAGGACACCTTGATGATTAGCCGAA signal ; CACCAGAAGTAACCTGTGTGGTCGTAGACGTAAGTCACGAAGATCCAGAGGTCAAG

BOLD TTCAACTGGTATGTCGACGGCGTTGAAGTGCACAACGCCAAGACTAAGCCCCGGGA

ITALICS GGAGCAGTACAATTCAACCTATCGGGTCGTCTCTGTGCTCACAGTCCTCCACCAAG

encodes MAR ATTGGCTTAATGGGAAGGAGTACAAATGTAAGGTGTCTAACAAAGCACTTCCCGCT 5 ' region CCAATCGAAAAGACTATAAGCAAGGCAAAGGGCCAACCGCGCGAACCACAGGTTTA from human TACACTGCCCCCAAGTAGGGACGAGCTGACGAAAAACCAGGTTTCCTTGACTTGTC IFN gene TCGTAAAGGGTTTTTATCCCTCAGACATCGCAGTTGAGTGGGAAAGCAATGGTCAG

CCTGAAAACAACTATAAAACAACACCTCCTGTCCTGGATTCTGATGGCTCCTTCTT TCTGTACAGTAAACTGACCGTAGATAAAAGCAGATGGCAGCAGGGGAATGTGTTCT CTTGCAGCGTCATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCACTCAGC TTGTCTCCAGGGAAGTAAGGATCCCCGGG^

TAAGATACA/TT^

T TATTTGjn^^

TAAAC^GTTAA^^

CAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAAATG TATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAAAATGGGAAA GAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGTGGGGATAGA CAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGATATATGTAA GTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGTCTTTTTCTT TTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGAACCCATATG TCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAGGCA AAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGACTTCAGTGTA GAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATTGTTAATTAG ATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAGAAAAGTCAG GCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATTGTAATATGC AGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGACAAAAATTTG AACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTCAATCTCAGA AATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAAAAGGCTAAG TAACATCTGTGGCTTAATTAAGGCGCGCCAT GAATT CTGCAGCCCGGGGGAT C CTTTGTTGATTTTTTCCACATAGATTATTTTTGACTGTTTTGGCACTTTATATAAA TGGAATCATATAGTAAATATATACATGTATATATGTATATATACACTATATATGTA TATATATAGTGTATATATATACATGTATATATGTATATTTACATATATACTGTATA TATGTATATTTACATATATACTGTATATATGTATATATACACGTATATACTGTATA TATACAGTATATACTGTATATATATACTGTATATATGTGTATATATATATACAGTA TATATACAGTATACATATATATACATGTATATATACTCAGTATATATACATGTATA TATACACAGTATATATACACTGTATATATGTATATATATACTGTGTATATATATAC AGTATATATACAGTATATATATACATGTATATGTATATATATACTGTATATATGTG TATATATACGTATATATACTGTATATATATATAGACACTTTTGTGTCTGGCTTCTT GCTCTCATCATAAAGCACTTGAAATCCATCCATGTTGTAGCTGGTATCAGTAGCTA GTTTCTTTTCATTGCTGAGAAGTATCACTTTTTATTGATGAGTAGTTTTACATTGT ATGAACATGCCCGAGTTTGTTTATGCATTCTACTAATAGACACCTGGGCTGTTTGC AGTTTTTGGCTATTACAAAGAAAATTTCTATATTTTTTTCCAACTTTTATTTTAGG TTCAGCGGGTACATGGGCAGGTTTGTTACATCGGTAAATTGCATGTCACTGGGGTT TGGTGTACAGATTATTTCACCACCCAGGTAATAAGCATAGTACCTGATAGGCAGGT TTTTGATCCTCACCCTCCTCCCTCAAGTAGGCCCCAGTGTCTATTGTTCTATTCCT TTTTTTTTTTTTTGAGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGT GCGATCTCGGCTCACTGCAAGGTCCACCTCCAGGGTTCAAGCGATTCTACTGCCGC AGCCTCCCGAGTAGCTGGGATTACAGGCACCCACCACCACGCCCGGCTAATTTTTG TATTTTTAGTAGAGACTGGGTTTCACCATGTTGGCCAGGATGGTCTCGAACTCCTC ACCTCAGGTGATCCGCCCGCTTCGGCCTCGCAAAGTACTGGGATTACAGGCGTGAG CCACGGTGCCTGGCCTATGGTTCCATTCTTTGTGTACACGTGTACTCAGTGTTTAG CTTCCACTTGTAAGTGAAAACATGCAGTATTTGGTTTTCTGAAATTCTTGTCTTCA TCTTTTTGTAGACAATCCACTACTTTTATTTTTAAATATTAGGAACAATAGCCTCT GTGTGTCCTGAATTGCAATGTTTTTTCTCTGATTTTGCTGCTCATCTTGTAACCTG TATGGTGTTTTTCTGTGTGCAAGCTTTTTACTTTACGTGGGCAATTTTATCATTCT TTTCTTTTATGGTTTCTGGGTTTCATGTCATGATTGGAGAAGCTTGATCTACCCTG ATACTATAAAAATATCCACCTTGGCTTTTTTTTTTTTTTTTAGATGGATTTTCACT CTTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTCGGCTCACCACAACCTCTGCC TCCCAGGTTCAAGTGATTCTCCTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGC ATGCGCCACCACGCTCAGCTAATTTTGTATTTTTAGTAGAGACAGGATTTCACCAT GTTGGTCAGGCTGGTCTTGAACTCCCCACCTCAGGTGATCTGTCTGCCTCGGCCTC CCAAAGTGCTGGGATTACAGGCGTGACCCACCACACCTGGCCCACGTTGTTTTCTG ATAGTGTTTTCACGTGTTATTTTTTCTGTCTTACTCTTTGAATCATCTAAATGTAT CTTAATGTCAGGAGTGAGGTAGAGGACCCAGTTGTTTGGTTTAAATGACTAGCCAG TTTTCCCAACACCAGGTGTTGAACATCCCTCACTGATGTGAGATGCTGCCTTTTTC ACAGTCTCAATCCCCATGTGCATTTCTATTTATTTTCCTTCCACTTTATTCACATC T T T T T CAGT AT C CACAAC CAT AT T GT T T TAAATAGCTT CAT GAT AT GT T T T AAT AT TTGGTAGAGCTAGAACCCTTTCTTGGTTCTTTTTCTTTTCTTCTTCTCCTCCTTTT CCTCCTCCTTCTTTCTCCTCCTCCTTCTTCTTTCTTTTTAGAAATTTCCTGGCTAT TCTTGTTTTGTAGTCTTCCAGATAAATAATTTTGAATCACTTTGTCATGTTCCAGA AAAT AAT AC T G C CAGAAT TTTTATTGT GAT C AC AGAAGT T T T AC AGAT T CAT C T G G G G GAAAAAAAC C CAT C T T C C C AGAAT AC AGAAAG G GT GAG G GAAAGAC AAAC AT C A AGGACAAGTCTCAGGTTCTGGACTTGGAGACCAAGAGGATTTGGGGCATCCGGGAG CAGGGCAGGGAGGTGTGATGGCTGCCTTTCACTGAGTAGGAGGATGCCCAGACTTG GGGGAACTATGGAAAGTTTGGTTCAGACTTTCTGAGTTGGAGATGTCTATGCGCAG TCCACAGGGCGAAGCCCTGGAGGTAGAGGCCCTCCTTACTGCCTCCCTCATTCAGT CTGAGGTCCATTCTCCACATGGACACTGAAGTGACTCAAGGCTTCCCTTGTTGGAT CAGGATAGTCTCCAGGGCCCTGGGTGGTCTGGGCCTGCCCACCTCCCCATCCCCTC TCCTGCCTGCCCTCCTCTTGCTCACTGGACCCCACCCACCAGCCTCCTTCTATTCC TCCTGACATTTGCACTTGCTGTTCCGTCTCTGCTTGGAACATCCTAGCTCTTTGCA TGGCTTGCTCCTTCCCATCTTTTAATCTAAGCTCAAATATCACTCCTTCAGATATG CCTTTCCCAAATATCCTAGCTAAAGAGAACTGCTCCCCACTTCCCACAAGCCCTAG CAAACTGGGACTCTCCTTCTTGCTTTCTTTCTCTCTGCCCCTTCTGTGTTTTTTCC ATAGACCTTAACTTGTTACTGTTTTATTTGTTGATGTGTTTACATCATTTGTCCCC CTGTGATGGTTAGTTTTATGTGTCAACTTGGCTAGGCTGTAGGGGCTGTTCGTCAA TCAAACACTGATCTATGTGTAGCTGTGAAGGGATTTTGTAGCTGTGATTAATAGTT GAC T T T AAGT AAG GAAGAT TAT C C T G GAGAAT GT G G G G GAAG G G G C AGAC C T C AG C C AAT CAGT T GAAAG G C C T T AAGAAC AAAG C T GAGAT T T C C C C GAG GAAGAAGAAAT TCTGCCTGTGGACTGCAGCATCAGCTCCTGCTTGAGAGGTTCCAGGCTGCCCTTCC TGACAGCCTGTCCTATGATTTTGGACTTGCCTTGCCAGGTTTTTCCTCTTTCTTGA CCCCCCAAAACCATATATATCTCCTGCTGGTTTGTTTCTCTGGTGTAATCCTGAGG GATGAACACTCCTCCCTCCCAGTTCACTCTGGGAGAGGTTGGGGATCATGTTTGTC TATTTCCCCATGCCTGGCCCATGCGTGTTTATTGAATCACTGAACAACCACGCATT AGGAAGCCAGTCATATGTGCTTCTTCAGAACCTCATGTCCATTGCCAGATCTCCCT GGCTTTTGTGGC T AGAG GAC AAGT GAGAGAT AGT AG C T T AC C C AC AGAC C T T G G C C CTGAGGCCCCCAGGGGCCTGAGCCTGCTGTAAGGGAGGAGGGAGCCCCTGAGGTCT CACACACCTCCCTGGGGATCTGGCATTTTCCCCTGGGGCTGGCCTCAGAGCTGGGC GGGGGCAGATTATGGAGTGGGTTGTAGAGGGAGCACCTCCCCACTAGCAATCATGG TTTTTTCTGCGCCTCTCATAGGGAGGCCTACAGGCCTCCACCTTCAGCTCTTCAGG AG C C AC AG G C AG C T GAGT GAC T T C T G CAT C AC AGT C T T C T C AGAGAC AAAC T T G C A GTAAGAAAGAGGGGCCCGTGAGGGACCCCAGAGAAGGCTGTTGTCAAAGCAGGATG AGAGTGAACTCTTCCATGGGGGACACCCAGCGTCTCCAAGCTCTTTTATGCTGTCT T C AAG G G GT C T AGAGAG C T T C AG C C C AAC AT GAGAC C CAGT C C AG C AG CAT T T C C C T G C GAAGT GAAAGT TAG GAC C C T GAC T AGAT AC AC C AC G C T GAC C T C AG C CAGAAT ATCAAGATGCTGAGGCGCTGAGATGCTGGGATGCTAAGGTGCTAAGGTGCTGGGGT GCTGGGGTGCTAGGATGCTGAGGTTTTGTGATACTGGGTCGCTGAGATGCTGGGAT ACTGGGGTGCTGAAGTGCTGGGGTACTGGGGTGTTGCCATGCTGAAATGCTGGGGT GTTGGGATGCTGAGGTGCTGGGTTGCTGGGATGCTGGGATAGTCCTTGGATGCTGC GGTGCTGAGATGCTGGTCTGCTGGTCTGCTGGGGTGTTGGGATGCTGGGATTTTGG GAT GCTGGAGTGCTGCAGTGCTGCAGTGCT GAGAT GCTGGAGTGCTGGGGTGCTGG AATACTGTAGTACTGGTGTGCTGGAGCATTGAGATGCTAGGGCACTGGGATGCTAA GGTGCTGAGATGCTGCAGTGCTGGGGTGTTGGGATGCCGAGGTGTTGGGATGCTTA AGTGCTGGGGTGTTGGGATGCTGGGGTGCTGGAACACTATGGTGCTGGGGTGCTGG AGTGTTGAGATACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGTGTTGGG ATGCCTAGGTGCTGGGGTGCCGAGATGCTGGAGTACTAGTGTGCTGGGATGCTGAA GTGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGA GTGTTGGGGTGCTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGA ATACTATGGTGCTGGGGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGAA GTGCTGAGATCCTGGAGTGCTGGGCTGCTGGGCCACAGGCTCTTGAATCCATTCGT C T G C C C AG G G GAAGAAAC C AGAAGAT AAAGAG C T AAT GAAG GAG CTTTGGTT GAGA G G GAG GAAGT AAT G GAAG GAG C AAC AT C T T GT G GAG GAG C AG GAGAGAAT G GAC C T C AG GT T G G GAGAGAG G G C C AG G C T AC AG G C C AGAGAG G C AGAAG GAT T C C AG C AGA GTGTGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTCCAGTAC TGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAG GAG GAAT T T G GAAC T GT GAC T AGAGT AC T T AG G G G GAAG GAG G C AAGAC T G GAG C C AGATTGCTCTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCACTCCTT TGAAGGGTGGCAGAGAGCTGGAATTGGTGCTGGATGGGCTGTGGGGTGACAGGGTC ATGTGGAAAGCCCCTGGGGGGCACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGA G GAC AG C T AAG G GAAAG C G GAAT G G C AGAAC AC G C AC T G C C AG GAG GAAT GAG GAT AGGGTCAGGAGTGCCAGGGGCAGTGAGGCCAGCCTGGGGTCAGGTGGCAGGACGTG TCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCTTGAGGTTCCCTGT GTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGCTCTGCG AGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGGGA CAACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGGGCTTCATT TTCCCAGCGGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTGGGTCCAACACAGAG CCAGAGAAGGGTGAATGGGCCTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAG TGATGGAGGGGGACAGCACAGCTGAGCCCAAGTGCTTTGGTGTGGCCCTGAGGGAA AGCTGCAGCCTGCCTGGGGCCTGGCATGGATGGGACACTTGAGGCAGAGGGACAAT AGTGGGCGCTGCAGTGAGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCTGCCTG AGACGGGCAGGGAGAACAGAGACAAAGTT GGT GACAGGGAAT GAAAGCT GACT GAA G GAC T T T AC C C AGAC C TAT GAG GAT AT C T C T C T C AG C AG GAAG C AG GAG G G GAC T G TGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCTCCGACCCCT CTGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCACCCCCATTGGTCGA TCGTTGTCCCTTGTCACAGCCATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGC ATTTTTAGGCTCTGCGGGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAG CTCCTGAAAGCCACTGATCAAATTGGAACATTTTGTACCTTAGGGATGAGGATATC AAC T C T C C C AG C C AC T T AGAG G GAT AAAT GT GAT GAT G CAT T C AAT T GT GAC T AC A TCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGC ATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCATCCATGAAACCTG GGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAAGAAGGG CATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCGTCTAGACCCTC AGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGTCTTTGTGACCTCCCATC CCCCTCTGTTTCATCCTCTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTC TTTGCCATGATGCAAAACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCA GCCTCTGAAAGAACCAAAGCCACTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACT GAATACTCCCTTGAAAAATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTATTA C AGAAC GAAGAAAAAAC AC AC T G G C T T GAAAC AGAC G C C AGAT T T C AAAT GT AGAG GT GAAAT AC GAG GT G G C AAT T AAAAT GT GAT T AC AGAAAGT C T G GAC AC T GAGAAA AGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATATACATGAC GAT AAT T G C AGAT AGAAAC CAT C AAAGAC AAAC C C C AAAT C AAC T AAT AAT GT T T A C AGAT GTTCCCCCC C AAAC C AC AGAG C C T T AC AT C AAAAC AAAT AC T GAAAG G C T T TAAACCAGGAACAGCTCGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTT CTCTCGGTCTGAATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGC TGAGACAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACT CTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCT G C T GT AAAG C T CAT TAG GAAAGAG C T AG C G GAGAC T GAAAG GT T C G C AAAAGAGAT T AAGAAT C AC AC AAG G C AAT AG GAT T T T T AGT GAAC AT AGAAAT AAAT G G C C AAGT GGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGTCATATCCACATT G C T CAT T G CAT T AAAAC CAT AAG C GAC T C AG C C AC C T AG C T T AAC AAG GT AT C AC T G GAG C AAAC AAC AC G GT C T G CAT AT T T GT AAC AT T GT AT AAT AAAC AC AAAAC AAT G CAT AGT AAAC AC AAC T C T AC T GAAAC AAAAG CCGTCGCTT TAT T T AC AAAGT C AC AAAAT GAAGT AT AAAT AC T T C T GT CAT T AAT GT T T AG GAAAAC CAT T T AC AAAAT T T T C AAAT AT GT AC AC GT AG C T T GAAAAAT C AC C AG C T T T C CAT T T T GT C AC AG GT A GAGAGAG G GAT AAG CAT G G G C T GAC AAC AC C AC T C AAAT T GT AAC G G GAGAC AAC T GCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCTATGG GCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGCCCAGAATTCT GGGCTGTGGCATTTTGAGCAGCAGCATGCTGTTCCAAAATGTCGTCGATCAGCCTC AAGTTGCACACCCAGTCTTCATCTGGGCTCACACAGGAGCCTTTCAAGAGAGCTTC AAT GAAAT C T AC C T CAT T G C AGT C AG GT GAC GAAAT C AGAT CAT TTAGTGGGGGTT GGGGCTGGCGCAAAAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCG AACGGACCTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGC AGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGC TGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCTGT ACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGGTTGTTGGC CAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCCCAATCAGATGGCC TCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTGAACGCTATTATCCACTTGG CTGAGGGGTGTTTTCCCCGAAACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCA TGCAGCATTGTTGAACGCAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGT GGGGACTCAAGGACTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACT TCTGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCTGCC CAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCTGGAGGTGAAAA TTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCTCGCAGCTCTGG TGCCTGGTGAGTGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCAGTTCT GGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCGGCTACACTTGG AG GT GAC TTGTTCTTG GAC G G C T AC AT AC AGAAAGAGAGAAGT G G G GAT GAGT T C C AAAGGCATCCTCGACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACG GACTTCTCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTC CTCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGGGAAG CTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAGCAGATCACACTGGACTGAAAT GCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATG TCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCCTTTGACA GGTGGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGGCTGG TGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGTGACCTGGGAC AGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCAT AGCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGA ACGGTAATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTC C C T GAGT T C T GAAGAG C C T G G G GAT G GAGAGAC AC G GAG C AGAAGAT G GAAG GT AG AGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCAC TCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACTTTTTTGGGGTGGACACT GAGTCATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAGT C AGAG C AC AAG C C C AGT GT GT C T GAC C C AC C C AAGAAT C CAT G C T C GT AT C T AC AA AAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATCATGGCATGTGT ATGTTTTTCAACTAGGTTACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCAC CAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGG CACTCCAGCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGT C T T C C AT AAGAC C AGAAGAG C AAT G GT GTAGAGGT GT CAT G G G C T AC AAT AAAGAT GCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCTGGCCCTCAGACAAATGCTGAG TGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGT TGCTGAACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACAT C AG G CAT T T T G C T AAC T G C T GT AT C C T AG G C C AAT AAAT GT T GAT C AC AT T TAT AG CTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCC TGACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCT GAC AGAT G G C C C AG GAAC C CAT T AGAAAT G GAAG GT C AC T G C AG C AGT AT GT GAAT GTGCGTGTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGTG ATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAG GGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAAGTGGTGATTTTGGAGTCATTT AGACCAGGCCTTCAATTGGGATCCACTAGTTCTAGAGCGGCCGGGCCCAGGGAACC CCGCAGGCGGGGGCGGCCAGTTTCCCGGGTTCGGCTTTACGTCACGCGAGGGCGGC AGGGAGGACGGAATGGCGGGGTTTGGGGTGGGTCCCTCCTCGGGGGAGCCCTGGGA AAAGAGGACTGCGTGTGGGAAGAGAAGGTGGAAATGGCGTTTTGGTTGACATGTGC CGCCTGCGAGCGTGCTGCGGGGAGGGGCCGAGGGCAGATTCGGGAATGATGGCGCG GGGTGGGGGCGTGGGGGCTTTCTCGGGAGAGGCCCTTCCCTGGAAGTTTGGGGTGC GATGGTGAGGTTCTCGGGGCACCTCTGGAGGGGCCTCGGCACGGAAAGCGACCACC TGGGAGGGCGTGTGGGGACCAGGTTTTGCCTTTAGTTTTGCACACACTGTAGTTCA TCTTTATGGAGATGCTCATGGCCTCATTGAAGCCCCACTACAGCTCTGGTAGCGGT AAC CAT G C GT AT T T GAC AC AC GAAG GAAC T AG G GAAAAG G CAT T AG GT CAT T T C AA GCCGAAATTCACATGTGCTAGAATCCAGATTCCATGCTGACCGATGCCCCAGGATA TAGAAAATGAGAATCTGGTCCTTACCTTCAAGAACATTCTTAACCGTAATCAGCCT CTGGTATCTTAGCTCCACCCTCACTGGTTTTTTCTTGTTTGTTGAACCGGCCAAGC TGCTGGCCTCCCTCCTCAACCGTTCTGATCATGCTTGCTAAAATAGTCAAAACCCC GGCCAGTTAAATATGCTTTAGCCTGCTTTATTATGATTATTTTTGTTGTTTTGGCA ATGACCTGGTTACCTGTTGTTTCTCCCACTAAAACTTTTTAAGGGCAGGAATCACC GCCGTAACTCTAGCACTTAGCACAGTACTTGGCTTGTAAGAGGTCCTCGATGATGG T T T GT T GAAT GAAT AC AT T AAAT AAT T AAC C AC T T GAAC C C T AAGAAAGAAG C GAT T C TAT T T CAT AT T AG G CAT T GT AAT GAC T T AAG GT AAAGAG C AGT G C TAT T AAC G G AGTCTAACTGGGAATCCAGCTTGTTTGGGCTATTTACTAGTTGTGTGGCTGTGGGC AACTTACTTCACCTCTCTGGGCTTAAGTCATTTTATGTATATCTGAGGTGCTGGCT ACCTCTTG GAGT TAT T GAGAG GAT T AT AAGAC AGT C TAT GT GAAT C AG C AAC C C T T G CAT GGCCCCTGGCGGG GAAC AGT AAT AAT AG C CAT CAT CAT GT T T AC T T AC AT AG T C C T AAT T AGT C T T C AAAAC AG CCCTGTAG C AAT G GT AT GAT TAT T AC CAT T T T AC AGAT GAG GAAC C T T T GAAG C C T C AGAGAG G C T AAC AGAC AT AC C C T AG GT CAT AC A GTTATTAAGAGAAGGAGCTCTGTCTCGAACCTAGCTCTCTCTCTCTCGAGTAATAC CAGTTAAAAAATAGGCTACAAATAGGTACT CAAAAAAAT GGTAGT GGCT GTT GTTT TTATTCAGTTGCTGAGGAAAAAATGTTGATTTTTCATCTCTAAACATCAACTTACT TAATTCTGCCAATTTCTTTTTTTTGAGACAGGGTCTCACTCTGTCACCTAGGATGG AGTGCAGTGGCACAATCACTGCTCACTGCAGCCTCGACTTCCCGGGCTCGGGTGAT TCTCCCCAGGCTCAGGGGATTCTCCCACTTCAGCCTCCCAAGTAGCTGGGACTACA GGTGCGCACCACCATCCCTGGCTAATATTTGTACTTTATTTTATTTATTTATTTAT TTATTTTTTGAGATGGAGTTTCGCTCTTGTTGCCCGGGCTGGAGTACAGTGGCATG ATCTCGGCTCAGTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTACCTCATC CCCCTGAGTAGCTGGGATTACAGGCGCCTGCCACCATGCCTGGCTAATTTTTTGTA TTTTTAATAGAGACGAGGTTTCACCATGTTGGCCAGGCTACTCTCGAACTCCTGAT CTCAGGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCC ACTGCGCCCGGCCTAATATTTGTATTTTTTGTAGAGATGGTGTTTTGCCATGTTGT CCAGGCTGGTCTTGAACTCCTGAGCTCAAGCGATCTGCCCGCCTCTGCTTCCCAAA GTGCTGGGATTACAGGCATGAGCCACCGTGCCTGGCCTAGGTAGACGCTTTTAGCT TTGGGGTGTGATGCCTGCCCCAGTATATAGTGAATTTAATTATTGCTAGAGCTGGC TGTTTGTTAGTTTTCTTTGAACATAAGATACTCATTGTTTTTAGTTTGCAAATCCC TCTTCCTTTTTAAAAAATTTCTTTCCCTTAAATTGTTTGCATGTTAGCAATAACAA ATGCTTAAATGGTGCTATGTGCTAGATACTCTTCTAAGCCCTGTTATGTATATTAA C T AAT T T T T TAAAT T AC AC AAAT CAGAGAGGTTAAGTAACTTGCC CAAGAT T AC C C AACAATACTAGGATTTGAACCTAAGTTTGTCTCACCCCAGATTCTGCTCTTAATCT CTAAACTTTTAAGTTAGTAGTGACAATAGTAGGTATTTATTGAATACTTAACTATG TTTTAGGCGTT GAAGT AAAT AT T T T G C AG G CAT TAT C T AAT GT AAAC AC C C T AAAG T T AC AT AAC AG GT AC C C T T T AG GT AAAT AAAC AC T AGT AT GAC C T T G GAG G C AC AG AT AGT T GAAGT AAC T T G C C C AAT AT C AC T T AC AT GAAAT T G G C C C T C AAAT GT GT C TGATACAACCCATGCTGCTTGTAACTATCGTTTTAAACTGCCAGGGTAAACTTGGA C AC AC T T GAG C T AAGAAAAAG C T T T T AGAT T T T T G C AAAT T AAT GT GAAAGAT AT G CTTTATGTGGATATAATATCTTCTAAATTTCGGGGATGGTAGTCCTAGAAATGTAA TCCTGCCCTAGCCGAGCTTACCCTGCCAATAATTTTTTACAGAATTGGTAAAACGG AGCACCTTTTTTTTGTCCTTGGCCACACTGTTATCAACAGGGTGTAGATTGACATC AATCTGTAGGTGTAAACCAGAATTACTCTTTGTGACCACCAGGAAATAGAGCAGTT CAGTTCAGGGGTTTCTTTCTGTGAATTTAGCACTGTGACCTGCATACTACAAGTCT ACTTTGTTTTCTATCCATTGTTTGTATCTGGGTATTGCAAAAGGTAGGAAAAGGAC CAACCAGATCAGCAGAGAAGAGTTGCCTTGGAGTTTTCTTTTAGTTTTCTGCAGTT CATTAGATAGTAACTAGGCCATGTCATTTTACTCCCTTGTAGTGAAGATATGTTGA AGTTGTACTGGTATACTCTTCTACCTTTCTGTAATTTTATATTGTGTAGACTTGAT AAAAT T T AT GT GT C AAT C AC C AC CAT T AAT AT C AAT AT T GAG C C T C AAT T C T T AT T TTTCTGCC C AGT G G C T G C C AAAT T AC T AAC AT T T AC AAT AAT T C AC T AC T AC T AAG AT AAT CTACTAGTTC GAT C AC AT AC T T C AAAT T GT TAT G GAAC TACTGTCTT C AG C AT T GT GCT T CT GAT AACT GAT AAGT AT AAT T T T T T T T T T GT C CAGAGT GAACAT GT C TAT T C T T C C AC T GT AC AC AC T AAT AAAAG GAAAAAT T GT AAT AT T G G GT AAAT T C AT GT C CT T ACACAT GT AGT AGT TAT GAGCC CAT GT C C CT AGAAT GAGT AAT AAT T T ATCCCTCCCTTGGTTGAATAGTCAAGAATGCTGATTTTAATTCTTCTAACAGCTTT AT C C C T C AGAAG G GAAG G C AAG C AAGT TAT AT AT GT AGT T TAT T T GT AAGAC T GAT AT GAAAT T G GAAGAT GAAT C T AC TAT T AG C T T T AAT TAT T T T T AC AT T T AG GAAT A T T G CAT C AGT AAC T CAT AAT TTTGGTTTTCTGT TAT C C T GAGT T AAC AC AAAT TAT CCAAGGAGATGGCGGATCATCTGCTTTGAGGTGTTTTTTTTTGAGAATTTTAATGT AT C T GAAT AT AAAAG GTAAAAAT AT G C C AAC TAG C AAT T T C T G C C CAT T C C AGAAG T T T G GAAAT AT TACT CAT T AC TAG GAAT TAAAT AAAAT AT G GT T TAT C TAT T GT T A TACCTCTTTTAATTCACATAGCTCATTTTTATCTTTTATTTTTGTTTGTTTTTTTT GAGATGGAGTCTTGCTCTGTCACCAGGCAGGAGTGCAGTGATGCAATCTCGGCTCA CTCTAGCCACCGACTCCCTGGTTCAAGCGATTCTCCTGCCTGAGCCTTCTGAGTAG C T G G GAT T AC AG G C AG G C AC C AC C AC G C C C AG C T AAT T T T T GT AGAGAC AG GAT T T CACCGTGTTGGCCAGGATGGTCTCCATCTCCTGACCTCATGATCTGCCTGCTTCGG CCTCCCAAAGTGCTGGGATTACAGGTGGGAGCCACTACGCCTGGCCCACATAGCTC ATTTTTAGACTCACTTCCATTAAGTCTTGTTTGGACCCACGAACATTGTCTTTTTT TTTTTAAGATGGAGTTTCACTTTTGTTGCCCAGACTGTAGTGCAATGGTGCAATCT CAGCTCACTGCAATCTCTGCCTCCTGGGTTCTAGCAATTCTCCTGCCTCAGCCTCC CGAGTAGCTGGAATTACAGGCGCCCGCCACCACGCCCAGCTAATTTTTGTGTTTTT AGTAGAGACGGGGTTTCACCATGTTGGGCAGGCCAGGGGTGATCCGCCCACCTCAG CCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGCATCTGGCCAACATGTCTT TTTTTTTTTTTTCCTTTTTAACCACAAAGAGACTTAAGCAGTCCTTGTCACAGATG ATGAATTGATGTTGCAAGTATTGTCTTAGCTTGGATTAATTTTCTTGCTTACTGTA AT T T T AGAT AAT AT AGCT T T GT AAT T AGAGAT T T TAT GT GTAAAC CACAAAAAT GT T T ACAT GAAGGC CAT TAT T ACAGAT GT GAC GT GCAT AAT TAT T AGT AAT T T GT AT G T T T AC AT G G GT C AGT C T G G C AAAAAAT TAT GAAGT T T T AAAAAT T AAAAAAAAT T A T AAT GCCAGTTTTACTG GAAAGT AAAAT T AT T T C AGT AAT C GAT TAT AG CAAAAGT AT T GAT T T T CAT T C C AGAC AAAAGT CAGAAT GAAAG GT AAT T T C T C AAT AC T C T T T CAGAT T AAT AAAAGT AC CT GT AGC GAT T T T TAT CAT T CACAAGT AT AT CACAAGT A AGT T AGAAT T T GAGAAC T GT GT T C T AGAT C T C T GAG GAGAT G C AGT C AGAT T T C T G AACTGTCTCAGCAAATGGTAAGTAACTTAGAGCTAGTAATTAATAACCTGTCCTTT GAT T T C T GAT T C AG C C AAGAAT G G C CAT AT T T G G GAAAG G CAGAT C T G GAGAGT AA CCACGTTTTCATTCATTTACCACTTCTAGGCCCCTCCAGAGCTCTCAGATATTTTG GGGTTGAGCCCTTCCCCAAAGCCATACAGGACCTTTTTTTTGTGATCTGTTCTAGC CATTTTTATGTTGGGTGCTTGTTATGGACTGAGCATTTATGTCCTCCCACACCCCC CCCATACCTTTTTTGAAGTCCTAACCCCCAGTGTGATGGTATTTGGAGACAGGGCC TTTGGAAGGTAATTACAGTTAGAAGAAGTCGGGAGGGTTGGGCCCAGGTCTGATTG GAT TAGTGCCCT TAT AT GAAAAGAC AC C AG GAC G G G C G C AGT G G C T C AC AC C T GT A ATCCCAGCACTTTGGGAGGCCAAGGTGGGTGGATCACGAGGTCAGGAGTTTGAGAC C AG C C T G G C C AAT GT AGT GAAAC AC CAT C T C T AC T AAAAAT AC AAAAAT T AG C T G G GTGTGGTAGCGGGCTCCTGTCATCCAAGCTACTCGGGAGGGTGAGGCATGAGAATC ACTTGAACCCGGGAGTTGGAGGTTGCAGTGAGCCCAGATTGTGCCACTGTACTCCA GCCTGGGT GACAGAGT GAGACT CT GT CT CAAAAAAGAAAAAAAAAAAAAAAGAGAC AC C AGAGAG C T T GT T AGAAGAG GT CAT GT GAG C AC AC AGT T AGAAGAC C T T C AAG C CAAAGAAGAGGCCTGAGATTGAAACCTACCTTGCAGGTACCTTAATTTTGGACTTC CCAGCCTCCAAAACTGTGAGAAATAAGTTTCTGTTAAGTCACTCAGTCTGTGGTAT TTTGTTATGGCAGCCTGAGCAGGTAGTTGTTCTTTCAGAAGGTGTTGATAATAACC ACAT G C AAC AC C AAGT C AC AAAT AAT AAAAC AGAT GTAACTTATATT CAT AC AGAA AGTTGGGCACTGCCATTGCCTTGTTGGTTTACACGGCTGTGCTAGTTCAGTAGCAG AAAGGTGCTGGTCTCCTTTACTCAGTTTACAATCTAGGCAGTAGAATGTAATCACT GCTTTAAACTTGATACTGCTTAGGGAGAGAATCATTGGTGCTGGGTAACTTTGGGT TCTAGGTTTACTTTTTGTGTATATATAACTGTTTTTGGTAAATCACAAGTTTCTGG G C T T GT C GAAT T AGAT T T T GT T AC AGAT TAT GAG C T T TAT TAT G C TAT AC AGT TAG TTGTATGTATATATGCCTTTCCCACTAGATTTTAAGCTTTTTTTTTTTTTTTTTTT TTGTGACGGAGTCTTGCTCTTGTCGCCCAGGCTGAAGTGGAGTGCAGTGGCACAAT CTCGGCTCACTGCAGCCTCCACCTCCTAGGTTCAAGCGATTCTCCTGCCTCGGCCT CCCAAGTAACTGGGACTACAGGCACGTGCCACCACACCCGGCTAATTTTTGTATTT TTTGTAGAGACAGGGTTTCGCCATGTTGGCTAGGCTGGTCTTGAACTTCTGGCCTC AGGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTTACAGGCATGAGCCAC CACGCCCAGCTATAAGCTCTTTAAGGGTTGTAAATTTATAATCATTCTTTTACTCT CCTGCAAATTCTGTTGCACACTGCCTTAATCAAGGTAGATGCTGAATGCATTTTTG TATAATTGAATATGTTGCAATCCCCAACTCTCTCCAACTGTTCCTGTCAAAGCAGC C AC T G GAT T GT T AAC T AAT C CAT AT T AGAT G G G GT T AAT T AAT AT CAGAT G G GAC A AGT AAG G G C T AAT AAGAT TAT AG G C C AC C AAGT AGAT TTCTGTCTAGCTCT TAT AG AGATTGAGTTTATTGGACCTGTTTGATAGGAAGTTTTGGTGTTTGGGATGATTAAA ACT GAAGT TCCTATTTATT GAAT TAT AC C TAT T TAT AT TAT T T CAT AT C AGT G GT C C AC AT G C AAGT GAG G C T T C T GAGAC AGAGT T T GAGT TCTCTCTT C AAC T AC C AT AA CACTTAACCTGTATCTTTTTTTTTTTTTTTTTTTTTAGACGGAGTCTCGCTCTGTC ACTCAGGCTGGAGTGTAGTGGTATGATCTCGGCTCACTGTAACCTCTGCCTCCTGG ATTCAAGCAGTTCTCCATGTCTCAGCCTCCCTAGTAGCTGGGATTACAGGCCTGTG CCACCATGCCTGGCTAATTTTTTTTTTGTATTTTTAGTAGAGACGGGGTTTTACCA CGTTGGCCAGGCTGGTCTCGAACTCTTGACCTCGAGCGATCAACTTGCCTTGGCCT CCCAAAGTGCTGGGATTACAGGCATGAGCCACAGCGCCCAGCCGTCTTTTTTTTTA AAT AG C AAT T T AAC AC T GT T C AC AGT TACT CAT GT AC AT GT CAT G C CAT C TAT T AC ACTGTAAGTTCTGTGAGGGTAGCTGTATCAAATTTATCTAACTCTCTCTAGTATGC AT GACAT AGT AAGT AT T CAAT AAAT AT T T GCAT AT T AGT GAT AAGGAT ACAGGT T C TGAATAGTGGGTCCTTACCATTTAAGAATTAGTATTTGATGGCCGGGCGGGGTGGC TCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCATGAGATCAG GAGAT C GAGAC CAT C C T G G C T AAC AT GGT GAAAT CCCGTCTT T AC AAAAAAAAT AC AAAAGAATTAACCAAGTGTGGTGGTGGGTGCCTGTAGTCCCAGCTACTGCTTTGTG AG G C T GAG G C AG G C AGAT C AC C T GAG GT G G GAAAT T C AAGAC C AG C C T GAC C AAC A TGGAGAAACCCCATCTCTACTAAAAATACAAAATTAGCCGGGCGTGGTGGCGCATG TCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAG GCGGAGCTTGCAGTGAGCCAGGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAG CGAGACTCCGTCT C AAAAAAAAAAAAAAAAAAAAAAT T AGT AT T T GAT AT T T GAT C AT T AAAT AT GAAT T AAGAG GAC T T AGAC T T T T T GT T AAAT GT C AAG C T G G GAAAAG TTGTCATTTAAATGAATTGCCTCTTATTTAATTTCGTCTGATGATACATTTTGTTT TTATTTTGTAAAAAATTATTTTTTTTCTTTTTGGAGACAGGGTCTTGCTCTGTTGC CCAGGCTGGTCACAAACTCCTGACCTCAAGCAATCCTCCTGCCTTAGCCTCCCAAA ATGCTGGGATTACAGGCGTGAGCCACCTCGCCCGGCCTTGTATTATGATACATTTT GAAC AAC T AC AAGT AGAC T T G GT AT AAT GAAC C T G C AC GT AC C CAT T G C C AAGT T C TGACAACTGTCTGTCTATAGCCAATTATGCATTTCTTAAATTAGAACCCCCCCAAT AT AC C CAAAT AT AT AT AT AT GT GT GCAT AT AT AT AGT AAGT T GT AACAAAGT T GT G AAT T CAT AC C T GAAGT AT C T C AAGT GAT G C AAGT T T TAT GAAT T T T T GT T TAT G C C T T T T G G GAAGAGT T GT AT T GAC AAAT TTTTTATGCT T AAAGT AAAC CAT AAAT C AA AAAAATAAAAT CTAGGAT GCAATAAAACAAAACAACTT CTT GACATAAGTAT GGTA TGTAAATCTGTTTTGATTGGAAATCAATTTGTTATATTGCCAGAATTCCTGTTTTA GAATACATCTCTGCTGATCTGTCTGTATTCTTAGACTGCATATCTGGGATGAACTC TGGGCAGAATTCACATGGGCTTCCTTTGAAATAAACAAGACTTTTCAAATTCTTAG T C GAT C T G C AGAAC C T GT AG C C AG G C AC T GAAC CAT T T T GAT AGAT G C AGT AAT C G TTGCAAGTGTATATTTCAAGGAGTTCTGGCTGGGTCCTAGTTTATGCTTGTGGCAG AAGCAGT GAGTAACT GGGAGGAAGTT GGT GAGTAAGCTT CAAGGAAGAAGT CATTT TTAGTACTCTGGATCTTCCTGATTTTAAAGCACTACAAAATGGTGCATTTTCATTC T T GT C AAGT GAT AAC AGAT AT AT T C T GAT GAG C C T GAAAT GAAT AT AT AT T GT AT C AT T T T TAT AAT AT C T AG C AAG GT T T GT AT T T T C C T AGAAC T T GAAC T AAAT T T C AG T T CAT AAAAT T T AT AAAAT ACT T AGT T GT T GT AAAAT AT T T T TGGAAT GT T CACAT AG GT GAC AC AC AAAT GT C C CAT T T T CAT T C T T T C TAT AGT AAAT AT GT T C T GAT AT GT GAAG GT T T AG C AGAT G CAT C AG CAT T T AAT C C T AGAG GAT C T G G CAT AAT C T T T T C C C C C AAGAAT AGAAAT T T T T T C T G C T T AT GAAAGT AGT AC AT GT T T C T T T AAAA ACAAATCAATATTGACTTCTGCCTGCTGTATAGCACTATGCCTCCACCTGGCCATG ACCAGGGGCATGTCCTGGTCCACCTACCTGAAAATGTTTGCAACCAGCCTCCTGGC CAT GT G C AC AG G G G C T GAAGT T GT C C C AC AG GT AT T AC G G G C C AAC C T GAC AAT AC AT GAAGT T C C AC C AAAGT C T GAGAAC T C AGAAC T GAG C T T T G G G GAC T GAAAGAC A G C AC AAAC C T CAAAT T T C T C AG C AC T G GAAAC C T C AAAAT AT AAC T GAAT T C CAT A AAT AAGAT T T T AAGT C T T AAAT AT GT AT T T T T AAAT GT AT T AAAAGT C AAG C T G C T T GT AT T T AAG C AC C T AAT AC AAT GCTTAGGTT GT AAAAG GAGAT G C T C AAT AG GT A C T AAC T GAT AT AT T GAGAT T T AAT TAT G GT T T GAC C AAT AT T TAT T G GAAAC C G C C AAAGCTTAAATCATCAGCTTCTTGAATGTGATTTGAAAGGTAATTTAGTATTGAAT AGCATGTGAGCTAGAGTATTTCATTCTTTCTGGTTTATTTCTTCAAATAGACTTTG AATATAAT GGT GAAT GGGTATTATAAATTAACTAATAAAAAT GACATT GAAAAT GA AAAAAT AT AT AT AT T AAAGT GTAGAAAGT GACCAGGCGT GGT GGCT CACACCT GTA ATCCAAGCACCTTGGGAGGCTGAGGCAGGAGGATCTCTTGATCCCAGGAGTTCAAG AC C AG C C T G G G C AAC AT AG C GAGAC TTCGTCTC T AAAAAAAAAAAAGAGAGAGAAA AAAAT T T T T T T TAT T TAAAAAAAGT GTAGAAAGT GT CAAGAC C C CACT T CT T AC CA TTATTTGGTATATTTCTCTATACCCACCCACCCTTCCTCCTTACTCCCTCCCTCCC TTCCCAATCTTTTTATCTTTTTGTATTCTGATTTTTTGTTTGTATATTTTGCTTTA AT T T AAT GT AT C C T T T AAAAAT T T C C CAT AC AT T T T AT AT GT AT AT AT AAAAAC G C AT G C T G C C AAAGAT AAT T T AT AAGAAAGAC CAT T GAAT T T T T T T AAAAGT GAT AT A T AT T CAT T GAAAAAAAT T T AGAAT AT AT AG CAAAG C AAT AAAGAAC T AAAT AAAAT TGCTGTAACTCCTCTTTCAAAGATAAGTGCTTTTATGATTTTGTTGTATTTTTTTC T GT AT AT AGGT ACAT AT AT AGT AT T T AT AAAGCT GT ACT CAT AGT ACAT T T T CACA T C AC AG GT AC CAT AT C AGT GT TAT T AAAT AT T T T GT AT G C C AG G G G C T AGAC AT AC CAAGAC AAC C AAT AT GTGGTTCTACT T AAAT AAT AT TAGAGTATCTTTTAT GAT GA C AC T T CAT GAGT T GAC TAT AAT AAT C T T AGAC T T C T AAGAGT TTGGGTTTT C AAAA GATCACTTAGCTTTTTTGGGTGATTTTTCCCCCTTACTGTGAGATGAGAGAGGCTG TTTGGATTTGGGATTGGGGTAGCGGGGACAGCAACTTTTCTTTTCTTTTTCTTTTT TATTTTGAGGTAGGGTATTGCTGTGTCACCCAGGCTGGAGTGCAGTGGTGTGATCT CGGCTCACTGCAACCTCCACCTCCCGGGCTCAGGTGATCCTCCTGCTTCAGCCTCC CCAGTAACTGGGACTACAGGCGCGTGCCACATGCCTGGCTAATTTTGTATTTTTAG TAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCTAACTCCTGACCTCAGGT GATACGCCCACCTGGGCCTCCCAAAATACTGGGATTACAGGCATGAGCCGCTGCAT CAGCCAGCAGTTTTTCTTGTGGTTTTTTTTGTTTGTTTTGTTTTGTTTTGTTTTTG AGATAGGGTCTTACTCTGTTGTCCACGCTGGAGTGCTGTGGTATGATCGTAGCTCA CTGCAGCCTCAAACTCCTGGGCTCAAGTGATTCCTTCTGCCTCCGCCTCCCGAGTA GCTGGGACTACAGGTATGCACCACCATACCTGGCAAATTTTTACAAAGTTTTTTGT AGGGACGGGGTCTTGCTACATTCCCCATGTCGGTCTTGAACTCCTGGCCTCAAGCA ACTCTCCTGTCTCAGCCTCCCAAAGCACTGGGATTACAAGTGTGAGCCACCACACC ATGCCAGTTTTTCCTGTTCAGTGTGATATTTTATCTTGTTAGACTACAGTGTGTTA AAAC TTGTTTTAC TAAAT T T T C AAAC AT AC T CAAAAGT G GAGAGAAT AGT AT AAT G AAT AC C C GT AT GT T CAT CAC C CAT GT T T AGAAT AT TAT TAAAT AT AAAGAT T T T GC TGCGTTTGTCTTAGCTCTTTAAAATTTTTCTTTTTCTCTTTGTGACCTAAAGGAAA T T C CAT AT C T TAT CAC TTTACTTC T AC AT T C T T GAC T AAGAT GAC T AAGAC AT AT A GTTACATGGTTTTTTGTTTTGTTTTTTGTTTTTTAAAGACGAAATCTCGCTCTTGT CCCCCAGGCTGGAGTGCAATGGTGCCATCTCAGCTCAGTGCAACCTCTGCCTTCTG GGTACAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCTCCTG CCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGCGGGGGGAGGTTTCA CCATGTTGACAAGGCTGGTCTGGAACTCCTGACCTCAGGTGATCCACCCGCCTCGG CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCTGTTTTTTTG TTTGTTTGTTTTGTTTTTTTTGAGACAGAGTCTTGCTCTGTTTCCCAGGCTGGAGT GAAGTGGCGCATTCTTGGCTCACTGCAACCTTCACCTCCCAGGTTCAAGTGATTCT CCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGTGTCACCACACCCGGCT AATTTTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTGCCCAGGCTGGTCT CGAACTCCTGAGCTCAGGCAGTCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTA CACGTGTGAACCAACCCGCCCGGCCTGTTGTTTTCTTACATAATTCATTATCATAC C T AC AAAGT T AAC AGT T AC T AAT AT CAT C T T AC AC C TAAAT T T C T C T GAT AGAC T A AGGTTATTTTTTAACATCTTAATCCAATCAAATGTTTGTATCCTGTAATGCTCTCA T T GAAAC AG C TAT AT TTCTTTTT C AGAT T AGT GAT GAT GAAC C AG GT TAT GAC C T T GATTTATTTTGCATACCTAATCATTATGCTGAGGATTTGGAAAGGGTGTTTATTCC T CAT G GAC T AAT TAT G GAC AG GTAAGT AAGAT C T T AAAAT GAG GT TTTTTACTTTT TCTTGTGTTAATTTCAAACATCAGCAGCTGTTCTGAGTACTTGCTATTTGAACATA AACTAGGCCAACTTATTAAATAACTGATGCTTTCTAAAATCTTCTTTATTAAAAAT AAAAGAGGAGGGCCTTACTAATTACTTAGTATCAGTTGTGGTATAGTGGGACTCTG T AG G GAC CAGAAC AAAGT AAAC AT T GAAG G GAGAT G GAAGAAG GAAC T C T AG C C AG AGTCTTGCATTTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAA GGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGT CAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTA TTCTGTGTTTT TAT T T T AAAAT TAT AAT AT C AAAAT AT T T GT GT TAT AAAAT AT T C TAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCTGTA CAC AT T T C T T C T C AAG CAC T G G C TAT G CAT GT AT AC TAT AT G CAAAAGT AC AT AT A T ACAT T TAT AT T T T AAC GT AT GAGT AT AGT T T TAAAT GT TAT T GGACACT T T T AAT ATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTT AT G GAGAT AC CAT AAC T GAT T T AAC C AGT C CAC TAT T GAT AGAC AC TATTTTGTTC TTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTTT ATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTG C CAAAT T T T C C T C T AAGAAGT T GAAT CAT TTTATACTCCT GAT GT T AT AT GAGAGT ACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGT TGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACC TCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGT GTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGT TACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTA C AGT C T C T C T T AAT AT T GT AT AT TAT C C AAGAAAT T T CAT T T AAT CAGAAC C T G C C AGTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAATTATGA TAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGA GGGCAAAGGATGTGTTACGTGGAAGATTTAAAAAAAATTTTTAATGCATTTTTTTG AGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCA CTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGC TGGGACTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCAT GGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTT GT TAT AAAGT T T AAT GT AT GAAAC T T T C TAT TAAAT T C C T GAT T T TAT T T C T GT AG GAC T GAAC GTCTTGCTC GAGAT GT GAT GAAG GAGAT G G GAG G C CAT CAC AT T GT AG

Figure imgf000053_0001
β globin CTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGA gene ; CAATGACATCCACTTTGCCTTTCTCTCCACAGGCGCGCACTCCGACATACAAATGA

ITALICS CCCAGTCTCCCTCCAGTCTGAGTGCTAGCGTAGGAGACCGGGTCACCATTACCTGC

encodes CMV AGGGCTAGTCAGGACGTGAATACCGCCGTCGCCTGGTACCAGCAGAAGCCAGGGAA

Enhancer; GGCCCCAAAGCTGCTGATTTACTCCGCCAGTTTCCTCTACTCAGGTGTACCCAGCA

HEAVY GATTCTCTGGCTCACGGTCTGGAACCGATTTCACTCTGACGATCAGCTCTCTGCAG

UNDERLINE CCGGAAGATTTCGCCACATACTACTGTCAGCAACACTACACCACACCCCCAACATT

encodes CGGACAGGGGACCAAAGTTGAGATCAAACGCACTGTGGCTGCCCCGAGTGTATTTA human EFl TATTTCCTCCCAGCGACGAGCAGCTGAAAAGCGGCACTGCATCCGTGGTGTGCCTG promotor ; CTGAATAACTTCTATCCACGGGAAGCAAAGGTCCAATGGAAAGTCGACAATGCTCT

DASHED GCAATCCGGCAACTCACAGGAGAGCGTCACCGAGCAGGACTCCAAAGATTCAACCT

UNDERLINE ACTCACTTTCTAGCACTTTGACCCTGTCTAAAGCTGACTATGAGAAGCATAAAGTG

encodes a TATGCCTGTGAGGTAACCCATCAGGGACTTTCCTCCCCAGTTACGAAAAGTTTTAA multiple TCGGGGCGAATGTCGGGCTAAGCGCGCACCTGTGAAACAGACACTCAATTTTGACC cloning TTCTGAAGCTGGCCGGTGATGTTGAGAGCAATCCTGGGCCTGGATGGAGCTGTATC site ; ATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGTTAACAGTAGCAGGCTTGAG lower case GTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGC encodes GCGCACTCCGAAGTCCAGTTGGTGGAGTCAGGCGGGGGTCTGGTGCAGCCAGGAGG synthetic CTCCCTTCGGCTTTCTTGTGCAGCAAGCGGTTTCAATATTAAGGACACCTATATTC intron ; ACTGGGTGAGACAAGCCCCAGGGAAAGGCCTCGAGTGGGTAGCAAGGATCTACCCT

BOLD ACTAACGGCTACACTCGATACGCAGACTCCGTCAAGGGGCGGTTCACTATTTCAGC

encodes AGACACATCCAAAAACACTGCTTATTTGCAGATGAACTCCCTGAGAGCCGAAGATA transgene CAGCTGTCTATTATTGTTCTAGGTGGGGTGGGGACGGCTTCTATGCCATGGATTAC

(HER Mut) TGGGGACAGGGAACACTTGTCACCGTGAGCAGTGCTTCTACCAAAGGACCTTCAGT from ATG to GTTCCCACTCGCTCCTTCTTCAAAGAGTACCTCCGGAGGCACCGCCGCGCTTGGGT stop codon; GTCTGGTAAAGGATTACTTTCCCGAGCCGGTGACCGTTTCCTGGAATTCCGGTGCT

CTCACGTCCGGAGTCCATACCTTTCCCGCCGTCCTGCAGTCTAGTGGCCTTTATTC

UJiDJ^RLTNE CTTGAGTAGCGTGGTGACCGTGCCAAGCAGCTCACTGGGCACCCAAACTTACATCT encodes GCAACGTGAACCACAAACCATCCAACACCAAGGTGGATAAAAAGGTTGAACCTAAA

SV40 poly AGTTGCGACAAAACACACACCTGCCCTCCGTGCCCTGCCCCCGAGCTCCTGGGAGG

Adenylation ACCCTCCGTGTTCCTCTTCCCCCCAAAGCCAAAAGACACTTTGATGATAGCACGCA signal ; CACCCGAAGTGACCTGCGTCGTAGTGGATGTTTCACACGAAGACCCCGAGGTCAAA

BOLD TTTAATTGGTACGTGGATGGTGTCGAAGTCCATAACGCCAAAACCAAGCCTCGAGA

ITALICS GGAACAGTACAATAGCACATACCGGGTGGTCTCAGTGCTCACCGTACTGCATCAAG

encodes MAR ATTGGCTTAACGGCAAAGAATATAAATGTAAGGTGAGTAACAAAGCCCTCCCTGCG

5 ' region CCTATCGAAAAGACAATTTCAAAAGCTAAGGGACAGCCCCGGGAGCCCCAGGTGTA from human TACTCTGCCCCCTAGTAGGGACGAATTGACTAAGAATCAGGTGTCACTCACCTGCC

IFN gene TGGTCAAGGGTTTCTACCCTTCTGATATTGCCGTGGAGTGGGAGTCCAACGGCCAG

CCTGAGAACAATTACAAAACCACGCCACCCGTGCTCGATTCTGACGGTAGCTTCTT

CCTGTACAGCAAGCTCACAGTCGACAAGAGCAGATGGCAGCAGGGAAACGTGTTTT

CCTGCTCAGTCATGCACGAGGCTCTTCACAATCACTATACTCAGAAGTCCCTCTCC

CTGTCCCCCGGTAAATAAGGATCCCCGGGAGATATCCTAGGCTTGGCCAGACATGA

TAAGATACATTGJ^^

ITIA^ITTG^^

^AAACAAGJI AACAA^

TGTGJGJAGj^i^

CAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAAATG

TATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAAAATGGGAAA

GAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGTGGGGATAGA

CAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCCATTGATATATGTAA

GTGACCTATGAAAAAAATATGGCATTTTACAATGGGAAAATGATGGTCTTTTTCTT

TTTTAGAAAAACAGGGAAATATATTTATATGTAAAAAATAAAAGGGAACCCATATG

TCATACCATACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAGGCA

AAACTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGACTTCAGTGTA

GAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATTGTTAATTAG

ATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAGAAAAGTCAG

GCCATAGAATGACAGAAAATATTTGCAACACCCCAGTAAAGAGAATTGTAATATGC

AGATTATAAAAAGAAGTCTTACAAATCAGTAAAAAATAAAACTAGACAAAAATTTG

AACAGATGAAAGAGAAACTCTAAATAATCATTACACATGAGAAACTCAATCTCAGA

AATCAGAGAACTATCATTGCATATACACTAAATTAGAGAAATATTAAAAGGCTAAG

TAACATCTGTGGCTTAATTAAGGCGCGCCAT GAATT CTGCAGCCCGGGGGAT C CTTTGTTGATTTTTTCCACATAGATTATTTTTGACTGTTTTGGCACTTTATATAAA TGGAAT CATATAGTAAATATATACAT GTATATAT GTATATATACACTATATAT GTA T AT AT AT AGT GT AT AT AT AT ACAT GTATATAT GT AT AT T T ACAT AT AT ACT GT AT A TAT GT AT AT T T ACAT AT AT ACT GTATATAT GT AT AT AT ACAC GT AT AT ACT GT AT A TAT ACAGTAT AT ACT GTATATAT AT ACT GTATATAT GT GTATATATATATACAGTA TATATACAGTATACATATATATACAT GTATATAT ACT CAGTATATATACAT GTATA TAT ACACAGTAT AT AT ACACT GTATATAT GTATATAT AT ACT GT GTATATAT AT AC AGTAT AT AT ACAGTAT AT AT AT ACAT GTATAT GTATATAT AT ACT GTATATAT GT G TAT AT AT AC GTATATAT AC T GT AT AT AT AT AT AGAC AC TTTTGTGTCTGGCTTCTT G C T C T CAT C AT AAAG C AC T T GAAAT C CAT C CAT GTTGTAGCT G GT AT C AGT AG C T A GTTTCTTTTCATTGCTGAGAAGTATCACTTTTTATTGATGAGTAGTTTTACATTGT ATGAACATGCCCGAGTTTGTTTATGCATTCTACTAATAGACACCTGGGCTGTTTGC AGTTTTTGGCTATTACAAAGAAAATTTCTATATTTTTTTCCAACTTTTATTTTAGG TTCAGCGGGTACATGGGCAGGTTTGTTACATCGGTAAATTGCATGTCACTGGGGTT TGGTGTACAGATTATTTCACCACCCAGGTAATAAGCATAGTACCTGATAGGCAGGT TTTTGATCCTCACCCTCCTCCCTCAAGTAGGCCCCAGTGTCTATTGTTCTATTCCT TTTTTTTTTTTTTGAGATGGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGT GCGATCTCGGCTCACTGCAAGGTCCACCTCCAGGGTTCAAGCGATTCTACTGCCGC AGCCTCCCGAGTAGCTGGGATTACAGGCACCCACCACCACGCCCGGCTAATTTTTG TATTTTTAGTAGAGACTGGGTTTCACCATGTTGGCCAGGATGGTCTCGAACTCCTC ACCTCAGGTGATCCGCCCGCTTCGGCCTCGCAAAGTACTGGGATTACAGGCGTGAG CCACGGTGCCTGGCCTATGGTTCCATTCTTTGTGTACACGTGTACTCAGTGTTTAG CTTCCACTTGTAAGTGAAAACATGCAGTATTTGGTTTTCTGAAATTCTTGTCTTCA T C T T T T T GT AGAC AAT C C AC T AC T T T TAT T T T T AAAT AT TAG GAAC AAT AG C C T C T GTGTGTCCTGAATTGCAATGTTTTTTCTCTGATTTTGCTGCTCATCTTGTAACCTG TATGGTGTTTTTCTGTGTGCAAGCTTTTTACTTTACGTGGGCAATTTTATCATTCT TTTCTTTTATGGTTTCTGGGTTTCATGTCATGATTGGAGAAGCTTGATCTACCCTG ATACTATAAAAATATCCACCTTGGCTTTTTTTTTTTTTTTTAGATGGATTTTCACT CTTGTTGCCCAGGCTGGAGTGCAATGGCGTGATCTCGGCTCACCACAACCTCTGCC TCCCAGGTTCAAGTGATTCTCCTGCCTCGGCCTCCTGAGTAGCTGGGATTACAGGC AT G C G C C AC C AC G C T C AG C T AAT T T T GT AT T T T T AGT AGAGAC AG GAT T T C AC CAT GTTGGTCAGGCTGGTCTTGAACTCCCCACCTCAGGTGATCTGTCTGCCTCGGCCTC CCAAAGTGCTGGGATTACAGGCGTGACCCACCACACCTGGCCCACGTTGTTTTCTG ATAGTGTTTTCACGTGTTATTTTTTCTGTCTTACTCTTTGAATCATCTAAATGTAT CTTAATGTCAGGAGTGAGGTAGAGGACCCAGTTGTTTGGTTTAAATGACTAGCCAG TTTTCCCAACACCAGGTGTTGAACATCCCTCACTGATGTGAGATGCTGCCTTTTTC ACAGTCTCAATCCCCATGTGCATTTCTATTTATTTTCCTTCCACTTTATTCACATC T T T T T CAGT AT C CACAAC CAT AT T GT T T T AAAT AGCT T CAT GAT AT GT T T T AAT AT TTGGTAGAGCTAGAACCCTTTCTTGGTTCTTTTTCTTTTCTTCTTCTCCTCCTTTT CCTCCTCCTTCTTTCTCCTCCTCCTTCTTCTTTCTTTTTAGAAATTTCCTGGCTAT TCTTGTTTTGTAGTCTTCCAGATAAATAATTTTGAATCACTTTGTCATGTTCCAGA AAAT AAT AC T G C C AGAAT TTTTATTGT GAT C AC AGAAGT T T T AC AGAT T CAT C T G G G G GAAAAAAAC C CAT C T T C C C AGAAT AC AGAAAG G GT GAG G GAAAGAC AAAC AT C A AGGACAAGTCTCAGGTTCTGGACTTGGAGACCAAGAGGATTTGGGGCATCCGGGAG CAGGGCAGGGAGGTGTGATGGCTGCCTTTCACTGAGTAGGAGGATGCCCAGACTTG GGGGAACTATGGAAAGTTTGGTTCAGACTTTCTGAGTTGGAGATGTCTATGCGCAG TCCACAGGGCGAAGCCCTGGAGGTAGAGGCCCTCCTTACTGCCTCCCTCATTCAGT CTGAGGTCCATTCTCCACATGGACACTGAAGTGACTCAAGGCTTCCCTTGTTGGAT CAGGATAGTCTCCAGGGCCCTGGGTGGTCTGGGCCTGCCCACCTCCCCATCCCCTC TCCTGCCTGCCCTCCTCTTGCTCACTGGACCCCACCCACCAGCCTCCTTCTATTCC TCCTGACATTTGCACTTGCTGTTCCGTCTCTGCTTGGAACATCCTAGCTCTTTGCA TGGCTTGCTCCTTCCCATCTTTTAATCTAAGCTCAAATATCACTCCTTCAGATATG CCTTTCCCAAATATCCTAGCTAAAGAGAACTGCTCCCCACTTCCCACAAGCCCTAG CAAACTGGGACTCTCCTTCTTGCTTTCTTTCTCTCTGCCCCTTCTGTGTTTTTTCC ATAGACCTTAACTTGTTACTGTTTTATTTGTTGATGTGTTTACATCATTTGTCCCC CTGTGATGGTTAGTTTTATGTGTCAACTTGGCTAGGCTGTAGGGGCTGTTCGTCAA TCAAACACTGATCTATGTGTAGCTGTGAAGGGATTTTGTAGCTGTGATTAATAGTT GAC T T T AAGT AAG GAAGAT TAT C C T G GAGAAT GT G G G G GAAG G G G C AGAC C T C AG C C AAT CAGT T GAAAG G C C T T AAGAAC AAAG C T GAGAT T T C C C C GAG GAAGAAGAAAT TCTGCCTGTGGACTGCAGCATCAGCTCCTGCTTGAGAGGTTCCAGGCTGCCCTTCC TGACAGCCTGTCCTATGATTTTGGACTTGCCTTGCCAGGTTTTTCCTCTTTCTTGA CCCCCCAAAACCATATATATCTCCTGCTGGTTTGTTTCTCTGGTGTAATCCTGAGG GATGAACACTCCTCCCTCCCAGTTCACTCTGGGAGAGGTTGGGGATCATGTTTGTC TATTTCCCCATGCCTGGCCCATGCGTGTTTATTGAATCACTGAACAACCACGCATT AGGAAGCCAGTCATATGTGCTTCTTCAGAACCTCATGTCCATTGCCAGATCTCCCT GGCTTTTGTGGC T AGAG GAC AAGT GAGAGAT AGT AG C T T AC C C AC AGAC C T T G G C C CTGAGGCCCCCAGGGGCCTGAGCCTGCTGTAAGGGAGGAGGGAGCCCCTGAGGTCT CACACACCTCCCTGGGGATCTGGCATTTTCCCCTGGGGCTGGCCTCAGAGCTGGGC GGGGGCAGATTATGGAGTGGGTTGTAGAGGGAGCACCTCCCCACTAGCAATCATGG TTTTTTCTGCGCCTCTCATAGGGAGGCCTACAGGCCTCCACCTTCAGCTCTTCAGG AG C C AC AG G C AG C T GAGT GAC T T C T G CAT C AC AGT C T T C T C AGAGAC AAAC T T G C A GTAAGAAAGAGGGGCCCGTGAGGGACCCCAGAGAAGGCTGTTGTCAAAGCAGGATG AGAGTGAACTCTTCCATGGGGGACACCCAGCGTCTCCAAGCTCTTTTATGCTGTCT T C AAG G G GT C T AGAGAG C T T C AG C C C AAC AT GAGAC C C AGT C C AG C AG CAT T T C C C T G C GAAGT GAAAGT TAG GAC C C T GAC T AGAT AC AC C AC G C T GAC C T C AG C C AGAAT ATCAAGATGCTGAGGCGCTGAGATGCTGGGATGCTAAGGTGCTAAGGTGCTGGGGT GCTGGGGTGCTAGGATGCTGAGGTTTTGTGATACTGGGTCGCTGAGATGCTGGGAT ACTGGGGTGCTGAAGTGCTGGGGTACTGGGGTGTTGCCATGCTGAAATGCTGGGGT GTTGGGATGCTGAGGTGCTGGGTTGCTGGGATGCTGGGATAGTCCTTGGATGCTGC GGTGCTGAGATGCTGGTCTGCTGGTCTGCTGGGGTGTTGGGATGCTGGGATTTTGG GATGCTGGAGTGCTGCAGTGCTGCAGTGCTGAGATGCTGGAGTGCTGGGGTGCTGG AATACTGTAGTACTGGTGTGCTGGAGCATTGAGATGCTAGGGCACTGGGATGCTAA GGTGCTGAGATGCTGCAGTGCTGGGGTGTTGGGATGCCGAGGTGTTGGGATGCTTA AGTGCTGGGGTGTTGGGATGCTGGGGTGCTGGAACACTATGGTGCTGGGGTGCTGG AGTGTTGAGATACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGTGTTGGG ATGCCTAGGTGCTGGGGTGCCGAGATGCTGGAGTACTAGTGTGCTGGGATGCTGAA GTGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGA GTGTTGGGGTGCTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGA ATACTATGGTGCTGGGGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGAA GTGCTGAGATCCTGGAGTGCTGGGCTGCTGGGCCACAGGCTCTTGAATCCATTCGT C T G C C C AG G G GAAGAAAC C AGAAGAT AAAGAG C T AAT GAAG GAG CTTTGGTT GAGA G G GAG GAAGT AAT G GAAG GAG C AAC AT C T T GT G GAG GAG C AG GAGAGAAT G GAC C T C AG GT T G G GAGAGAG G G C C AG G C T AC AG G C C AGAGAG G C AGAAG GAT T C C AG C AGA GTGTGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTCCAGTAC TGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAG GAG GAAT T T G GAAC T GT GAC T AGAGT AC T T AG G G G GAAG GAG G C AAGAC T G GAG C C AGATTGCTCTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCACTCCTT TGAAGGGTGGCAGAGAGCTGGAATTGGTGCTGGATGGGCTGTGGGGTGACAGGGTC ATGTGGAAAGCCCCTGGGGGGCACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGA G GAC AG C T AAG G GAAAG C G GAAT G G C AGAAC AC G C AC T G C C AG GAG GAAT GAG GAT AGGGTCAGGAGTGCCAGGGGCAGTGAGGCCAGCCTGGGGTCAGGTGGCAGGACGTG TCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCTTGAGGTTCCCTGT GTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGCTCTGCG AGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGGGA CAACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGGGCTTCATT TTCCCAGCGGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTGGGTCCAACACAGAG CCAGAGAAGGGTGAATGGGCCTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAG TGATGGAGGGGGACAGCACAGCTGAGCCCAAGTGCTTTGGTGTGGCCCTGAGGGAA AGCTGCAGCCTGCCTGGGGCCTGGCATGGATGGGACACTTGAGGCAGAGGGACAAT AGTGGGCGCTGCAGTGAGGCTGGCTCTTGGAGAGGTTTCCTGAGGAGTGCTGCCTG AGACGGGCAGGGAGAACAGAGACAAAGTT GGT GACAGGGAAT GAAAGCT GACT GAA G GAC T T T AC C C AGAC C TAT GAG GAT AT C T C T C T C AG C AG GAAG C AG GAG G G GAC T G TGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCTCCGACCCCT CTGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCACCCCCATTGGTCGA TCGTTGTCCCTTGTCACAGCCATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGC ATTTTTAGGCTCTGCGGGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAG CTCCTGAAAGCCACTGATCAAATTGGAACATTTTGTACCTTAGGGATGAGGATATC AAC T C T C C C AG C C AC T T AGAG G GAT AAAT GT GAT GAT G CAT T C AAT T GT GAC T AC A TCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGC ATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCATCCATGAAACCTG GGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAAGAAGGG CATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCGTCTAGACCCTC AGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGTCTTTGTGACCTCCCATC CCCCTCTGTTTCATCCTCTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTC TTTGCCATGATGCAAAACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCA GCCTCTGAAAGAACCAAAGCCACTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACT GAATACTCCCTTGAAAAATAAAAACATAGAGGCACTTTTCTCCTGGCTGTTTATTA C AGAAC GAAGAAAAAAC AC AC T G G C T T GAAAC AGAC G C C AGAT T T C AAAT GT AGAG GT GAAAT AC GAG GT G G C AAT T AAAAT GT GAT T AC AGAAAGT C T G GAC AC T GAGAAA AGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATATACATGAC GAT AAT T G C AGAT AGAAAC CAT C AAAGAC AAAC C C C AAAT C AAC T AAT AAT GT T T A C AGAT GTTCCCCCC C AAAC C AC AGAG C C T T AC AT C AAAAC AAAT AC T GAAAG G C T T TAAACCAGGAACAGCTCGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTT CTCTCGGTCTGAATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGC TGAGACAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACT CTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCT G C T GT AAAG C T CAT TAG GAAAGAG C T AG C G GAGAC T GAAAG GT T C G C AAAAGAGAT T AAGAAT C AC AC AAG G C AAT AG GAT T T T T AGT GAAC AT AGAAAT AAAT G G C C AAGT GGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGTCATATCCACATT G C T CAT T G CAT T AAAAC CAT AAG C GAC T C AG C C AC C T AG C T T AAC AAG GT AT C AC T G GAG C AAAC AAC AC G GT C T G CAT AT T T GT AAC AT T GT AT AAT AAAC AC AAAAC AAT G CAT AGT AAAC AC AAC T C T AC T GAAAC AAAAG CCGTCGCTT TAT T T AC AAAGT C AC AAAAT GAAGT AT AAAT AC T T C T GT CAT T AAT GT T T AG GAAAAC CAT T T AC AAAAT T T T C AAAT AT GT AC AC GT AG C T T GAAAAAT C AC C AG C T T T C CAT T T T GT C AC AG GT A GAGAGAG G GAT AAG CAT G G G C T GAC AAC AC C AC T C AAAT T GT AAC G G GAGAC AAC T GCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCTATGG GCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGCCCAGAATTCT GGGCTGTGGCATTTTGAGCAGCAGCATGCTGTTCCAAAATGTCGTCGATCAGCCTC AAGTTGCACACCCAGTCTTCATCTGGGCTCACACAGGAGCCTTTCAAGAGAGCTTC AAT GAAAT C T AC C T CAT T G C AGT C AG GT GAC GAAAT C AGAT CAT TTAGTGGGGGTT GGGGCTGGCGCAAAAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCG AACGGACCTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGC AGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGC TGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCTGT ACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGGTTGTTGGC CAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCCCAATCAGATGGCC TCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTGAACGCTATTATCCACTTGG CTGAGGGGTGTTTTCCCCGAAACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCA TGCAGCATTGTTGAACGCAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGT GGGGACTCAAGGACTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACT TCTGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCTGCC CAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCTGGAGGTGAAAA TTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCTCGCAGCTCTGG TGCCTGGTGAGTGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCAGTTCT GGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCGGCTACACTTGG AG GT GAC TTGTTCTTG GAC G G C T AC AT AC AGAAAGAGAGAAGT G G G GAT GAGT T C C AAAGGCATCCTCGACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACG GACTTCTCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTC CTCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGGGAAG CTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAGCAGATCACACTGGACTGAAAT GCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATG TCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCCTTTGACA GGTGGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGGCTGG TGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGTGACCTGGGAC AGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCAT AGCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGA ACGGTAATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTC C C T GAGT T C T GAAGAG C C T G G G GAT G GAGAGAC AC G GAG C AGAAGAT G GAAG GT AG AGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCAC TCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACTTTTTTGGGGTGGACACT GAGTCATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAGT C AGAG C AC AAG C C C AGT GT GT C T GAC C C AC C C AAGAAT C CAT G C T C GT AT C T AC AA AAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATCATGGCATGTGT ATGTTTTTCAACTAGGTTACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCAC CAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGG CACTCCAGCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGT C T T C C AT AAGAC C AGAAGAG C AAT G GT GTAGAGGT GT CAT G G G C T AC AAT AAAGAT GCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCTGGCCCTCAGACAAATGCTGAG TGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGT TGCTGAACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACAT C AG G CAT T T T G C T AAC T G C T GT AT C C T AG G C C AAT AAAT GT T GAT C AC AT T TAT AG CTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCC TGACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCT GAC AGAT G G C C C AG GAAC C CAT T AGAAAT G GAAG GT C AC T G C AG C AGT AT GT GAAT GTGCGTGTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGTG ATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAG GGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAAGTGGTGATTTTGGAGTCATTT AGACCAGGCCTTCAATTGGGATCCACTAGTTCTAGAGCGGCCGGGCCCAGGGAACC CCGCAGGCGGGGGCGGCCAGTTTCCCGGGTTCGGCTTTACGTCACGCGAGGGCGGC AGGGAGGACGGAATGGCGGGGTTTGGGGTGGGTCCCTCCTCGGGGGAGCCCTGGGA AAAGAGGACTGCGTGTGGGAAGAGAAGGTGGAAATGGCGTTTTGGTTGACATGTGC CGCCTGCGAGCGTGCTGCGGGGAGGGGCCGAGGGCAGATTCGGGAATGATGGCGCG GGGTGGGGGCGTGGGGGCTTTCTCGGGAGAGGCCCTTCCCTGGAAGTTTGGGGTGC GATGGTGAGGTTCTCGGGGCACCTCTGGAGGGGCCTCGGCACGGAAAGCGACCACC TGGGAGGGCGTGTGGGGACCAGGTTTTGCCTTTAGTTTTGCACACACTGTAGTTCA TCTTTATGGAGATGCTCATGGCCTCATTGAAGCCCCACTACAGCTCTGGTAGCGGT AAC CAT G C GT AT T T GAC AC AC GAAG GAAC T AG G GAAAAG G CAT T AG GT CAT T T C AA GCCGAAATTCACATGTGCTAGAATCCAGATTCCATGCTGACCGATGCCCCAGGATA TAGAAAATGAGAATCTGGTCCTTACCTTCAAGAACATTCTTAACCGTAATCAGCCT CTGGTATCTTAGCTCCACCCTCACTGGTTTTTTCTTGTTTGTTGAACCGGCCAAGC TGCTGGCCTCCCTCCTCAACCGTTCTGATCATGCTTGCTAAAATAGTCAAAACCCC GGCCAGTTAAATATGCTTTAGCCTGCTTTATTATGATTATTTTTGTTGTTTTGGCA ATGACCTGGTTACCTGTTGTTTCTCCCACTAAAACTTTTTAAGGGCAGGAATCACC GCCGTAACTCTAGCACTTAGCACAGTACTTGGCTTGTAAGAGGTCCTCGATGATGG T T T GT T GAAT GAAT AC AT T AAAT AAT T AAC C AC T T GAAC C C T AAGAAAGAAG C GAT T C TAT T T CAT AT T AG G CAT T GT AAT GAC T T AAG GT AAAGAG C AGT G C TAT T AAC G G AGTCTAACTGGGAATCCAGCTTGTTTGGGCTATTTACTAGTTGTGTGGCTGTGGGC AACTTACTTCACCTCTCTGGGCTTAAGTCATTTTATGTATATCTGAGGTGCTGGCT ACCTCTTG GAGT TAT T GAGAG GAT TAT AAGAC AGT C TAT GT GAAT C AG C AAC C C T T G CAT GGCCCCTGGCGGG GAAC AGT AAT AAT AG C CAT CAT CAT GT T T AC T T AC AT AG T C C T AAT T AGT C T T C AAAAC AG CCCTGTAG C AAT G GT AT GAT TAT T AC CAT T T T AC AGAT GAG GAAC C T T T GAAG C C T C AGAGAG G C T AAC AGAC AT AC C C T AG GT CAT AC A GTTATTAAGAGAAGGAGCTCTGTCTCGAACCTAGCTCTCTCTCTCTCGAGTAATAC CAGTTAAAAAATAGGCTACAAATAGGTACT CAAAAAAAT GGTAGT GGCT GTT GTTT TTATTCAGTTGCTGAGGAAAAAATGTTGATTTTTCATCTCTAAACATCAACTTACT TAATTCTGCCAATTTCTTTTTTTTGAGACAGGGTCTCACTCTGTCACCTAGGATGG AGTGCAGTGGCACAATCACTGCTCACTGCAGCCTCGACTTCCCGGGCTCGGGTGAT TCTCCCCAGGCTCAGGGGATTCTCCCACTTCAGCCTCCCAAGTAGCTGGGACTACA GGTGCGCACCACCATCCCTGGCTAATATTTGTACTTTATTTTATTTATTTATTTAT TTATTTTTTGAGATGGAGTTTCGCTCTTGTTGCCCGGGCTGGAGTACAGTGGCATG ATCTCGGCTCAGTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTACCTCATC CCCCTGAGTAGCTGGGATTACAGGCGCCTGCCACCATGCCTGGCTAATTTTTTGTA TTTTTAATAGAGACGAGGTTTCACCATGTTGGCCAGGCTACTCTCGAACTCCTGAT CTCAGGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCC ACTGCGCCCGGCCTAATATTTGTATTTTTTGTAGAGATGGTGTTTTGCCATGTTGT CCAGGCTGGTCTTGAACTCCTGAGCTCAAGCGATCTGCCCGCCTCTGCTTCCCAAA GTGCTGGGATTACAGGCATGAGCCACCGTGCCTGGCCTAGGTAGACGCTTTTAGCT TTGGGGTGTGATGCCTGCCCCAGTATATAGTGAATTTAATTATTGCTAGAGCTGGC TGTTTGTTAGTTTTCTTTGAACATAAGATACTCATTGTTTTTAGTTTGCAAATCCC TCTTCCTTTTTAAAAAATTTCTTTCCCTTAAATTGTTTGCATGTTAGCAATAACAA ATGCTTAAATGGTGCTATGTGCTAGATACTCTTCTAAGCCCTGTTATGTATATTAA C T AAT T T T T T AAAT T AC AC AAAT CAGAGAGGTTAAGTAACTTGCC CAAGAT T AC C C AACAATACTAGGATTTGAACCTAAGTTTGTCTCACCCCAGATTCTGCTCTTAATCT CTAAACTTTTAAGTTAGTAGTGACAATAGTAGGTATTTATTGAATACTTAACTATG TTTTAGGCGTT GAAGT AAAT AT T T T G C AG G CAT TAT C T AAT GT AAAC AC C C T AAAG T T AC AT AAC AG GT AC C C T T T AG GT AAAT AAAC AC T AGT AT GAC C T T G GAG G C AC AG AT AGT T GAAGT AAC T T G C C C AAT AT C AC T T AC AT GAAAT T G G C C C T C AAAT GT GT C TGATACAACCCATGCTGCTTGTAACTATCGTTTTAAACTGCCAGGGTAAACTTGGA C AC AC T T GAG C T AAGAAAAAG C T T T T AGAT T T T T G C AAAT T AAT GT GAAAGAT AT G CTTTATGTGGATATAATATCTTCTAAATTTCGGGGATGGTAGTCCTAGAAATGTAA TCCTGCCCTAGCCGAGCTTACCCTGCCAATAATTTTTTACAGAATTGGTAAAACGG AGCACCTTTTTTTTGTCCTTGGCCACACTGTTATCAACAGGGTGTAGATTGACATC AATCTGTAGGTGTAAACCAGAATTACTCTTTGTGACCACCAGGAAATAGAGCAGTT CAGTTCAGGGGTTTCTTTCTGTGAATTTAGCACTGTGACCTGCATACTACAAGTCT ACTTTGTTTTCTATCCATTGTTTGTATCTGGGTATTGCAAAAGGTAGGAAAAGGAC CAACCAGATCAGCAGAGAAGAGTTGCCTTGGAGTTTTCTTTTAGTTTTCTGCAGTT CATTAGATAGTAACTAGGCCATGTCATTTTACTCCCTTGTAGTGAAGATATGTTGA AGTTGTACTGGTATACTCTTCTACCTTTCTGTAATTTTATATTGTGTAGACTTGAT AAAAT T T AT GT GT C AAT C AC C AC CAT T AAT AT C AAT AT T GAG C C T C AAT T C T T AT T TTTCTGCC C AGT G G C T G C C AAAT T AC T AAC AT T T AC AAT AAT T C AC T AC T AC T AAG AT AAT CTACTAGTTC GAT C AC AT AC T T C AAAT T GT TAT G GAAC TACTGTCTT C AG C AT T GT GCT T CT GAT AACT GAT AAGT AT AAT T T T T T T T T T GT C CAGAGT GAACAT GT C TAT T C T T C C AC T GT AC AC AC T AAT AAAAG GAAAAAT T GT AAT AT T G G GT AAAT T C AT GT C CT T ACACAT GT AGT AGT TAT GAGCC CAT GT C C CT AGAAT GAGT AAT AAT T T ATCCCTCCCTTGGTTGAATAGTCAAGAATGCTGATTTTAATTCTTCTAACAGCTTT AT C C C T C AGAAG G GAAG G C AAG C AAGT TAT AT AT GT AGT T TAT T T GT AAGAC T GAT AT GAAAT T G GAAGAT GAAT C T AC TAT T AG C T T T AAT TAT T T T T AC AT T T AG GAAT A T T G CAT C AGT AAC T CAT AAT TTTGGTTTTCTGT TAT C C T GAGT T AAC AC AAAT TAT CCAAGGAGATGGCGGATCATCTGCTTTGAGGTGTTTTTTTTTGAGAATTTTAATGT AT C T GAAT AT AAAAG GTAAAAAT AT G C C AAC TAG C AAT T T C T G C C CAT T C C AGAAG T T T G GAAAT AT TACT CAT T AC TAG GAAT T AAAT AAAAT AT G GT T TAT C TAT T GT T A TACCTCTTTTAATTCACATAGCTCATTTTTATCTTTTATTTTTGTTTGTTTTTTTT GAGATGGAGTCTTGCTCTGTCACCAGGCAGGAGTGCAGTGATGCAATCTCGGCTCA CTCTAGCCACCGACTCCCTGGTTCAAGCGATTCTCCTGCCTGAGCCTTCTGAGTAG C T G G GAT T AC AG G C AG G C AC C AC C AC G C C C AG C T AAT T T T T GT AGAGAC AG GAT T T CACCGTGTTGGCCAGGATGGTCTCCATCTCCTGACCTCATGATCTGCCTGCTTCGG CCTCCCAAAGTGCTGGGATTACAGGTGGGAGCCACTACGCCTGGCCCACATAGCTC ATTTTTAGACTCACTTCCATTAAGTCTTGTTTGGACCCACGAACATTGTCTTTTTT TTTTTAAGATGGAGTTTCACTTTTGTTGCCCAGACTGTAGTGCAATGGTGCAATCT CAGCTCACTGCAATCTCTGCCTCCTGGGTTCTAGCAATTCTCCTGCCTCAGCCTCC CGAGTAGCTGGAATTACAGGCGCCCGCCACCACGCCCAGCTAATTTTTGTGTTTTT AGTAGAGACGGGGTTTCACCATGTTGGGCAGGCCAGGGGTGATCCGCCCACCTCAG CCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGCATCTGGCCAACATGTCTT TTTTTTTTTTTTCCTTTTTAACCACAAAGAGACTTAAGCAGTCCTTGTCACAGATG ATGAATTGATGTTGCAAGTATTGTCTTAGCTTGGATTAATTTTCTTGCTTACTGTA AT T T T AGAT AAT AT AGCT T T GT AAT T AGAGAT T T TAT GT GT AAAC CACAAAAAT GT T T ACAT GAAGGC CAT TAT T ACAGAT GT GAC GT GCAT AAT TAT T AGT AAT T T GT AT G T T T AC AT G G GT C AGT C T G G C AAAAAAT TAT GAAGT T T T AAAAAT T AAAAAAAAT T A T AAT GCCAGTTTTACTG GAAAGT AAAAT T AT T T C AGT AAT C GAT TAT AG CAAAAGT AT T GAT T T T CAT T C C AGAC AAAAGT C AGAAT GAAAG GT AAT T T C T C AAT AC T C T T T CAGAT T AAT AAAAGT AC CT GTAGC GAT T T T TAT CAT T CACAAGT AT AT CACAAGT A AGT T AGAAT T T GAGAAC T GT GT T C T AGAT C T C T GAG GAGAT G C AGT C AGAT T T C T G AACTGTCTCAGCAAATGGTAAGTAACTTAGAGCTAGTAATTAATAACCTGTCCTTT GAT T T C T GAT T C AG C C AAGAAT G G C CAT AT T T G G GAAAG G CAGAT C T G GAGAGT AA CCACGTTTTCATTCATTTACCACTTCTAGGCCCCTCCAGAGCTCTCAGATATTTTG GGGTTGAGCCCTTCCCCAAAGCCATACAGGACCTTTTTTTTGTGATCTGTTCTAGC CATTTTTATGTTGGGTGCTTGTTATGGACTGAGCATTTATGTCCTCCCACACCCCC CCCATACCTTTTTTGAAGTCCTAACCCCCAGTGTGATGGTATTTGGAGACAGGGCC TTTGGAAGGTAATTACAGTTAGAAGAAGTCGGGAGGGTTGGGCCCAGGTCTGATTG GAT TAGTGCCCT TAT AT GAAAAGAC AC C AG GAC G G G C G C AGT G G C T C AC AC C T GT A ATCCCAGCACTTTGGGAGGCCAAGGTGGGTGGATCACGAGGTCAGGAGTTTGAGAC C AG C C T G G C C AAT GT AGT GAAAC AC CAT C T C T AC T AAAAAT AC AAAAAT T AG C T G G GTGTGGTAGCGGGCTCCTGTCATCCAAGCTACTCGGGAGGGTGAGGCATGAGAATC ACTTGAACCCGGGAGTTGGAGGTTGCAGTGAGCCCAGATTGTGCCACTGTACTCCA GCCTGGGT GACAGAGT GAGACT CT GT CT CAAAAAAGAAAAAAAAAAAAAAAGAGAC AC C AGAGAG C T T GT T AGAAGAG GT CAT GT GAG C AC AC AGT T AGAAGAC CTT C AAG C CAAAGAAGAGGCCTGAGATTGAAACCTACCTTGCAGGTACCTTAATTTTGGACTTC CCAGCCTCCAAAACTGTGAGAAATAAGTTTCTGTTAAGTCACTCAGTCTGTGGTAT TTTGTTATGGCAGCCTGAGCAGGTAGTTGTTCTTTCAGAAGGTGTTGATAATAACC AC AT G C AAC AC C AAGT C AC AAAT AAT AAAAC AGAT GTAACTTATATT CAT AC AGAA AGTTGGGCACTGCCATTGCCTTGTTGGTTTACACGGCTGTGCTAGTTCAGTAGCAG AAAGGTGCTGGTCTCCTTTACTCAGTTTACAATCTAGGCAGTAGAATGTAATCACT GCTTTAAACTTGATACTGCTTAGGGAGAGAATCATTGGTGCTGGGTAACTTTGGGT TCTAGGTTTACTTTTTGTGTATATATAACTGTTTTTGGTAAATCACAAGTTTCTGG G C T T GT C GAAT T AGAT T T T GT T AC AGAT TAT GAG CTT TAT TAT G C TAT AC AGT TAG TTGTATGTATATATGCCTTTCCCACTAGATTTTAAGCTTTTTTTTTTTTTTTTTTT TTGTGACGGAGTCTTGCTCTTGTCGCCCAGGCTGAAGTGGAGTGCAGTGGCACAAT CTCGGCTCACTGCAGCCTCCACCTCCTAGGTTCAAGCGATTCTCCTGCCTCGGCCT CCCAAGTAACTGGGACTACAGGCACGTGCCACCACACCCGGCTAATTTTTGTATTT TTTGTAGAGACAGGGTTTCGCCATGTTGGCTAGGCTGGTCTTGAACTTCTGGCCTC AGGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTTACAGGCATGAGCCAC CACGCCCAGCTATAAGCTCTTTAAGGGTTGTAAATTTATAATCATTCTTTTACTCT CCTGCAAATTCTGTTGCACACTGCCTTAATCAAGGTAGATGCTGAATGCATTTTTG TATAATTGAATATGTTGCAATCCCCAACTCTCTCCAACTGTTCCTGTCAAAGCAGC C AC T G GAT T GT T AAC T AAT C CAT AT T AGAT G G G GT T AAT T AAT AT C AGAT G G GAC A AGT AAG G G C T AAT AAGAT TAT AG G C C AC C AAGT AGAT TTCTGTCTAGCTCT TAT AG AGATTGAGTTTATTGGACCTGTTTGATAGGAAGTTTTGGTGTTTGGGATGATTAAA ACT GAAGT TCCTATTTATT GAAT TAT AC C TAT T TAT AT TAT T T CAT AT C AGT G GT C C AC AT G C AAGT GAG G C T T C T GAGAC AGAGT T T GAGT TCTCTCTT C AAC T AC C AT AA CACTTAACCTGTATCTTTTTTTTTTTTTTTTTTTTTAGACGGAGTCTCGCTCTGTC ACTCAGGCTGGAGTGTAGTGGTATGATCTCGGCTCACTGTAACCTCTGCCTCCTGG ATTCAAGCAGTTCTCCATGTCTCAGCCTCCCTAGTAGCTGGGATTACAGGCCTGTG CCACCATGCCTGGCTAATTTTTTTTTTGTATTTTTAGTAGAGACGGGGTTTTACCA CGTTGGCCAGGCTGGTCTCGAACTCTTGACCTCGAGCGATCAACTTGCCTTGGCCT CCCAAAGTGCTGGGATTACAGGCATGAGCCACAGCGCCCAGCCGTCTTTTTTTTTA AAT AG C AAT T T AAC AC T GT T C AC AGT TACT CAT GT AC AT GT CAT G C CAT C TAT T AC ACTGTAAGTTCTGTGAGGGTAGCTGTATCAAATTTATCTAACTCTCTCTAGTATGC AT GACAT AGT AAGT AT T CAAT AAAT AT T T GCAT AT T AGT GAT AAGGAT ACAGGT T C TGAATAGTGGGTCCTTACCATTTAAGAATTAGTATTTGATGGCCGGGCGGGGTGGC TCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCATGAGATCAG GAGAT C GAGAC CAT C C T G G C T AAC AT G GT GAAAT CCCGTCTT T AC AAAAAAAAT AC AAAAGAATTAACCAAGTGTGGTGGTGGGTGCCTGTAGTCCCAGCTACTGCTTTGTG AG G C T GAG G C AG G C AGAT C AC C T GAG GT G G GAAAT T C AAGAC C AG C C T GAC C AAC A TGGAGAAACCCCATCTCTACTAAAAATACAAAATTAGCCGGGCGTGGTGGCGCATG TCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAG GCGGAGCTTGCAGTGAGCCAGGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAG CGAGACTCCGTCT C AAAAAAAAAAAAAAAAAAAAAAT T AGT AT T T GAT AT T T GAT C AT T AAAT AT GAAT T AAGAG GAC T T AGAC T T T T T GT T AAAT GT C AAG C T G G GAAAAG TTGTCATTTAAATGAATTGCCTCTTATTTAATTTCGTCTGATGATACATTTTGTTT TTATTTTGTAAAAAATTATTTTTTTTCTTTTTGGAGACAGGGTCTTGCTCTGTTGC CCAGGCTGGTCACAAACTCCTGACCTCAAGCAATCCTCCTGCCTTAGCCTCCCAAA ATGCTGGGATTACAGGCGTGAGCCACCTCGCCCGGCCTTGTATTATGATACATTTT GAAC AAC T AC AAGT AGAC T T G GT AT AAT GAAC C T G C AC GT AC C CAT T G C C AAGT T C TGACAACTGTCTGTCTATAGCCAATTATGCATTTCTTAAATTAGAACCCCCCCAAT AT AC C CAAAT AT AT AT AT AT GT GT GCAT AT AT AT AGT AAGT T GT AACAAAGT T GT G AAT T CAT AC C T GAAGT AT C T C AAGT GAT G C AAGT T T TAT GAAT T T T T GT T TAT G C C T T T T G G GAAGAGT T GT AT T GAC AAAT TTTTTATGCT T AAAGT AAAC CAT AAAT C AA AAAAATAAAAT CTAGGAT GCAATAAAACAAAACAACTT CTT GACATAAGTAT GGTA TGTAAATCTGTTTTGATTGGAAATCAATTTGTTATATTGCCAGAATTCCTGTTTTA GAATACATCTCTGCTGATCTGTCTGTATTCTTAGACTGCATATCTGGGATGAACTC TGGGCAGAATTCACATGGGCTTCCTTTGAAATAAACAAGACTTTTCAAATTCTTAG T C GAT C T G C AGAAC C T GT AG C C AG G C AC T GAAC CAT T T T GAT AGAT G C AGT AAT C G TTGCAAGTGTATATTTCAAGGAGTTCTGGCTGGGTCCTAGTTTATGCTTGTGGCAG AAGCAGT GAGTAACT GGGAGGAAGTT GGT GAGTAAGCTT CAAGGAAGAAGT CATTT TTAGTACTCTGGATCTTCCTGATTTTAAAGCACTACAAAATGGTGCATTTTCATTC T T GT C AAGT GAT AAC AGAT AT AT T C T GAT GAG C C T GAAAT GAAT AT AT AT T GT AT C AT T T T TAT AAT AT C T AG C AAG GT T T GT AT T T T C C T AGAAC T T GAAC T AAAT T T C AG T T CAT AAAAT T T AT AAAAT ACT T AGT T GT T GT AAAAT AT T T T TGGAAT GT T CACAT AG GT GAC AC AC AAAT GT C C CAT T T T CAT T C T T T C TAT AGT AAAT AT GT T C T GAT AT GT GAAG GT T T AG C AGAT G CAT C AG CAT T T AAT C C T AGAG GAT C T G G CAT AAT C T T T T C C C C C AAGAAT AGAAAT T T T T T C T G C T T AT GAAAGT AGT AC AT GT T T C T T T AAAA ACAAATCAATATTGACTTCTGCCTGCTGTATAGCACTATGCCTCCACCTGGCCATG ACCAGGGGCATGTCCTGGTCCACCTACCTGAAAATGTTTGCAACCAGCCTCCTGGC CAT GT G C AC AG G G G C T GAAGT T GT C C C AC AG GT AT T AC G G G C C AAC C T GAC AAT AC AT GAAGT T C C AC C AAAGT C T GAGAAC T C AGAAC T GAG C T T T G G G GAC T GAAAGAC A G C AC AAAC C T C AAAT T T C T C AG C AC T G GAAAC C T C AAAAT AT AAC T GAAT T C CAT A AAT AAGAT T T T AAGT C T T AAAT AT GT AT T T T T AAAT GT AT T AAAAGT C AAG C T G C T T GT AT T T AAG C AC C T AAT AC AAT GCTTAGGTT GT AAAAG GAGAT G C T C AAT AG GT A C T AAC T GAT AT AT T GAGAT T T AAT TAT G GT T T GAC C AAT AT T TAT T G GAAAC C G C C AAAGCTTAAATCATCAGCTTCTTGAATGTGATTTGAAAGGTAATTTAGTATTGAAT AGCATGTGAGCTAGAGTATTTCATTCTTTCTGGTTTATTTCTTCAAATAGACTTTG AAT AT AAT GGT GAAT G G GT AT TAT AAAT T AAC T AAT AAAAAT GAC AT T GAAAAT GA AAAAAT AT AT AT AT T AAAGT GTAGAAAGT GACCAGGCGT GGT GGCT CACACCT GTA ATCCAAGCACCTTGGGAGGCTGAGGCAGGAGGATCTCTTGATCCCAGGAGTTCAAG AC C AG C C T G G G C AAC AT AG C GAGAC TTCGTCTC T AAAAAAAAAAAAGAGAGAGAAA AAAAT T T T T T T TAT T TAAAAAAAGT GTAGAAAGT GT CAAGAC C C CACT T CT T AC CA TTATTTGGTATATTTCTCTATACCCACCCACCCTTCCTCCTTACTCCCTCCCTCCC TTCCCAATCTTTTTATCTTTTTGTATTCTGATTTTTTGTTTGTATATTTTGCTTTA AT T T AAT GT AT C C T T T AAAAAT T T C C CAT AC AT T T T AT AT GT AT AT AT AAAAAC G C AT G C T G C C AAAGAT AAT T TAT AAGAAAGAC CAT T GAAT T T T T T T AAAAGT GAT AT A T AT T CAT T GAAAAAAAT T T AGAAT AT AT AG CAAAG C AAT AAAGAAC T AAAT AAAAT TGCTGTAACTCCTCTTTCAAAGATAAGTGCTTTTATGATTTTGTTGTATTTTTTTC T GT AT AT AGGT ACAT AT AT AGT AT T T AT AAAGCT GT ACT CAT AGT ACAT T T T CACA T C AC AG GT AC CAT AT C AGT GT TAT T AAAT AT T T T GT AT G C C AG G G G C T AGAC AT AC CAAGAC AAC C AAT AT GTGGTTCTACT T AAAT AAT AT TAGAGTATCTTTTAT GAT GA C AC T T CAT GAGT T GAC TAT AAT AAT C T T AGAC T T C T AAGAGT TTGGGTTTT C AAAA GATCACTTAGCTTTTTTGGGTGATTTTTCCCCCTTACTGTGAGATGAGAGAGGCTG TTTGGATTTGGGATTGGGGTAGCGGGGACAGCAACTTTTCTTTTCTTTTTCTTTTT TATTTTGAGGTAGGGTATTGCTGTGTCACCCAGGCTGGAGTGCAGTGGTGTGATCT CGGCTCACTGCAACCTCCACCTCCCGGGCTCAGGTGATCCTCCTGCTTCAGCCTCC CCAGTAACTGGGACTACAGGCGCGTGCCACATGCCTGGCTAATTTTGTATTTTTAG TAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCTAACTCCTGACCTCAGGT GATACGCCCACCTGGGCCTCCCAAAATACTGGGATTACAGGCATGAGCCGCTGCAT CAGCCAGCAGTTTTTCTTGTGGTTTTTTTTGTTTGTTTTGTTTTGTTTTGTTTTTG AGATAGGGTCTTACTCTGTTGTCCACGCTGGAGTGCTGTGGTATGATCGTAGCTCA CTGCAGCCTCAAACTCCTGGGCTCAAGTGATTCCTTCTGCCTCCGCCTCCCGAGTA GCTGGGACTACAGGTATGCACCACCATACCTGGCAAATTTTTACAAAGTTTTTTGT AGGGACGGGGTCTTGCTACATTCCCCATGTCGGTCTTGAACTCCTGGCCTCAAGCA ACTCTCCTGTCTCAGCCTCCCAAAGCACTGGGATTACAAGTGTGAGCCACCACACC ATGCCAGTTTTTCCTGTTCAGTGTGATATTTTATCTTGTTAGACTACAGTGTGTTA AAAC TTGTTTTAC T AAAT T T T C AAAC AT AC T C AAAAGT G GAGAGAAT AGT AT AAT G AAT AC C C GT AT GT T CAT CAC C CAT GT T T AGAAT AT TAT T AAAT AT AAAGAT T T T GC TGCGTTTGTCTTAGCTCTTTAAAATTTTTCTTTTTCTCTTTGTGACCTAAAGGAAA T T C CAT AT C T TAT CAC TTTACTTC T AC AT T C T T GAC T AAGAT GAC T AAGAC AT AT A GTTACATGGTTTTTTGTTTTGTTTTTTGTTTTTTAAAGACGAAATCTCGCTCTTGT CCCCCAGGCTGGAGTGCAATGGTGCCATCTCAGCTCAGTGCAACCTCTGCCTTCTG GGTACAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCTCCTG CCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGCGGGGGGAGGTTTCA CCATGTTGACAAGGCTGGTCTGGAACTCCTGACCTCAGGTGATCCACCCGCCTCGG CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCTGTTTTTTTG TTTGTTTGTTTTGTTTTTTTTGAGACAGAGTCTTGCTCTGTTTCCCAGGCTGGAGT GAAGTGGCGCATTCTTGGCTCACTGCAACCTTCACCTCCCAGGTTCAAGTGATTCT CCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGTGTCACCACACCCGGCT AATTTTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTGCCCAGGCTGGTCT CGAACTCCTGAGCTCAGGCAGTCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTA CACGTGTGAACCAACCCGCCCGGCCTGTTGTTTTCTTACATAATTCATTATCATAC C T AC AAAGT T AAC AGT T AC T AAT AT CAT C T T AC AC C T AAAT T T C T C T GAT AGAC T A AGGTTATTTTTTAACATCTTAATCCAATCAAATGTTTGTATCCTGTAATGCTCTCA T T GAAAC AG C TAT AT TTCTTTTT C AGAT T AGT GAT GAT GAAC C AG GT TAT GAC C T T GATTTATTTTGCATACCTAATCATTATGCTGAGGATTTGGAAAGGGTGTTTATTCC T CAT G GAC T AAT TAT G GAC AG GT AAGT AAGAT C T T AAAAT GAG GT TTTTTACTTTT TCTTGTGTTAATTTCAAACATCAGCAGCTGTTCTGAGTACTTGCTATTTGAACATA AACTAGGCCAACTTATTAAATAACTGATGCTTTCTAAAATCTTCTTTATTAAAAAT AAAAGAGGAGGGCCTTACTAATTACTTAGTATCAGTTGTGGTATAGTGGGACTCTG T AG G GAC C AGAAC AAAGT AAAC AT T GAAG G GAGAT G GAAGAAG GAAC T C T AG C C AG AGTCTTGCATTTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAA GGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGT CAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTA TTCTGTGTTTT TAT T T T AAAAT TAT AAT AT C AAAAT AT T T GT GT TAT AAAAT AT T C TAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCTGTA C AC AT T T C T T C T C AAG C AC T G G C TAT G CAT GT AT AC TAT AT G C AAAAGT AC AT AT A T ACAT T TAT AT T T T AAC GT AT GAGT AT AGT T T TAAAT GT TAT T GGACACT T T T AAT ATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTT AT G GAGAT AC CAT AAC T GAT T T AAC CAGT C C AC TAT T GAT AGAC AC TATTTTGTTC TTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTTT ATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTG C CAAAT T T T C C T C T AAGAAGT T GAAT CAT TTTATACTCCT GAT GT T AT AT GAGAGT ACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGT TGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACC TCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGT GTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGT TACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTA CAGT C T C T C T T AAT AT T GT AT AT TAT C C AAGAAAT T T CAT T T AAT C AGAAC C T G C C AGTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAATTATGA TAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGA GGGCAAAGGATGTGTTACGTGGAAGATTTAAAAAAAATTTTTAATGCATTTTTTTG AGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCA CTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGC TGGGACTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCAT GGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTT GT TAT AAAGT T T AAT GT AT GAAAC T T T C TAT TAAAT T C C T GAT T T TAT T T C T GT AG GAC T GAAC GTCTTGCTC GAGAT GT GAT GAAG GAGAT G G GAG G C CAT C AC AT T GT AG CCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATC AAAG C AC T GAAT AGAAAT AGT GAT AGAT C CAT T C C T AT GAC T GT AGAT T T T AT C AG ACT GAAGAG C TAT T GT GT GAGT AT AT T T AAT AT AT GAT TCTTTTTAGTGG C AAC AG TAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCATTTCAC T T G GT T AC AGT GAGAT T T T T C T AAC AT AT T C AC TAGTACTT T AC AT C AAAG C C AAT ACTGTTTTTTTAAAACTAGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGT GTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTT TTCACTATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGG AGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTC C T AGAAAG C AC AT G GAGAG C T AGAGT AT T G G C GAAT T GAAGAAAT CCCCCTTTTTT T T T AAC AC AC T T AAGAAAG G G GAC T G C AG GT AT AC T C AAGAGAGT AAGT C G C AC C A GAAAC C AC T T T T GAT C C AC AGT CTGCCTGTGT C AC AC AAT T GAAAT G CAT C AC AAC AT T GACACT GT GGAT GAAACAAAAT CAGT GT GAAT T T T AGT AGT GAAT T T CAT T CA TAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTT TATGTTGGGTTGGTATTTCCTGTGAGTTACTGTTTTCCTTTAAAATAGGAATTTTT CAT AC T C T T CAAAGAT T AGAAC AAAT GT C CAGT T T T T G C T GT T T CAT GAAT GAGT C CTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAATAAGACC ACT TAT C T AC AT T T AAC TAT C AAC C T CAT C C T C T C CAT T AAT CAT C TAT T T T AGT G ACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGC CAT T T AAGT AT TAT TAT AGAC AT TTTCACTATCC CAT TAAAAC CCTTTATGCC CAT ACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGCT TGAATTAAAGACTTGTTTACGGTATCGATAAGCTTGATATCAAAACGCCAACTTTG ACCCGGAACGCGGAAAACACCTGAGAAAAACACCTGGGCGAGTCTCCACGTAAACG GT C AAAGT CCCCGCGGCCC T AGAC AAAT AT T AC G C G C TAT GAGT AAC AC AAAAT T A TTCAGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTC GGTTACGCCCAAATTTACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTT CCTGTGTACACCGGCGCACACCAAAAACGTCACTTTTGCCACATCCGTCGCTTACA TGTGTTCCGCCACACTTGCAACATCACACTTCCGCCACACTAGTAGGTCACC0GGC

Fab GAGGTACAGCTTGTGGAAAGCGGCGGAGGGTTGGTGCAGCCCGGCAGATCACTCCG 29 fragment of GCTCTCTTGTGCAGCCTCCGGCTTCACCTTCGACGACTATGCCATGCACTGGGTGC adalimumab GACAAGCTCCCGGAAAGGGACTGGAATGGGTATCCGCCATTACCTGGAACTCCGGC contained CACATTGATTATGCTGACTCAGTGGAGGGCCGCTTCACAATAAGCCGGGATAATGC within TNF5 TAAAAATAGCCTCTACTTGCAGATGAATAGCCTCAGAGCCGAAGACACAGCTGTAT cassette ATTATTGCGCCAAGGTGTCCTACCTGTCTACGGCTTCCAGCCTGGACTACTGGGGC

CAAGGAACTCTGGTCACCGTTTCATCCGCCTCAACAAAGGGACCATCCGTCTTTCC TCTTGCTCCTAGCTCCAAGAGCACTTCCGGGGGGACAGCCGCCCTCGGATGCCTGG TGAAGGACTACTTTCCTGAGCCAGTGACTGTAAGTTGGAACTCCGGTGCCCTGACC TCCGGTGTTCACACCTTTCCCGCTGTGCTGCAGAGCAGCGGTCTCTACTCCCTTAG CTCCGTCGTTACCGTTCCCAGCTCATCTCTGGGAACACAGACTTACATCTGTAACG TGAATCATAAACCGTCCAATACAAAGGTGGACAAAAAGGTGGAACCAAAATCTTGT GACAAGACTTAA

Light chain GATATACAGATGACACAGAGCCCCAGCAGCCTCAGCGCCTCCGTCGGCGACAGGGT 30 sequence of CACGATTACCTGTCGGGCCAGCCAGGATGTTAATACGGCAGTGGCATGGTATCAGC trastuzumab AAAAGCCCGGTAAGGCTCCTAAGTTGCTCATTTACAGCGCTTCCTTCCTTTATTCA

GGAGTTCCATCCAGGTTTAGCGGCAGCCGGTCTGGTACCGATTTTACGTTGACCAT CTCTAGTCTGCAGCCTGAGGACTTTGCGACCTACTATTGTCAGCAGCACTACACAA CTCCCCCCACCTTCGGCCAGGGCACCAAGGTCGAGATCAAAAGGACCGTGGCAGCC CCGTCTGTATTCATTTTCCCCCCCTCAGACGAGCAGCTTAAGAGTGGAACCGCCTC CGTTGTGTGCCTCCTGAATAACTTCTATCCACGAGAAGCGAAGGTTCAATGGAAGG TAGATAATGCTCTGCAGAGCGGCAATAGTCAGGAGAGCGTCACAGAGCAGGACAGC AAAGACAGTACCTATTCACTGTCCTCAACCTTGACATTGTCAAAGGCCGACTACGA AAAGCATAAGGTCTACGCATGCGAAGTTACCCATCAGGGACTCTCCAGCCCGGTCA CCAAATCATTTAACAGAGGCGAGTGC

Heavy chain GAGGTGCAGCTTGTGGAATCCGGAGGCGGTCTTGTGCAGCCTGGGGGCTCTCTCCG 31 sequence of CCTCTCTTGCGCTGCTTCCGGCTTTAACATAAAAGACACCTACATTCATTGGGTCA wt GGCAGGCGCCGGGTAAGGGATTGGAGTGGGTCGCCCGGATCTACCCGACAAATGGG trastuzumab TACACTAGGTATGCAGATTCTGTAAAAGGAAGATTCACCATCTCAGCTGACACGTC

AAAGAATACCGCATATCTTCAGATGAATTCTCTGAGGGCGGAGGATACTGCCGTGT ACTACTGCTCAAGGTGGGGCGGCGATGGCTTTTACGCTATGGACTATTGGGGCCAG GGCACACTGGTGACAGTGAGCTCCGCCTCTACGAAGGGACCCTCAGTTTTCCCACT GGCCCCTAGTTCTAAAAGCACCTCTGGGGGCACAGCTGCACTCGGATGCCTTGTTA AAGACTACTTCCCTGAACCTGTGACTGTCTCCTGGAACAGTGGAGCATTGACCTCA GGGGTGCATACCTTTCCTGCTGTCCTCCAGAGTTCAGGACTCTACTCACTTTCTTC CGTTGTTACAGTACCCTCATCATCCCTCGGAACCCAGACTTATATCTGTAACGTCA ATCATAAGCCCAGCAATACAAAAGTGGATAAAAAGGTGGAACCAAAGTCATGTGAT AAAACCCATACGTGCCCGCCCTGCCCAGCACCTGAGCTGCTGGGCGGGCCTTCTGT ATTCCTGTTTCCACCAAAGCCAAAGGACACCTTGATGATTAGCCGAACACCAGAAG TAACCTGTGTGGTCGTAGACGTAAGTCACGAAGATCCAGAGGTCAAGTTCAACTGG TATGTCGACGGCGTTGAAGTGCACAACGCCAAGACTAAGCCCCGGGAGGAGCAGTA CAATTCAACCTATCGGGTCGTCTCTGTGCTCACAGTCCTCCACCAAGATTGGCTTA ATGGGAAGGAGTACAAATGTAAGGTGTCTAACAAAGCACTTCCCGCTCCAATCGAA AAGACTATAAGCAAGGCAAAGGGCCAACCGCGCGAACCACAGGTTTATACACTGCC CCCAAGTAGGGACGAGCTGACGAAAAACCAGGTTTCCTTGACTTGTCTCGTAAAGG GTTTTTATCCCTCAGACATCGCAGTTGAGTGGGAAAGCAATGGTCAGCCTGAAAAC AACTATAAAACAACACCTCCTGTCCTGGATTCTGATGGCTCCTTCTTTCTGTACAG TAAACTGACCGTAGATAAAAGCAGATGGCAGCAGGGGAATGTGTTCTCTTGCAGCG TCATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCACTCAGCTTGTCTCCA GGGAAGTAA

Heavy chain GAGGTACAATTAGTTGAGTCGGGAGGGGGCTTGGTGCAACCCGGCGGGAGTCTTCG 32 sequence of TTTGTCTTGCGCCGCAAGCGGTTTCAATATCAAGGATACCTATATACATTGGGTGA wt GGCAAGCACCCGGCAAGGGGCTGGAATGGGTAGCAAGAATATACCCCACTAATGGC trastuzumab TATACTCGATATGCGGACTCCGTCAAGGGAAGGTTCACAATTTCCGCGGATACGAG with S254A TAAAAATACCGCGTACTTACAGATGAATAGCCTCAGAGCCGAGGATACCGCCGTTT mutation ACTACTGCTCACGGTGGGGGGGAGACGGGTTTTACGCAATGGATTATTGGGGACAA GGTACGCTTGTTACCGTTTCGTCTGCCTCTACAAAAGGGCCCTCAGTTTTCCCTTT AGCACCGTCTAGTAAGAGTACTTCTGGAGGAACAGCAGCACTCGGTTGCTTAGTCA AGGATTACTTCCCAGAGCCCGTTACCGTATCTTGGAACTCGGGCGCACTTACCTCC GGAGTCCACACTTTTCCAGCGGTACTGCAGAGTTCTGGCCTGTACAGCCTATCGTC CGTGGTGACAGTGCCCTCATCAAGTCTGGGGACGCAAACCTATATTTGTAATGTGA ATCACAAGCCGAGTAACACTAAGGTAGATAAAAAAGTCGAACCTAAGTCTTGTGAT AAAACTCATACCTGTCCACCATGTCCGGCTCCGGAGCTTCTAGGAGGCCCATCGGT ATTTTTGTTCCCACCAAAGCCGAAGGACACGTTGATGATCGCCCGGACACCAGAAG TCACTTGTGTCGTGGTGGACGTATCGCATGAAGATCCGGAAGTAAAATTCAATTGG TACGTGGATGGAGTTGAGGTTCATAATGCGAAGACCAAACCGCGAGAGGAGCAATA TAATAGCACATATCGCGTCGTAAGTGTGCTAACGGTTCTCCACCAGGATTGGTTAA ATGGTAAAGAATATAAGTGTAAAGTTAGCAACAAGGCCTTACCAGCTCCAATAGAA AAAACAATTTCTAAGGCCAAGGGCCAGCCGAGGGAACCCCAAGTCTATACTTTACC GCCTTCAAGAGACGAATTGACTAAGAATCAGGTATCGCTGACTTGCCTGGTGAAAG GCTTTTATCCCAGCGATATCGCTGTCGAATGGGAGAGCAATGGGCAGCCTGAGAAC AATTACAAAACAACTCCACCAGTTCTTGATAGCGATGGGAGCTTCTTTCTGTACAG CAAACTGACGGTTGATAAGTCACGCTGGCAACAAGGCAACGTGTTTTCCTGCTCTG TGATGCATGAAGCGCTGCATAACCATTACACGCAGAAAAGCCTGTCCTTATCACCT GGAAAG

Optimized GATATACAGATGACACAGAGCCCCAGCAGCCTCAGCGCCTCCGTCGGCGACAGGGT 33 light chain CACGATTACCTGTCGGGCCAGCCAGGATGTTAATACGGCAGTGGCATGGTATCAGC sequence of AAAAGCCCGGTAAGGCTCCTAAGTTGCTCATTTACAGCGCTTCCTTCCTTTATTCA trastuzumab GGAGTTCCATCCAGGTTTAGCGGCAGCCGGTCTGGTACCGATTTTACGTTGACCAT

CTCTAGTCTGCAGCCTGAGGACTTTGCGACCTACTATTGTCAGCAGCACTACACAA CTCCCCCCACCTTCGGCCAGGGCACCAAGGTCGAGATCAAAAGGACCGTGGCAGCC CCGTCTGTATTCATTTTCCCCCCCTCAGACGAGCAGCTTAAGAGTGGAACCGCCTC CGTTGTGTGCCTCCTGAATAACTTCTATCCACGAGAAGCGAAGGTTCAATGGAAGG TAGATAATGCTCTGCAGAGCGGCAATAGTCAGGAGAGCGTCACAGAGCAGGACAGC AAAGACAGTACCTATTCACTGTCCTCAACCTTGACATTGTCAAAGGCCGACTACGA AAAGCATAAGGTCTACGCATGCGAAGTTACCCATCAGGGACTCTCCAGCCCGGTCA CCAAATCATTTAACAGAGGCGAGTGC

Optimized GAGGTGCAGCTTGTGGAATCCGGAGGCGGTCTTGTGCAGCCTGGGGGCTCTCTCCG 34 heavy chain CCTCTCTTGCGCTGCTTCCGGCTTTAACATAAAAGACACCTACATTCATTGGGTCA sequence of GGCAGGCGCCGGGTAAGGGATTGGAGTGGGTCGCCCGGATCTACCCGACAAATGGG trastuzumab TACACTAGGTATGCAGATTCTGTAAAAGGAAGATTCACCATCTCAGCTGACACGTC

AAAGAATACCGCATATCTTCAGATGAATTCTCTGAGGGCGGAGGATACTGCCGTGT ACTACTGCTCAAGGTGGGGCGGCGATGGCTTTTACGCTATGGACTATTGGGGCCAG GGCACACTGGTGACAGTGAGCTCCGCCTCTACGAAGGGACCCTCAGTTTTCCCACT GGCCCCTAGTTCTAAAAGCACCTCTGGGGGCACAGCTGCACTCGGATGCCTTGTTA AAGACTACTTCCCTGAACCTGTGACTGTCTCCTGGAACAGTGGAGCATTGACCTCA GGGGTGCATACCTTTCCTGCTGTCCTCCAGAGTTCAGGACTCTACTCACTTTCTTC CGTTGTTACAGTACCCTCATCATCCCTCGGAACCCAGACTTATATCTGTAACGTCA ATCATAAGCCCAGCAATACAAAAGTGGATAAAAAGGTGGAACCAAAGTCATGTGAT AAAACCCATACGTGCCCGCCCTGCCCAGCACCTGAGCTGCTGGGCGGGCCTTCTGT ATTCCTGTTTCCACCAAAGCCAAAGGACACCTTGATGATTAGCCGAACACCAGAAG TAACCTGTGTGGTCGTAGACGTAAGTCACGAAGATCCAGAGGTCAAGTTCAACTGG TATGTCGACGGCGTTGAAGTGCACAACGCCAAGACTAAGCCCCGGGAGGAGCAGTA CAATTCAACCTATCGGGTCGTCTCTGTGCTCACAGTCCTCCACCAAGATTGGCTTA ATGGGAAGGAGTACAAATGTAAGGTGTCTAACAAAGCACTTCCCGCTCCAATCGAA AAGACTATAAGCAAGGCAAAGGGCCAACCGCGCGAACCACAGGTTTATACACTGCC CCCAAGTAGGGACGAGCTGACGAAAAACCAGGTTTCCTTGACTTGTCTCGTAAAGG GTTTTTATCCCTCAGACATCGCAGTTGAGTGGGAAAGCAATGGTCAGCCTGAAAAC AACTATAAAACAACACCTCCTGTCCTGGATTCTGATGGCTCCTTCTTTCTGTACAG TAAACTGACCGTAGATAAAAGCAGATGGCAGCAGGGGAATGTGTTCTCTTGCAGCG TCATGCACGAAGCTCTGCATAACCACTACACTCAGAAGTCACTCAGCTTGTCTCCA GGGAAGTAA

Optimized GAAGTCCAGTTGGTGGAGTCAGGCGGGGGTCTGGTGCAGCCAGGAGGCTCCCTTCG 35 heavy chain GCTTTCTTGTGCAGCAAGCGGTTTCAATATTAAGGACACCTATATTCACTGGGTGA sequence of GACAAGCCCCAGGGAAAGGCCTCGAGTGGGTAGCAAGGATCTACCCTACTAACGGC trastuzumab TACACTCGATACGCAGACTCCGTCAAGGGGCGGTTCACTATTTCAGCAGACACATC

CAAAAACACTGCTTATTTGCAGATGAACTCCCTGAGAGCCGAAGATACAGCTGTCT with S254A ATTATTGTTCTAGGTGGGGTGGGGACGGCTTCTATGCCATGGATTACTGGGGACAG mutation GGAACACTTGTCACCGTGAGCAGTGCTTCTACCAAAGGACCTTCAGTGTTCCCACT

CGCTCCTTCTTCAAAGAGTACCTCCGGAGGCACCGCCGCGCTTGGGTGTCTGGTAA AGGATTACTTTCCCGAGCCGGTGACCGTTTCCTGGAATTCCGGTGCTCTCACGTCC GGAGTCCATACCTTTCCCGCCGTCCTGCAGTCTAGTGGCCTTTATTCCTTGAGTAG CGTGGTGACCGTGCCAAGCAGCTCACTGGGCACCCAAACTTACATCTGCAACGTGA ACCACAAACCATCCAACACCAAGGTGGATAAAAAGGTTGAACCTAAAAGTTGCGAC AAAACACACACCTGCCCTCCGTGCCCTGCCCCCGAGCTCCTGGGAGGACCCTCCGT GTTCCTCTTCCCCCCAAAGCCAAAAGACACTTTGATGATAGCACGCACACCCGAAG TGACCTGCGTCGTAGTGGATGTTTCACACGAAGACCCCGAGGTCAAATTTAATTGG TACGTGGATGGTGTCGAAGTCCATAACGCCAAAACCAAGCCTCGAGAGGAACAGTA CAATAGCACATACCGGGTGGTCTCAGTGCTCACCGTACTGCATCAAGATTGGCTTA ACGGCAAAGAATATAAATGTAAGGTGAGTAACAAAGCCCTCCCTGCGCCTATCGAA AAGACAATTTCAAAAGCTAAGGGACAGCCCCGGGAGCCCCAGGTGTATACTCTGCC CCCTAGTAGGGACGAATTGACTAAGAATCAGGTGTCACTCACCTGCCTGGTCAAGG GTTTCTACCCTTCTGATATTGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAAC AATTACAAAACCACGCCACCCGTGCTCGATTCTGACGGTAGCTTCTTCCTGTACAG CAAGCTCACAGTCGACAAGAGCAGATGGCAGCAGGGAAACGTGTTTTCCTGCTCAG TCATGCACGAGGCTCTTCACAATCACTATACTCAGAAGTCCCTCTCCCTGTCCCCC GGTAAATAA

Amino acid EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG 36 sequence of YTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ WT GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS

trastuzumab GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSCD heavy KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI|RTPEVTCVWDVSHEDPEVKFNW chain . YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

Highlighted NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP S is GK

mutated to

A in

mutated

version

(SEQ ID NO:

37)

Amino acid EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG 37 sequence of YTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ mutated GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS trastuzumab GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD heavy KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI RTPEVTCVWDVSHEDPEVKFNW chain . YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

Highlighted NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP and GK

underlined

A is

substituted

in the

mutated

version (wt

is S) .

Amino acid DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS 38 sequence of GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAA WT PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS

trastuzumab KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

light

chain . Exemplary RAKR 39 amino acid

sequence of

furin

Exemplary APVKQTLNFDLLKLAGDVESNPGP 40 amino acid

sequence of

2A

Amino acid DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQS 41 sequence of GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAA light chain PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS adalimumab KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSG 42 sequence of HIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWG heavy chain QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT adalimumab SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSC

DESCRIPTION OF THE EMBODIMENTS

A. Micro-organs Producing Antibodies

[0059] We herein show successful antibody secretion from human dermal micro-organs. The antibody produced by the human dermal micro-organs is functional, systemically delivered to the serum at therapeutically relevant levels, and produced for extended periods of time after implantation. In one embodiment, transduction of a human dermal micro-organ with viral vector comprising a nucleic acid encoding full length or partial length heavy and light chain antibody sequences separated by a cleavage site that is cleaved post-translation, such as a Furin 2A site, leads to production of functional antibody that is secreted from the micro-organ, and can be detected in human serum at therapeutic levels for extended periods of time. The micro-organ is transduced in vitro and implanted into a human subject, and within the human subject, the heavy and light chain are translated as a single polypeptide separated by a cleavage site, wherein the cleavage site is cleaved after translation to produce heavy and light chain that can associate to form a functional antibody.

[0060] In some embodiments, a human dermal micro-organ is transduced with a viral vector comprising a nucleic acid encoding full length or partial length heavy and light chain antibody sequences separated by a stop codon and an internal ribosome entry site (IRES). In this embodiment, the heavy or light chain is translated as one polypeptide, and the IRES allows translation of the partner heavy or light chain as a second polypeptide. In this embodiment, two separate polypeptides are translated. After translation the polypeptides associate to form a functional antibody that is secreted from the micro-organ, and can be detected in human serum. The micro-organ is transduced in vitro and implanted into a human subject, and within the human subject, the heavy and light chains are translated as two separate polypeptides that associate to form functional antibodies.

[0061] Active adalimumab from transduced dermal micro-organs generated from human tummy tuck waste tissue transduced with HDAd vector comprising a construct comprising partial length heavy and light chains of adalimumab separated by a cleavable site such as furin 2a (F2A) successfully produced antibody capable of binding T Fa. The antibody expressed by the dermal micro-organ can be translated, processed, and secreted in its active form, as evidenced by the ability of secreted adalimumab from transduced dermal micro-organs to bind its appropriate target, T F-alpha. Expression of trastuzumab was also successful following transduction of micro-organs. Long-lasting secretion of antibody is possible in vivo, as

transplantation of transduced dermal micro-organs into severe combined

immunodeficient (SCID) mice led to production of active adalimumab for over a month.

[0062] As used herein "micro-organ," "microorgan," and "MO," are used interchangeably throughout to refer to an explant of human tissue that is retrieved from a donor and then maintained ex vivo for future transplantation. The donor may be the same individual into whom the transduced micro-organ is later implanted. The micro-organ may be generated from dermal tissue, in which case it is referred to as a "dermal micro-organ," or "DMO". In some cases, this dermal micro-organ is generated from donated skin, as from a tummy tuck procedure.

[0063] As used herein, "TARGT" refers to micro-organs, which have been transduced with a virus containing an expression construct using the TARGT

(Transduced Autologous Restorative Gene Therapy) technology. In short, the TARGT procedure involves harvesting of a micro-organ, culturing the micro-organ in vitro, ex vivo transduction of the micro-organ with a viral vector comprising a nucleic acid encoding a protein, peptide, or in this case, an antibody. The secretion of antibody from the micro-organ is quantitated and verified, and implanted into an experimental subject or patient. When a TARGT is used to generate antibody, it is termed

"TARGT-antibody". In one embodiment of the present invention, a nucleic acid encoding a heavy chain and light chain of an antibody is provided within a viral vector cassette, wherein the heavy and light chain are separated by a site cleavable after translation, such that the TARGT-antibody fulfills all the expression, folding, and secretion requirements to generate active antibody both in vitro and in vivo.

[0064] "Construct" and "cassette" are used interchangeably throughout.

[0065] As used herein, the term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule comprising at least complementarity-determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen. The term antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse, cynomolgus monkey, etc.

[0066] The term "heavy chain" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some

embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0067] The term "heavy chain variable region" refers to a region comprising a heavy chain complementary determining region (CDR) 1, framework region (FR) 2, CDR2, FR3, and CDR3 of the heavy chain. In some embodiments, a heavy chain variable region also comprises at least a portion of an FRl and/or at least a portion of an FR4. In some embodiments, a heavy chain CDR1 corresponds to Kabat residues 31 to 35; a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabat residues 95 to 102. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.).

[0068] The term "light chain" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence. The term "light chain variable region" refers to a region comprising a light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, a light chain variable region also comprises an FR1 and/or an FR4. In some embodiments, a light chain CDR1 corresponds to Kabat residues 24 to 34; a light chain CDR2 corresponds to Kabat residues 50 to 56; and a light chain CDR3 corresponds to Kabat residues 89 to 97. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.).

[0069] A "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc) and at least one constant region from a second species (such as human, cynomolgus monkey, etc). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.

[0070] A "humanized antibody" refers to an antibody in which at least one amino acid in a framework region of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is an Fab, an scFv, a (Fab'X etc.

[0071] A "human antibody" as used herein refers to antibodies produced in humans, antibodies produced in non-human animals that comprise human

immunoglobulin genes, such as XenoMouse®, and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences.

[0072] The antibodies produced from the micro-organs described herein may be chimeric, human, humanized, or recombinant. [0073] "Treatment," as used herein, covers any administration or application of a therapeutic for a disease (also referred to herein as a "disorder" or a "condition") in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, partially or fully relieving the disease, partially or fully relieving one or more symptoms of a disease, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.

a. Micro-organs

[0074] The generation and use of a dermal micro-organ for expression of polypeptides has been previously described (see US Application 20150118187). In the present invention, the polypeptides produced by the micro-organ are antibodies. In one embodiment, the micro-organ is a genetically modified dermal micro-organ. Dermal micro-organs may comprise a plurality of dermis components, wherein in one embodiment dermis is the portion of the skin located below the epidermis. These components may comprise fibroblast cells, epithelial cells, other cell types, bases of hair follicles, nerve endings, sweat and sebaceous glands, and blood and lymph vessels. In one embodiment, a dermal micro-organ may comprise some fat tissue, wherein in another embodiment, a dermal micro-organ may not comprise fat tissue. In some embodiments, the dermal micro-organ is generated from tissue collected from a tummy tuck procedure. In one embodiment the dermal micro-organ does not comprise epidermis.

[0075] In some embodiments, the dermal MO is used to generate a TARGT that expresses antibody (ie, TARGT-antibody).

[0076] In some embodiments, the dermal MO is derived from autologous tissue and is generated from tissue harvested from the same subject in which it is transplanted after transduction using the TARGT system.

b. Viral vectors transduced

[0077] Any methodology known in the art can be used for genetically altering the micro-organ explant to allow expression of functional antibody. Any one of a number of different vectors can be used in embodiments of this invention, such as viral vectors, plasmid vectors, linear DNA, etc., as known in the art, to introduce an exogenous nucleic acid fragment encoding a therapeutic agent into target cells and/or tissue. In some embodiments, viral vectors may be used to transduce the micro-organ, such as adenovirus vectors, helper-dependent adenovirus vectors (HDAd), adeno- associated virus vectors, and retroviral vectors (such as lentivirus vectors). In some embodiments, the viral vector is an HDAd that has been modified, such as being a gutless, gutted, mini, fully deleted, high-capacity, Δ, or pseudo adenovirus. In some embodiments, the HDAd has been deleted of all viral coding sequences, expresses no viral proteins, or is a non-replicating vector,

c. Expression Constructs

[0078] In one embodiment, expression constructs containing partial length light and heavy chains of antibodies with signaling sequences and a separation site cleavable after translation were cloned into the multiple cloning site of an HDAd viral vector MAR-EFla construct containing regulatory elements (see US Application 20150118187). The separation site allowed stoichiometric expression of both the light chain and heavy chain of the antibody from a single cassette. Expression constructs containing heavy chain and light chain with furin 2A elements were effective in producing active antibody secretion after viral transduction. In some embodiments, the expression construct of the viral construct is one of TNF1, T F2, T F3, or T F5 as shown in Figures 1 A, IB, 1C, and ID. In some embodiments, the expression construct of the viral construct is one of HER2 WT, Optimized HER2 WT, HER2 Mut, or Optimized HER2 Mut as shown in Figures 9 A and 9B. In some embodiments, the components of the expression construct are regulatory elements, separation sites (to allow stoichiometric expression), antibody elements, signal sequences, and/or a polyadenylation site.

i. Regulatory Elements

[0079] In some embodiments, the vector comprises a nucleic acid sequence encoding an antibody operably linked to an upstream MAR regulatory sequence. In some embodiments, at least one additional regulatory sequence to the MAR regulatory sequence is also present.

[0080] In some embodiments, the additional regulatory sequences may comprise a MAR sequence (or two MAR sequences), a CAG promoter sequence, an EFl -alpha promoter sequence, and/or a woodchuck hepatitis virus post-transcriptional regulation element (WPRE sequence). In certain embodiments, the sequence of the EFl-alpha promoter corresponds to SEQ ID NO: 7. In certain embodiments, the CpG free MAR from human beta globin gene (SEQ ID NO: 8) may be one or more of the MAR sequences. In certain embodiments, the MAR 5' region from human IFN-beta gene (SEQ ID NO: 9) may be one or more of the MAR sequences. In certain embodiments, the CMV enhancer (SEQ ID NO: 6) may be used as a regulatory sequence.

[0081] As regulatory sequences are well-known to those skilled in the art, the present invention is not limited by a specific regulatory sequences. Those skilled in the art would understand that regulatory sequences may be tested and selected based upon the optimal level of expression of the resulting antibody. Any regulatory sequence or set or regulatory sequences that allow expression of antibodies encoded by the sequences of the cassette would be appropriate, based upon the desired level of protein expression for a particular micro-organ.

ii. Separation Sites

Those skilled in the art of generation of recombinant antibodies would understand that stoichiometric expression of the light chain and heavy chain of an antibody may improve expression of the resulting antibody, as improper ratios of the light chain and heavy chain can lead to potential aggregation and glycosylation of the monoclonal antibody Ho SCL et al., (May 2013), PLoS One. 21;8(5):e63247. In some embodiments, the light chain and heavy chain are produced in a stoichiometric fashion. There are a number of means of generating stoichiometric expression of polypeptides from a single cassette, and therefore the invention is not limited by the means by which the antibodies are expressed in a stoichiometric fashion.

[0082] In certain embodiments, the light chain and heavy chain sequences are separated by an IRES sequence. Those skilled in the art would understand that there is a large range of IRES sequences, the list of which is diverse and constantly growing; therefore, the scope of the present invention is not limited by the particular IRES used within the construct. In some embodiments, the IRES is that contained within SEQ ID NO: 13. In other embodiments, the IRES is selected from known databases. The efficacy of any particular IRES element can be readily tested by detecting expression of the heavy and light chain using standard protocols. In certain embodiments, the antibody sequence upstream of the IRES contained a stop codon.

[0083] In some embodiments, the light chain and heavy chain sequences are separated by a 2A element or a 2A-like element. In certain embodiments, the 2A element is that of foot-and-mouth disease, as contained in SEQ ID NO: 12. In some embodiments the 2A or 2A-like element comprises or consists of the amino acids of SEQ ID NO: 40. In some embodiments, another 2A or 2A-like element is used. In certain embodiments, the 2A-like sequence is that from equine rhinitis A virus or thosea asigna virus. The efficacy of any particular 2A or 2A-like element can be readily tested by detecting expression of the heavy and light chain using standard protocols. In other embodiments, the construct does not contain a 2A element.

[0084] In certain embodiments, a furin cleavage sequence is upstream of the 2A element, to generate a furin 2A element (F2A) and eliminate the additional amino acids that would otherwise remain attached to the upstream protein after cleavage of the 2A element. In certain embodiments, the furin cleavage sequence is contained within SEQ ID NO: 11 or 39. In other embodiments, a pro-protein convertase other than furin is contained within the cassette. In some embodiments, the pro-protein convertase is one of PACE4, PCl/3, PC2, PC4, PC5/6, or PC7. In other embodiments, the construct does not contain a furin or other pro-protein cleavage site.

[0085] In certain embodiments, no method is employed to promote

stoichiometric expression of the heavy and light chains,

iii. Antibody Elements

[0086] As used herein, "antibody" refers to full length as well as functional fragments or variants thereof, so long as the functional fragment or variant is capable of binding antigen or epitope. For example, the term "antibody" refers to antibodies portions, fragments, regions, peptides, single chains, bispecific antibodies and derivatives thereof so long as they bind to antigen or epitope. In some embodiments, the "antibody" is a fragment antigen binding (Fab) fragment.

[0087] Bispecific antibodies may be expressed in the dermal micro-organs according to the recombinant techniques described herein. For example, the antibody elements of the cassettes may comprise a full length or partial length heavy and light chain of one antibody and a full length or partial length heavy and light chain of another antibody. The construct may be designed as follows: signal sequence, heavy chain, F2a, light chain, [(stop, IRES), or F2A] signal sequence, heavy chain, F2a, light chain, stop. Any length or variant of heavy and light chain sequences may be used as long as the bispecific antibody maintains binding to its two antigens.

[0088] Antibody fragments or variants thereof may lack the Fc region of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than a control antibody containing an Fc region. Portions of antibodies may be made by expressing a portion of the recombinant molecule. [0089] In one embodiment, the antibody may have an IgG, IgA, IgM, or IgE isotype. In one embodiment, the antibody is an IgG.

[0090] In some embodiments, the light chain and heavy chain sequences of an antibody are optimized. In certain embodiments, these optimized sequences are those of adalimumab and are contained within SEQ ID NO: 1-4. In other embodiments, the heavy and light chain sequences of a known antibody sequence are not optimized.

[0091] In some embodiments, the micro-organ expresses a Fab fragment of adalimumab, such as, for example, the nucleic acids of SEQ ID No: 29.

[0092] In some embodiments, the heavy chain and light chain of the antibody are those of trastuzumab (SEQ ID No: 30-35). In some embodiments, the heavy and light chain of the antibody are optimized from those of trastuzumab (SEQ ID No: 33- 35). In some embodiments, the heavy chain of trastuzumab contains an S254A mutation (SEQ ID No: 32 and 35). In some embodiments, the optimized sequence of trastuzumab contains an S254A mutation (SEQ ID No: 35).

[0093] In some embodiments, the heavy chain sequence is downstream of the light chain sequence. In some embodiments, the light chain sequence is downstream of the heavy chain sequence. Those skilled in the art could test for differences in expression based on placements of different components within the expression cassette.

[0094] In one embodiment, the antibody or functional part thereof comprises a VH domain comprising a CDR1, a CDR2, and a CDR3, and a VL domain comprising a CDR1, a CDR2, and a CDR3.

[0095] In one embodiment, the micro-organ secretes an antibody or functional part thereof comprising a VH domain and a VL domain.

[0096] In certain embodiments, an antibody of the disclosure may

immunospecifically bind to its target antigen and may have a dissociation constant (Kd) of less than about 3000 pM, less than about 2500 pM, less than about 2000 pM, less than about 1500 pM, less than about 1000 pM, less than about 750 pM, less than about 500 pM, less than about 250 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 75 pM as assessed using a method known to one of skill in the art (e.g., a BIAcore assay, ELISA) (Biacore International AB, Uppsala, Sweden).

iv. Signal sequences [0097] In certain embodiments, the light chain and heavy chain antibody sequences include a signal sequence, which may be defined as a sequence of amino acids at the amino terminus. In certain embodiments, use of signal sequences (also known as signal peptides) may improve secretion of antibody. As there are a wide variety of signal sequences known to those skilled in the art, the invention is not limited by the specific signal sequence incorporated into the cassette. In certain embodiments, the signal sequences may be included from databases. In other embodiments, the heavy chain signal sequence comprises an intron for stabilization, as noted in SEQ ID NO: 5. In some embodiments, the signal sequence is identical for the heavy chain and light chains, and in other embodiments the light and heavy chains contain different signal sequences. In one embodiment a heavy chain signal sequence is used in front of both the heavy chain and the light chain.

v. Polyadenylation signals

[0098] In one embodiment, a polyadenylation signal is used in the construct downstream of the heavy and light chain. A number of polyadenylation signals would be known to those in the art to promote polyadenylation of an mRNA transcript, and any known sequence could be tested. In certain embodiments, the simian virus 40 (SV40) poly-adenylation signal is used, corresponding to SEQ ID NO: 10.

v. Tags

[0099] In one embodiment, the antibody produced by the dermal micro-organ is flagged or tagged with a detectable moiety. The detectable moiety may be a fluorescent or enzymatic or other moiety that allows detection of the produced antibody.

B. Methods of Treatment and Prevention

[00100] Therapeutic antibodies have shown efficacy in model systems for a variety of human diseases and conditions. Therefore, the antibody produced by the methods described herein is not limited by the nature of the disease/condition, which is being treated or prevented.

[00101] In certain embodiments, the antibody produced by the dermal micro-organ is of use in treatment or prevention of a cancer or oncologic indication, such as breast cancer, colon or colorectal cancer, lymphoma, non-Hodgkin's lymphoma, acute myelogenous lymphoma, chronic lymphocytic leukemia, epithelial tumors, lung tumors, ovarian tumors, renal cell tumors, prostate carcinoma, neuroectodermal tumors, glioma, head and neck tumors, thyroid cancer, kidney tumors, melanoma, hematologic malignancies, bone metastases, neuroblastoma, sarcoma, metastatic brain cancer, diagnosis of colorectal cancer, non-small cell lung carcinoma, gastrointestinal cancers, solid tumors, adrenocortical carcinoma, clear cell renal carcinoma, stomach cancer, squamous cell carcinoma, nasopharyngeal cancer, acute lymphoblastic leukemia, follicular lymphoma, small cell lung cancer, T-cell lymphoma, or multiple myeloma, as well as diagnosis and detection of tumors.

[00102] In some embodiments, the antibody produced by the dermal micro-organ is for use in the treatment or prevention of an autoimmune or an inflammatory condition, such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, juvenile idiopathic arthritis, multiple sclerosis, asthma, severe allergic reactions, idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis, psoriatic arthritis, chronic spontaneous urticarial, systemic scleroderma, systemic lupus erythematous, dermatomyositis, polymyositis, or lupus nephritis.

[00103] In still other embodiments, the antibody produced by the dermal micro-organ of the invention may be for prevention/prophylaxis or treatment of infectious diseases, such as Clostridium difficile infection, sepsis, viral infections (such as influenza A, hepatitis B, respiratory syncytial virus), invasive Candida infection, Pseudomonas aeruginosa infection, cytomegalovirus infection,

Staphylococcus aureus infection, Escherichia coli infection, or other viral, bacterial, or fungal infections.

[00104] In certain embodiments, the antibody produced by the dermal micro-organs described herein may be for the treatment of platelet aggregation inhibition, Alzheimer's disease, hypercholesterolemia, hemolytic disease of the newborn, prevention of organ transplant rejections, retinopathy of prematurity, thromboembolism, osteoporosis, dyslipidemia, thrombocytopenic purpura, thrombosis, bleeding, paroxysmal nocturnal hemoglobinuria, heart attack, stroke, traumatic shock, sciatic and other forms of pain, reduction of scarring after glaucoma surgery, anthrax, diabetes mellitus type 1, amyotrophic lateral sclerosis, macular degeneration, uveitis, muscular dystrophy, osteomyelitis, or muscular atrophy.

[00105] The variables of the dosing schedule will be determined by one of skill in the art depending on the disorder being treated and choice of treatment. For example, for chronic conditions, such as autoimmune disorders, micro-organ transplantation may occur with regular frequency. In some embodiments, the level of antibody produced by the micro-organ in the serum determines the timing of subsequent implantations or removal of dermal micro-organs. In some embodiments, the levels of antibody produced by the micro-organ in vitro is used to determine the number that are implanted into a subject.

[00106] In some embodiments, the antibody production by the micro- organ is prophylactic or preventative. In certain embodiments, the micro-organ may be implanted before exposure or potential exposure to an infectious agent, whereby the resulting antibody can prevent infection or disease.

[00107] In certain embodiments, the antibody produced by the dermal micro-organ described herein is used for diagnostic or imaging purposes. In certain embodiments, the antibody produced is for diagnostic use over a sustained period, such that the regression or growth of a tumor. In certain embodiments, the therapeutic regimen for a patient may be determined or modified based upon the results of imaging using the antibody produced by the TARGT-antibody.

[00108] In some embodiments, the antibody production by the micro- organ is intended for short-term treatment. In certain embodiments, a measure of disease activity is used to determine when treatment with the antibody from a micro- organ has been successful. In certain embodiments, the micro-organ is removed when measures of disease activity indicate that treatment with therapeutic antibody from a micro-organ is no longer necessary, and the dermal micro-organ can be removed. In certain embodiments, regression of a tumor may be the measure of disease activity that indicates that treatment with therapeutic antibody from a micro-organ is no longer necessary, and the dermal micro-organ can be removed.

[00109] In some embodiments, measures of the serum antibody produced by the micro-organ are used to determine the optimal number of micro- organs to be used. In some embodiments, micro-organs secreting antibody may be removed or added based on measures of the serum antibody produced by the micro- organ.

[00110] In some embodiments, measures of disease activity are used to determine the optimal number of micro-organs to be used. In some embodiments, micro-organs secreting antibody may be removed or added based on measures of disease activity. In certain embodiments, the measures of disease activity to determine the optimal number of micro-organs may be tumor size, levels of disease biomarkers, or any other diagnostic of disease activity that may come, for example, from imaging, blood work, or other diagnostics known to those skilled in the art.

[00111] As used herein the term "combination" is used in its broadest sense and means that a subject is treated with at least two therapeutic regimens.

Treatment can be at the same time (e.g. simultaneously or concomitantly), or at different times (e.g. consecutively or sequentially), or a combination thereof. For the purposes of the present disclosure, administering at the same time (e.g.,

simultaneously) refers to administering the TARGT-antibody and other therapeutic, such as, for example, an anti-inflammatory agent, together via same TARGT-antibody or in separate delivery devices. As used herein administering at different times (e.g., sequentially) refers to administering the TARGT-antibody of the combination therapy a few hours to days, weeks and even months apart from the other therapeutic.

[00112] Therefore, in certain embodiments a subject undergoing combination antibody therapy can receive both TARGT-antibody and antiinflammatory at the same time (e.g., simultaneously) or at different times (e.g., sequentially, in either order, on the same day, or on different days), so long as the therapeutic effect of the combination of both substances is caused in the subject undergoing therapy. In some embodiments, the combination of TARGT-antibody and anti-inflammatory will be given simultaneously. Sequential administration may be performed regardless of whether the subject responds to the first administration.

[00113] In certain embodiments, the TARGT-antibody is administered as combination therapy with a non-biologic agent. In certain embodiments, the TARGT-antibody is administered in combination with a biologic agent, such as an antibody or antibody fragment that is not generated via a TARGT-antibody. In certain embodiments, this biologic or non-biologic agent for combination therapy with TARGT-antibody is a chemotherapeutic agent or anti-infective.

C. Modified Antibodies

[00114] Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an antibody or functional part thereof, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that are routinely used to generate amino acid substitutions. In one embodiment, the VH and/or VL derivatives may include about 25 or fewer amino acid substitutions, about 20 or fewer amino acid substitutions, about 15 or fewer amino acid substitutions, about 10 or fewer amino acid substitutions, about 5 or fewer amino acid substitutions, about 4 or fewer amino acid substitutions, about 3 or fewer amino acid substitutions, about 2 or fewer amino acid substitutions, or 1 amino acid substitution relative to the original VH and/or VL of the antibody or functional part thereof. In another embodiment, the VH and/or VL derivatives may have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues (e.g., amino acid residues which are not critical for the antibody or functional part thereof to specifically bind to its antigen). Mutations can also be introduced randomly along all or part of the VH and/or VL coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody or functional part thereof can be expressed and the activity can be determined. The percent identity of two amino acid sequences can be determined by any method known to one skilled in the art, including, but not limited to, BLAST protein searches.

[00115] In certain embodiments, an antibody or functional part thereof is an isotype switched variant (e.g., to a different isotype, such as an IgG2 to an IgGl, IgG3, or IgG4 human isotype, etc.).

[00116] In some embodiments, antibodies or functional parts thereof are modified with respect to effector function, so as to enhance the effectiveness of the antibody in treating B cell malignancies, for example. An exemplary effector function is antibody-dependent cell mediated cytotoxicity, or ADCC, which is a cell-mediated reaction in which non-specific cytotoxic cells recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The cytotoxic cells, or effector cells, may be leukocytes which express one or more FcRs. Effector cells express at least FcyRI, FOyRII, FcyRIII and/or FcyRIV in mouse. Some human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. Of these cells, the primary cells for mediating ADCC are NK cells, which express FcyRIII. Monocytes express FcyRI, FcyRII, FcyRIII and/or FcyRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991).

[00117] One method for enhancing effector function of antibodies is by producing engineered glycoforms. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat Biotechnol 17: 176-180. One or more amino acid substitutions can also be made that result in elimination of a glycosylation site present in the Fc region (e.g., Asparagine 297 of IgG).

[00118] An antibody can also be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.

[00119] In one embodiment, an antibody or functional part thereof comprises a variant Fc region that mediates enhanced antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, antibody or functional part thereof comprises an Fc region having complex N-glycoside-linked sugar chains linked to Asn297 in which fucose is not bound to N-acetylglucosamine in the reducing end, wherein said Fc region mediates enhanced antibody-dependent cellular cytotoxicity (ADCC).

[00120] In certain embodiments, an antibody or functional part thereof comprises an Fc variant, wherein said variant Fc domain has an affinity for Fc gamma receptor IIB that is at least about 2 fold, or at least about 3 fold, or at least about 5 fold, or at least about 7 fold, or at least about 10 fold, or at least about 20 fold, or at least about 30 fold, or at least about 40 fold, or at least about 50 fold, or at least about 60 fold, or at least about 70 fold, or at least about 80 fold, or at least about 90 fold, or at least about 100 fold, or at least about 200 fold greater than that of a comparable non-variant Fc domain.

[00121] In other embodiments, effector function may be altered by introducing cysteine residue(s) in the Fc region of the antibody, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased

complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, Caron et al, J. Exp Med., 176: 1191-1195 (1992) and Shopes, B., J. Immunol., 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research, 53 :2560-2565 (1993). An antibody can also be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al., Anti-Cancer Drug Design, 3 :219- 230 (1989).

[00122] Other methods of engineering Fc regions of antibodies so as to alter effector functions are known in the art (e.g., U.S. Patent Publication No.

20040185045 and PCT Publication No. WO 2004/016750, both to Koenig et al, which describe altering the Fc region to enhance the binding affinity for FcyRIIB as compared with the binding affinity for FCyRIIA; see, also, PCT Publication Nos. WO 99/58572 to Annour et at, WO 99/51642 to Idusogie et al., and U.S. Pat. No.

6,395,272 to Deo et al. Methods of modifying the Fc region to decrease binding affinity to FcyRIIB are also known in the art (e.g., U.S. Patent Publication No.

20010036459 and PCT Publication No. WO 01/79299, both to Ravetch et al.).

Modified antibodies having variant Fc regions with enhanced binding affinity for FcyRIIIA and/or FcyRIIA as compared with a wildtype Fe region have also been described (e.g., PCT Publication Nos. WO 2004/063351, to Stavenhagen et al.).

EXAMPLES

Example 1. Expression of light and heavy chains of adalimumab in the TARGT system leads to adalimumab secretion from human skin fibroblasts.

[00123] Adalimumab is an antibody that confers specificity to human

TNF-alpha with efficacy in treatment of multiple human autoimmune diseases. The TARGT system was tested to determine if it could be used to express functional antibody, using adalimumab as a proof of principle. The heavy and light chain variable regions of adalimumab were expressed in the TARGT system using a helper- dependent adenovirus (FID Ad). In these experiments, a MAR-EFl alpha cassette, as shown in Figure IE was utilized, and the constructs shown in Figures 1A-1D were inserted into the cassette at the multiple cloning site (mcs). The resultant cassette was associated with HDAd and used to transduce tummy tuck excised human micro- organs as well as human skin fibroblasts in vitro.

[00124] When monoclonal antibodies are produced recombinantly, production of monoclonal antibodies with separate polypeptides of the heavy chain and light chain using separate promoters or separate vectors can lead to non- expressing clones surviving drug selection, as described in Ho SCL et al., (May 2013), PLoS One. 21;8(5):e63247. In addition, the use of separate promoters or separate vectors for the light chain and heavy chain leads to a lack of control over the ratio of light chain to heavy chain, which can lead to potential aggregation and glycosylation of the monoclonal antibody. Therefore, constructs were designed using a single construct to produce separate polypeptides for the heavy chain and light chain of adalimumab. Expression cassette constructs that employed either a furin 2 A (F2A) cleavage site or an internal ribosome entry site (IRES) separating the heavy and light chain were investigated.

[00125] In one embodiment a furin 2A element, as described by Ho et al., 2013, was used to separate the heavy and light chain sequences. It has been described that when 2 genes are linked in one single open reading frame with a 2A element between them, there is cleavage co-translationally between the last 2 amino acids at the c-terminus of the 2A polypeptide, resulting in production of equal amounts of the 2 proteins. For example, the foot-and-mouth disease virus 2A element has been described (see Donnelly MLL et al. (May 2001) J Gen Virol. 82(Pt 5): 1027- 41). Recently, the addition of a furin cleavage sequence upstream of the 2A element (termed a furin 2A element, F2A) has been shown to eliminate the additional amino acids that would otherwise remain attached to the upstream protein after cleavage at the 2A element, as noted by Ho et al., 2013.

[00126] In another embodiment, an IRES separating site was placed between the heavy and light chain sequences. IRES elements have been described as being capable of expression of multiple genes in one mRNA. See, Ho et al., 2013. When IRES elements are used between two open reading frames (ORFs), the first ORF is translated via a canonical cap-dependent mechanism, while the second ORF is translated via a cap-independent mechanism. The IRES element follows the stop codon of the first gene, as noted in Ngoi SM et al., (Mar 2004) Curr Gene Ther.

4(1): 15-31.

[00127] In a first set of experiments, three antibody expression cassettes were tested as shown in Figures 1A, IB, and 1C. The cassettes differed in that TNF1 (SEQ ID: 14) used a furin 2 A (F2A) cleavage site between the antibody heavy and light chain, whereas TNF3 (SEQ ID: 15) utilized an internal ribosome entry site (IRES) separating the heavy and light chain sequences. T F2 was similar to TNF1 in that it also used a furin 2A (F2A) cleavage site between the antibody heavy and light chain, but the heavy and light chain ordering was reversed as compared to TNF 1. The light and heavy chain sequences of adalimumab were codon optimized in all of the TNF1, TNF2, and TNF3 cassettes. Additionally, as shown in Figure ID, a fragment antigen binding (Fab) fragment cassette, TNF5, was also designed (SEQ ID NO: 19) and tested.

[00128] The organization of the components of the TNFl cassette was as follows: start-signaling peptide-light chain-furin cleavage sequence-2A element- signaling peptide-heavy chain-stop. The sequence of the TNFl construct corresponds to SEQ ID: 14. The organization of the components of the TNF2 construct was as follows: start-signaling peptide-heavy chain-furin cleavage sequence-2A element- signaling peptide-light chain-stop. The organization of the components of the TNF3 cassette was as follows: start-signaling peptide-light chain-stop-IRES-start-signaling peptide-heavy chain-stop. The sequence of the TNF3 construct corresponds to SEQ ID: 15.

[00129] The TNFl and TNF3 construct were cloned into the HDAd-

MAR-EF1 alpha cassette using the multiple cloning site (MCS), as shown in Fig. IE. The sequence of pAd-MAR-EF la-opt hTNFl is SEQ ID: 16. The sequence of pAd- MAR-EF la-opt hTNF2 is SEQ ID: 20. The sequence of pAd-MAR-EF la-opt hTNF3 is SEQ ID: 17. The sequence of pAd-MAR-EF la-opt hTNF5 is SEQ ID: 21.

[00130] To test for expression of adalimumab by these constructs, fibroblast cells were extracted from human skin using standard procedures. These skin fibroblasts were nucleofected with the p Ad-MAR-EF 1 alpha construct expressing either TNFl or TNF3 cassettes. Cell and supernatant samples were collected at one and two days after nucleofection to measure adalimumab.

[00131] Nucleofection procedures were as follows. Human dermal fibroblast cells (HDF) were extracted from tummy tuck tissue using collagenase. Cells used for the study were taken from passage 5 and greater. Growth medium was DMEM-F-12 (ADCF) with phenol red (HyClone) supplemented with 10% DCS (Defined Calf serum Iron Supplemented HyQ); AmBisome 2^g/ml (Liposomal Amphotericin-B 50mg - GILEAD); Gentamycin sulfate 50μg/ml (Gentamicin-IKA 80mg Teva). Trypsin/EDTA, trypsin neutralizing solution (TNS) and HEPES buffered saline (HBS) used were from Lonza.

[00132] Five days before nucleofection, cells were seeded on 10cm2 plates with media exchange every three days. Cells were grown until they reached 90% confluency. Growth medium was removed from four plates and the cells were washed once with 10ml of HBS. In order to harvest the cells, 3ml of Trypsin/EDTA solution was added to the plate; the plate was gently swirled to ensure an even distribution of the solution and then the plate was incubated at 37°C for 3 minutes.

[00133] The plate was removed from the incubator, trypsin neutralizing solution (TNS) was added to inactivate the trypsin; and cells were gently resuspended and transferred by pipetting to a tube. Cells were counted three times, and a typical yield was found to be 13.5xl06 cells. The cells were pelleted at 500xg for lOmin in a 50ml tubes.

[00134] For the nucleofection, cells were resuspended with 1400μ1 of

Human Dermal Fibroblast Nucleofector™ Solution (final cone. 7.9xl05 cells/ΙΟΟμΙ). 100 μΐ of cells were mixed with 5μg DNA from each mAb expression cassette (see representative values in Table 2 below). The sample to be nucleofected was transferred into an Amaxa cuvette.

[00135] Manufacturer electroporation program U-23 on Amaxa®

Nucleofector® (Lonza) was used. Post-nucleofection, the cells were removed from the cuvette immediately by adding 500μ1 of pre-warmed culture medium. Reactions containing 7.9xl05 cells/ΙΟΟμΙ were seeded into 6 well plate containing 1.5ml of growth medium using plastic pipette. Media was collected (2ml) after 24 and 48 hours and kept at -80°C until ELISA analysis.

Figure imgf000084_0001

[00136] To determine the amount of total adalimumab that was secreted from skin fibroblasts, an ELISA for detection of total adalimumab was developed. ELISA plates were coated with anti-human FC antibody (Product # 109-005-098, Jackson ImmunoResearch) as the capture antibody. Plates were then blocked with 5% bovine serum albumin (BSA), and experimental samples were diluted and incubated with the coated plates. Plates were then incubated with horseradish peroxidase (HRP)- conjugated anti-human Kappa secondary antibody (Product # ab79115, AbCam). All incubations were for 1 hour and were followed by 3 washes.

[00137] As seen in Figure IF, levels of secreted adalimumab in supernatant samples were substantially higher for the TNFl construct versus the TNF3 construct at both day 1 and day 2. Levels of intracellular adalimumab were substantially lower than secreted levels, regardless of cassette, indicating the efficient secretion of adalimumab when expressed by the cassettes.

Example 2. Transduction of human micro-organs with HDAd-MAR-EFl alpha expressing the TNFl cassette generates TARGT-adalimumab that secretes active adalimumab.

[00138] Because the TNFl construct led to efficient secretion of adalimumab in skin fibroblast cells in vitro, it was tested in the TARGT system in vitro. We have previously found that dermal micro-organs (MOs) could effectively secrete therapeutic polypeptides, such as erythropoietin (see US Application

20150118187).

[00139] Dermal micro-organs (MOs) were harvested using the

Derma Vac harvester from human skin using the NOUVAG chuck driller set at 7,000 rpm and equipped with 14G coring needle. Double hump 3mm DermaVac was used to position the skin. Harvested MOs were incubated for one minute in saline. Then all the MOs were washed 3 times with DMEM F-12 media without serum in a Petri dish. Every wash was performed in a new Petri dish. All washed MOs were placed in 24 well/plate (SARSTEDT for Suspension Cells) containing 1ml production medium. The plates were incubated for 24 hours at 5% CO2, 32°C incubator.

[00140] After a latency period, the MOs were transduced with viral vector expressing TNFl cassette to generate TARGTs. In this experiment an FID Ad vector was used carrying the cassette of Figure IE with the construct of Figure 1 A inserted at the multiple cloning site. The MOs were transduced with 250 microliter of production media containing 1.5X1010 viral particles. The mixture was placed on an orbital shaker for 4 hours (300rpm) followed by 20 hours with no shaking in a 5% C02, 32°C incubator. To stop the transduction, the solution containing the viral vector was removed and discarded, and six media exchanges with fresh production medium were performed in order to remove residual, unabsorbed viral particles. Following the washes, 1ml of media was added, and the TARGTs were incubated in a 5% C02, 32°C incubator for 3 days. The micro-organs that were transduced with HD Ad-MAR-EF 1 alpha expressing the TNF1 cassette will be referred to throughout as "TARGT-adalimumab". At the TARGTs maintenance phase, the production media was exchanged every 3-4 days and the spent media was collected for TARGT secreted mAb secretion using ELISA analysis.

[00141] After the media was exchanged, levels of secreted adalimumab were measured in the spent media by ELISA. Active adalimumab levels were measured by ELISA (Product # SQ-M1885, Sanquin). Briefly, ELISA plates were coated with anti-TNF-alpha antibody followed by blocking with 5% BSA overnight at room temperature. TNF-alpha was then incubated with the coated plate followed by incubation with TARGT spent media. In in vivo experiments described later, mouse serum samples were used in this assay. Following incubation of samples, secondary biotinylated anti-human IgG antibody was incubated, followed by HRP-conjugated streptavidin. The duration of all incubations was 1 hour, except for the streptavidin incubation that was 30 minutes. All antibodies and washes were performed using reagents in the kit.

[00142] The amount of secreted active adalimumab per day was measured for each TARGT-adalimumab at day 6, 9, 16, 20, 27, 34, and 44 days after harvesting of the micro-organ by collection of micro-organ supernatants from four separate TARGT-adalimumabs. As shown in Figure 2, secretion of active

adalimumab could be detected as early as six days after harvesting, and secretion persisted for the 44 days of the experiment. The concentration of active adalimumab secreted from TARGT-adalimumab was in the range of microgram per TARGT per day.

[00143] In order to investigate potential skin-to-skin variability in secretion of adalimumab from TARGT-adalimumab, we generated micro-organs from two different donors (HA308 and HA305). TARGT-adalimumabs were generated using the transduction process described above for micro-organs from both donors, and the results were compared. As shown in Figure 3, the amount of secreted active adalimumab per TARGT-adalimumab were measured per day for four separate TARGT-adalimumabs from each donor at 6, 9, 16, 20, 27, 34, and 44 days after harvesting of the micro-organ. For TARGT-adalimumabs from both donor HA308 and donor HA305, secretion of active adalimumab could be detected by day 6 after harvesting. Secretion of active adalimumab from TARGT-adalimumabs was maintained through the longest period of measurement (44 days for those from donor HA308 and 34 for those from donor HA305) and was in the microgram per per TARGT per day range for both donors.

[00144] Further results are shown in Figure 4 A, which characterized the

TNF2 (heavy chain first, SEQ ID NO: 18), , and TNF1 (light chain first, SEQ ID No.: 14) constructs in three independent nucleofections. These results indicate higher secretion of adalimumab from the TNFl construct. Figure 4B further shows that secretion following nucleofection of the TNFl construct was also higher than secretion following nucleofection of the TNF5 (Fab, SEQ ID NO: 21) construct comprising Fab fragments.

[00145] Results from these experiments suggest that adalimumab can be successfully secreted from transduced dermal micro-organs. In addition, results from ELISAs with the target for adalimumab, TNF-alpha, confirm that the adalimumab secreted from TARGT-adalimumab is active and capable of binding to its appropriate ligand. Expression of active adalimumab from TARGT-adalimumab persisted for weeks after harvesting and transduction of the micro-organs, indicating that TARGT- adalimumab allows antibody secretion in vitro for extended periods. This secretion of TARGT-adalimumab was consistent across skin donors.

Example 3. Western blot analysis of spent media from TARGT-adalimumab confirms the production of adalimumab.

[00146] The adalimumab secreted from TARGT-adalimumab was properly expressed, folded, and secreted. Both non-reducing and reducing western blot analysis were done to characterize the adalimumab produced from TARGT- adalimumab and to ensure that similar results were reproducibly seen for micro- organs generated from different donors. The adalimumab produced from TARGT- adalimumab was also compared to commercially available adalimumab (HUMIRA®).

[00147] To characterize adalimumab folding, TARGT-adalimumab spent media samples were analyzed by SDS PAGE (12% precast Tris/Glycine gel) followed by western blot. Prior to loading on the gel, the molecular weight standard, TARGT-adalimumab media, and negative control samples were diluted with reducing sample buffer or with non-reducing sample buffer and incubated for 5 minutes at 95°C. The running conditions were as follows: 100V, 1 hour in cooled MOPS buffer. We used 5μ1 of pre-stained molecular weight marker.

[00148] Following electrophoresis, the proteins separated in the gel were electroblotted to a 0.2um nitrocellulose membrane (Product # IB23001, Lite Technologies) using a dry transfer system (Product # IB21001 Life Technologies). Blotting was done as recommended by the manufacturer for antibodies transfer: 20V for 1 minute, 23V for 4 minutes, and 25V for 2 minutes. Next, the blotted membrane was blocked with PBS 0.2% Tween (PBST)/10% skim milk solution for overnight at 4°C, followed by incubation with HRP-conjugated goat anti-human IgG (heavy chain plus light chain) in PBST/1% milk for 4 hours at room temperature. After three 5- minute washes with PBST, detection was carried out using either DAB substrate or chemiluminescent substrate. Imaging for chemiluminescent substrate was conducted by the LAS 500 chemiluminescent imager (General Electric).

[00149] Under non-reducing conditions, as shown in Figure 5, the whole antibody of adalimumab could be detected in supernatant samples at day 9 from TARGT-adalimumab generated from two separate donors (Skin #1 and Skin #2). Lanes 3-5 on the gel in Figure 5 show three separate TARGT-adalimumabs that were generated from micro-organs from donor #1. Similarly, lanes 6-8 of this gel show three separate TARGT-adalimumabs that were generated from micro-organs from donor #2. Whole antibody, heavy chain, and light chain of adalimumab were detected for all TARGT-adalimumabs tested. These results show that multiple TARGT-adalimumabs can be generated from the same donor with similar profiles. In addition, results were consistent for TARGT-adalimumab from two different donors.

[00150] The profile of secreted adalimumab from TARGT- adalimumabs to the profile of commercially available adalimumab was compared. A commercial adalimumab standard was run in lanes 1 1 and 12 of the gel in Figure 5. The whole antibody produced from TARGT-adalimumab ran at the same molecular weight as that of commercially available adalimumab. We noted that heavy and light chain molecules for adalimumab were also detected separately for the TARGT- adalimumab samples. This may be due to the high sample concentrations that were run on the gel.

[00151] To confirm that the antibody signal on non-reducing gels was specific to the successful expression of adalimumab, we tested a negative control of a TARGT expressing erythropoietin instead of expressing adalimumab. As expected, spent media samples from a TARGT expressing erythropoietin did not produce any antibody signal on the non-reducing western blots, as shown in lane 10 of the gel in Figure 5. Molecular weight marker is shown in lane 1 to allow determination of molecular weights of the bands in experimental samples. [00152] The running pattern of spent media samples from TARGT- adalimumab samples from donor #1 and #2, under reducing conditions, was tested. In this experiment (Fig. 6A), lanes 1, 3, and 4 correspond to separate TARGT- adalimumabs generated from skin micro-organs from donor #1, while lanes 5-7 correspond to separate TARGT-adalimumab generated from skin micro-organs from donor #2. As shown in Figure 6A, similar results were seen for TARGT-adalimumabs from both donors compared with the commercial adalimumab standard (lanes 10-11) using chemiluminescent substrate. For TARGT-adalimumab, heavy chain and light chain could be detected at 50 kDa and 25 kDa, respectively. In addition, a band at 75 kDa could be detected in both the TARGT-adalimumab and the commercial adalimumab samples, which may be due to incomplete reducing of the antibody. In the TARGT-adalimumab samples, a fraction of incomplete F2A cleavage also cannot be ruled out. The negative control, spent samples from a TARGT expressing erythropoietin (lane 9), did not produce any antibody signal on reducing western blots. Molecular weight marker is shown in lane 2 to allow determination of molecular weights of the bands in experimental samples.

[00153] As shown in Figure 6B, similar results were seen for the same membrane using the DAB substrate. All lane designations are the same as in Figure 6A. Results are consistent between membranes when developed using DAB or chemiluminescent substrates, with chemiluminescent have a more robust signal as expected.

[00154] Together, the data from non-reducing and reducing western blot analyses indicate that the profile of adalimumab secreted by TARGT- adalimumab is consistent with the profile of commercially available adalimumab. In addition, the profile of separate TARGT-adalimumabs produced from the same donor was consistent, as was the profile of adalimumab produced from TARGT- adalimumabs from different donors. These results show that the TARGT system is a reproducible means of producing secreted adalimumab that has a western blot profile consistent with the profile of commercially available adalimumab.

Example 4. In vivo profile of TARGT-adalimumab in SCID mice.

[00155] In-vivo experiments were conducted in SCID mice in order to verify that TARGT-adalimumab secretes detectable levels of adalimumab in vivo. In addition, we analyzed the extent to which the secreted adalimumab is active in mice sera. [00156] For the in vivo experiments, SCID mice of CB-

17Prkdcscid/NCr Hsd strain were used at 11-12 weeks of age. TARGT-adalimumabs were produced using the procedure outlined in Example 2. The TARGT-adalimumabs were implanted subcutaneously in the backs of 5 individual mice using 10G implantation needles. Implantation was done at day 7 after transduction of the TARGT with HDAd-MAR-EFl alpha-expressing the TNF1 cassette (as described in Example 2). Each mouse received four TARGT-adalimumabs. Mice were treated with DepoMedrol (methylprednisolone) every 2 weeks.

[00157] In order to measure the concentrations of adalimumab secreted into the blood by the TARGT-adalimumabs, mouse blood was collected a day before TARGT implantation (baseline) and then approximately weekly for the duration of the experiment. Level of total adalimumab was measured using the IgG Kappa ELISA, and active adalimumab was measured using the commercial active adalimumab ELISA (as described in Examples 1 and 2, respectively).

[00158] As shown in Figure 7 A, both total and active adalimumab secreted from TARGT-adalimumab could be detected in SCID mice sera starting at 7 days after implantation. Secretion of both total and active adalimumab could be detected in the μg/ml range at days 7, 14, 21, 28, 35, 42, and 49 after implantation. These data show that implantation of TARGT-adalimumabs produced long-lasting secretion of adalimumab in SCID mice. At baseline, no adalimumab could be measured in mice sera (data not shown).

[00159] We also tested the active fraction of adalimumab over time in the sera of SCID mice, as measured by the ratio of active to total serum adalimumab values. As shown in Figure 7B, the fraction of adalimumab that was active peaked in samples at days 14 and 21 after implantation. However, high levels of active adalimumab were measured for the duration of the experiment, indicating that therapeutic levels of adalimumab can be produced in vivo for extended periods of time.

[00160] These experiments show the in vivo efficacy of TARGT- adalimumab to stably produce adalimumab in serum when the micro-organs are transplanted in vivo. Therefore, the TARGT-adalimumab system fulfills all the expression, folding, and secretion requirements for producing active adalimumab in vivo. Example 5: TARGT-antibody is effective in treating disease

[00161] Next, the efficacy of TARGT-adalimumab was assessed in a mouse model of rheumatoid arthritis. Dermal fibroblasts were extracted from C57B1 mice ears. The fibroblasts were transduced with HDAd-MAR-EFla-TNFl viral vector (SEQ ID No: 16) and were measured for adalimumab secretion levels.

Heterozygous Tgl97 transgenic mice were obtained from Biomedcode (Biomedical science research center, "Alexander Flemming"). Four experimental groups (n=5 or 6, gender matched) were used. Groups 1 and 2 were implanted with HDAd-MAR- EFla-TNFl transduced mouse dermal fibroblasts and embedded in Matrigel

(Cultrex®, Cat. # 3433-005-01, Trevigen) at dose of 3xl05 (0.3M) and lxlO6 cells (1M) per mouse. Group 3 was implanted with non-transduced (naive) mouse dermal fibroblast cells embedded in Matrigel at dose of lxlO6 cells per mouse. Group 4 was used as positive control, where mice were injected IP bi-weekly with 3 mg/kg anti- TNFa adalimumab. The study initiated when animals were 6.5 weeks of age, and the effect of treatments was monitored by weekly assessment of body weight and arthritic score. Statistical differences significance was assessed by 1-way ANOVA followed by Dunnett's range statistical test using GraphPad Prism 6 software. Differences between all groups were compared, and were considered significant at values of 0.05 > *p > 0.01 and **p < 0.01.

[00162] Results of the in vivo efficacy model are shown in Figures 8 A and 8B. Treatment with 0.3M, 1M, and standard (twice a week injected adalimumab at 3mg/kg concentration) all showed a decrease in arthritis score when compared to negative control mice implanted with naive non-transduced cells (Figure 8A). The decrease in RA score of the treated mice was found to be statistically significant (Figure 8B). Thus, treatment of mice with TARGT-adalimumab produced significant efficacy. These data confirm that adalimumab produced by TARGTs is an effective therapy for rheumatoid arthritis, as well as other diseases known to be treated with adalimumab. Therefore, production of adalimumab by TARGT-adalimumab s fulfills all requirements for translation, folding, assembly, and secretion to generate active antibody.

Example 6. Expression of light and heavy chains of trastuzumab

[00163] Trastuzumab (Herceptin®) is a humanized IgGl-Kappa directed against the extracellular domain of human epidermal growth factor receptor 2 (Her2). Trastuzumab light and heavy chains were inserted in the open reading frame of the HD Ad-MAR-EF 1 alpha cassette as shown in Figures 9 A and 9B. Furin cleavage sites adjacent to 2A element were used in order to produce two separate antibody chains (light and heavy) to enable correct antibody folding. The light chain was inserted with a heavy chain signal peptide bearing an intron for stabilization. Downstream to the light chain, the heavy chain of trastuzumab was inserted with its signal peptide.

[00164] Two cassettes were designed for trastuzumab. The Her WT cassette used the wildtype human IgGl Kappa sequence, identical to commercially- available trastuzumab (Herceptin®) (SEQ ID No: 36). An optimized HER WT cassette was also developed that has an identical amino acid sequence, but optimized nucleotide sequence for trastuzumab (SEQ ID No: 23). The HER Mut cassette used a single point mutation at position 254 of human IgGl heavy chain of trastuzumab in which a serine was replaced with alanine (S254A) in order to generate a trastuzumab variant with a potentially longer CNS half-life in addition to an optimized nucleotide sequence (SEQ ID No: 24). The human IgGi 254 position of trastuzumab is suspected to have a major influence on hlgGl-FcRn affinity; thus, mutation at this point should decrease affinity to FcRn and increase CNS half-life.

[00165] Transduction of MOs was performed as described in Example

1. Active trastuzumab concentrations were measured using commercial trastuzumab ELISA (Cat. # Q-TRAS, Matriks biotechnology) according to the manufacturer instructions. The micro-organs that were transduced with HD Ad-MAR-EF 1 alpha expressing the HER WT cassette (SEQ ID No: 25) will be referred to throughout as "TARGT -trastuzumab WT". The micro-organs that were transduced with HD Ad- MAR-EF 1 alpha expressing the HER Mut cassette (SEQ ID No: 26, optimized sequence with S254A mutation) will be referred to throughout as "TARGT- trastuzumab MUT".

[00166] In order to detect total IgG kappa in samples collected in-vitro, an in house ELISA was established. Primary Ab used was ^g/ml anti human FC (Cat. # 109-005-098, Jackson ImmunoResearch), secondary Ab used was HRP conjugated anti human Kappa (Cat. # ab79115, AbCam), and the blocking step was done with 3% BSA. All incubations were done for 1 hour followed by 3 washes. For Trastuzumab levels, IgGl Kappa (Product^ 5154- IMG, Sigma Aldrich) standard was used.

[00167] In order to detect total IgG kappa in samples collected in-vivo, an in house ELISA was established. Primary Ab used was 5μg/ml anti human FC (Cat. # 109-005-098, Jackson ImmunoResearch), secondary Ab used was biotin labeled anti human IgG Kappa antibody (Product # ab79114, AbCam) followed by the addition of HRP conjugated streptavidin (Product # 016-030-084 Jackson

ImmunoResearch). Plates block was done with 3% BSA. All incubations were done for 1 hour followed by 3 washes. For trastuzumab levels, IgGl Kappa (Product^ 5154- IMG, Sigma Aldrich) or commercial Herceptin® drug standard was used.

[00168] As shown in Figure 10, TARGTs transduced with HD Ad-

MAR-EFla-HER WT (SEQ ID No: 25) or HD Ad-M AR-EF 1 a-HER Mut (SEQ ID No: 26) vectors exhibit trastuzumab secretion levels of 2-10μg/TARGT/day. HA 407, HA 408, HA 409, and HA-416 refer to separate MOs collected from different donors that were independently transduced. The data suggest that the mutated version of trastuzumab with the point mutation at S254A may be secreted at higher level. These higher levels may not be attributed solely to the single point mutation in the IgG heavy chain, and may instead due to differences in nucleic acid sequence generated by the sequence optimization algorithm (without altering the amino acid sequence), as this may change mRNA stability. In addition, Figure 11 suggests that 80-100% of the TARGT secreted trastuzumab is active for both the TARGT-trastuzumab WT and TARGT-trastuzumab Mut, with no major differences between WT and Mut constructs seen in two separate experiments (HA408 and HA407).

A. SDS-PAGE and Western Blot Analysis for Trastuzumab

Characterization

[00169] SDS-PAGE and Western Blot analysis were used to

characterize the folding of trastuzumab HER WT and HER Mut secreted from

TARGTs transduced with SEQ ID No: 25 (HER WT) or SEQ ID No: 26 (HER Mut). Before loading trastuzumab-containing samples onto 4-12% precast Tris Glycine gels, the samples were diluted with reducing or with non-reducing Laemmli sample buffer (Cat. #1610747, Bio-Rad) and incubated 5 minutes at 95°C. Five microliters of pre- stained molecular weight was also loaded to the gel (Cat. # 1610374, Bio-Rad).

[00170] The electrophoresis running conditions were as follows: 100V,

1 hour in chilled MOPS buffer. Following electrophoresis, the proteins separated on the gel were electroblotted to 0.2μιη nitrocellulose membrane (Cat. # IB23001, Lite Technologies) using dry transfer system (Cat. # IB21001 Life Technologies). Blotting conditions used were as recommended by manufacturer for antibodies transfer: 20V for 1 minute, 23V for 4 minutes and then 25V for 2 minutes. The blotted membrane was blocked overnight at 4°C with PBS containing 0.2% Tween (PBST) and 10% skim milk solution. Post blocking the membrane was incubated for 1 hour at room temperature with HRP conjugated donkey anti human H+L suspended in PBS containing 0.2% Tween (PBST) and 1% skim milk solution. Post-blotting the membrane was washed three times (5 min each) with PBST and then exposed to HRP chemiluminescent substrate (Cat # 1705060, Bio-Rad). Imaging of chemiluminescent signal was conducted by Amersham 600 chemiluminescent imager (General Electric).

[00171] Reducing (Figure 12 A) and non-reducing (Figure 12B) western-blot analysis of trastuzumab WT (HER WT, lanes 5-6) and trastuzumab Mut (HER Mut, lanes 2-3) secreted from TARGTs suggests proper assembly of the mAb when compared to the commercial Herceptin® (lane 8), commercial Humira® (Lane 9), or TARGT-secreted Humira (Lanes 11-12, labeled as TNF-1).

[00172] Together, the data from non-reducing and reducing western blot analyses indicate that the profile of trastuzumab (Herceptin) secreted by TARGT - trastuzumab is consistent with the profile of commercially available trastuzumab. These results show that the TARGT system is a reproducible means of producing secreted trastuzumab that has a western blot profile consistent with the profile of commercially available trastuzumab.

B. Analysis of Trastuzumab Binding to Her2 Receptor Using Flow

Cytometry

[00173] BT-474 Her2 positive breast ductal carcinoma cells were expanded, trypsinized, and washed with 3mM EDTA PBS. Cells (lxlO6) were incubated with spent media of TARGT -trastuzumab WT (HER WT, transduced with SEQ ID No: 25) and TARGT-trastuzumab Mut (HER Mut, transduced with SEQ ID No: 26) at several concentrations. Commercial Trastuzumab (Herceptin®), at concentrations of 50, 10 and 5μg/ml, was also used as positive control. Commercial Adalimumab (HUMIRA®) was used as IgG kappa isotype control (negative control). Post-binding reaction, PE-labeled anti-human antibody was added (1 hour at 4°C) in order to label the antibodies bound to the cells. Finally, cells were washed and analyzed using a LSRII flow cytometer (BD Biosciences, San Jose, CA). In order to assess the binding specificity of TARGT-secreted trastuzumab to the Her2 positive cells, HEK cells were also used as negative control.

[00174] Results are shown in Figures 13A, 13B, 13C, and 13D. Flow cytometry analysis of trastuzumab secreted from TARGT-trastuzumab WT (WT Herceptin, Figure 13C) or TARGT-trastuzumab Mut (Mut Herceptin, Figure

13D)suggest specific binding of the secreted mAbs to Her2-expressing BT474 cell line at efficiency similar to commercial trastuzumab (Herceptin®, shown in Figure 13B) while not showing non-specific binding to HEK cells. In Figures 13A-D for BT- 474, the isotype control is shown as the left-most peak.

[00175] These data indicate that trastuzumab secreted from TARGTs is capable of binding to native Her-2 with a similar profile to commercially available trastuzumab (Herceptin).

EQUIVALENTS

[00176] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00177] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

What is Claimed is:
1. A genetically modified micro-organ that provides delivery of an antibody, comprising a vector comprising a nucleic acid sequence encoding an antibody.
2. A genetically modified micro-organ that provides delivery of an antibody, comprising a vector comprising a nucleic acid sequence encoding an antibody operably linked to an upstream MAR regulatory sequence, and comprising at least one additional regulatory sequence.
3. The genetically modified micro-organ of claim 1 or claim 2, wherein the
antibody is secreted from the micro-organ for a sustained period of time of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.
4. The genetically modified micro-organ of any of claims 1-3, wherein the
antibody binds to its antigen.
5. The genetically modified micro-organ of any of claims 1-4, wherein the
antibody can be detected in human serum after implantation to a human.
6. The genetically modified micro-organ of any of claims 1-5, wherein the
antibody can be detected in human serum after implantation to a human, wherein the antibody is detected in the serum for a sustained period of time of at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.
7. The genetically modified micro-organ of any of claims 1-6, wherein the
antibody binds tumor necrosis factor alpha (T Fa).
8. The genetically modified micro-organ of any of claims 1-7, wherein the
antibody comprises the light chain and heavy chain of adalimumab (SEQ ID NOs 41 and 42).
9. The genetically modified micro-organ of any of claims 1-8, wherein the
antibody comprises a portion of the light chain and a portion of the heavy chain of adalimumab (SEQ ID NOs 41 and 42), wherein the antibody produced from the micro-organ binds to TNFa.
10. The genetically modified micro-organ of any of claims 1-9, wherein the sequence of the antibody comprises or consists of:
i) the nucleic acids of one or more of SEQ ID NOs: 1-4, 14-15, 18, and 19;
ii) the heavy and/or light chain portions of one or more of SEQ ID NOs:
16-17, and 20-22;
iii) a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one or more of SEQ ID NOs: 1-4, 14-15, 18, and 19;
iv) a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one or more of the heavy and/or light chain portions of SEQ ID NOs: 16-17, and 20-22; v) nucleic acids encoding the amino acids of SEQ ID NO: 41 (light chain), and SEQ ID NO: 42 (heavy chain); and
vi) nucleic acids encoding the antigen binding portions of the amino acids of SEQ ID NO: 41 (light chain), and SEQ ID NO: 42 (heavy chain).
11. The genetically modified micro-organ of any of claims 1-6, wherein the
antibody binds Human Epidermal Growth Factor Receptor 2 (HER2).
12. The genetically modified micro-organ of claim 11, wherein the antibody comprises the light chain and heavy chain of trastuzumab.
13. The genetically modified micro-organ of any of claims 11-12, wherein the antibody comprises a portion of the light chain and a portion of the heavy chain of trastuzumab, wherein the antibody produced from the micro-organ binds to HER2.
14. The genetically modified micro-organ of any of claims 11-13, wherein the sequence of the antibody comprises or consists of:
i) the nucleic acids of one or more of SEQ ID NOs: 22-23, 30-31, and 33-35;
ii) the heavy and/or light chain portions of one or more of SEQ ID NOs:
25-28;
iii) a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one or more of SEQ ID NOs: 22-23, 30-31, and 33-35; iv) a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one or more of the heavy and/or light chain portions of SEQ ID NOs: 25-28; v) nucleic acids encoding the amino acids of SEQ ID NO: 38 (light chain), and SEQ ID NO: 36 or 37 (heavy chain); and
vi) nucleic acids encoding the antigen binding portions of the amino acids of SEQ ID NO: 38 (light chain), and SEQ ID NO: 36 or 37 (heavy chain).
15. The genetically modified micro-organ of any of claims 1- 14 wherein the antibody is an antibody portion, fragment, region, peptide or derivative that retains ability to bind antigen.
16. The genetically modified micro-organ of claim 15, wherein the antibody is a Fab fragment.
17. The genetically modified micro-organ of claim 16, wherein the sequence of the Fab fragment comprises the nucleic acids of SEQ ID NO: 29, or a nucleic acid having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to SEQ ID NO: 29.
18. The genetically modified micro-organ of any of claims 1-17, wherein the at least one additional regulatory sequence comprises a MAR sequence, a CMV sequence, a CAG sequence, an EFla promoter sequence, or a WPRE sequence.
19. The genetically modified micro-organ of any of claims 1-18, wherein the nucleic acid encoding the antibody is inserted at the multiple cloning site (mcs) shown in Figure IE.
20. The genetically modified micro-organ of any of claims 1-19, wherein the antibody comprises a light chain and a heavy chain, wherein the light and heavy chains are produced by a single cassette, and wherein a cleavable element is placed between the heavy and light chain in the cassette.
21. The modified micro-organ of claim 21, wherein the cleavable element is a IRES, 2A, furin, or furin 2A element.
22. The genetically modified micro-organ of any of claims 1-21, wherein the regulatory sequences and/or cleavable elements within the cassette are selected based upon secretion of antibody when the expression cassette is expressed in a heterologous cell system.
23. The genetically modified micro-organ of any of claims 1-22, wherein a cassette contained within the vector includes the optimized sequences for expression of adalimumab comprising one of SEQ ID NO: 1-4, or a sequence having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one of SEQ ID NO: 1-4.
24. The genetically modified micro-organ of any of claims 1-22, wherein a
cassette contained within the vector includes sequences for expression of trastuzumab comprising one of SEQ ID NO: 30-35, or a sequence having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, or 80% identity to one of SEQ ID NO: 30-35.
25. The genetically modified micro-organ of any of claims 1-24, wherein a
cassette contained within the vector includes sequences for expression of a Fab fragment of adalimumab comprising or consisting of SEQ ID NO: 29, or a sequence having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86,
85, 84, 83, 82, 81, or 80% identity to SEQ ID NO: 29.
26. The genetically modified micro-organ of any of claims 1-24, wherein a
cassette contained within the vector includes sequences for expression of a Fab fragment of tratuzumab comprising or consisting of the nucleic acids necessary to express an fab fragment from any one of SEQ ID NOs: 23 or 24, or a sequence having at least 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87,
86, 85, 84, 83, 82, 81, or 80% identity to any one of SEQ ID NOs: 23 or 24.
27. The genetically modified micro-organ of any of claims 1-26, wherein the vector is a helper dependent adenovirus (HDAd) vector or an adeno-associated virus (AAV) vector.
28. The genetically modified micro-organ of any of claims 1-27, wherein said genetically modified micro-organ is a genetically modified dermal micro- organ.
29. The genetically modified micro-organ of any of claims 1-27, wherein said genetically modified micro-organ is a genetically modified dermal micro- organ, wherein the dermal micro-organ does not comprise epidermis.
30. The genetically modified micro-organ of any of claims 1-29, wherein the TARGT system is employed to generate a TARGT-antibody.
31. A method of treating or diagnosing a disease in a human subject in need
thereof comprising the steps a) Providing at least one genetically modified micro-organ according to any of claims 1-30, wherein the micro-organ provides delivery of a therapeutic antibody;
b) Determining the secretion level of said antibody by at least one genetically modified micro-organ in vitro;
c) Implanting at least one genetically modified micro-organ in said human subject; and
d) Measuring antibody levels in the blood serum of said subject,
wherein implantation of said at least one genetically modified micro-organ increases in vivo serum antibody levels, thereby treating or diagnosing the disease in the human subject.
32. The method of claim 31, wherein the implanting of the genetically modified micro-organ results in sustained serum levels of antibody; optionally, where these levels are sustained for at least three months.
33. The method of any of claim 31-32, further comprising administering a
corticosteroid by subcutaneous injection in the vicinity of the genetically modified micro-organ, and optionally repeating administration of
corticosteroid about every 2 weeks post-implantation until removal of the genetically modified micro-organ.
34. The method of any of claims 31-33, wherein the antibody produced is for therapeutic, diagnostic, or imaging purposes, or to prevent infection with an infectious agent.
35. The method of any of claims 31-34, wherein the antibody treats or prevents an oncologic, autoimmune, or inflammatory disease, or a symptom of an oncologic, autoimmune, or inflammatory disease.
36. The method of any of claims 31-35, wherein the antibody produced by the genetically modified micro-organ is used in combination with another biologic or non-biologic agent for the treatment or prevention of a disease or a symptom of a disease.
37. The method of any of claims 31-35, further comprising removing the micro- organ or adding additional micro-organs, wherein the timing of removal of the genetically modified micro-organ(s) or the timing of subsequent implantations of genetically modified micro-organ(s) is determined by measurement of serum levels of the antibody.
38. The method of any one of claims 31-35, wherein the number of genetically modified micro-organs is optimized, optionally by implantation of additional micro-organs or removal of implanted micro-organs, based on serum levels of the antibody produced.
39. The method of any one of claims 31-38, whereby the antibody produced by the genetically modified micro-organ is capable of preventing infection by an infectious agent or preventing harmful effects of exposure to a toxin.
40. The method of any one of claims 31-39, wherein the timing of removal of the genetically micro-organ is determined based on remission or amelioration of disease symptoms or improvements in diagnostic markers, biomarkers, imaging, or tumor regression.
41. The method of any one of claims 31-40, whereby the antibody produced by the genetically modified micro-organ is of use as a diagnostic for imaging of a tumor(s) within a subject; optionally, wherein the change in tumor size over time can be monitored by imaging with said antibody produced by the genetically modified micro-organ; optionally where the therapeutic regimen for said subject is determined or modified based on imaging results with said antibody produced by the genetically modified micro-organ.
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