AAV/XBP1S-HA VIRUS, GENE THERAPY METHOD AND USE THEREOF IN THE OPTIMISATION AND IMPROVEMENT OF LEARNING, MEMORY AND COGNITIVE CAPACITIES

Abstract

This invention presents a sequence of the virus AAV/XBP1s-HA, method and its use in the improvement of cognitive functions, of memory and of learning, as presented in the in vivo studies in FIG. 12/17 right panel.

Claims

1. An adeno-associated vector (AAV) CHARACTERIZED in that it comprises a recombinant viral genome where that genome comprises an expression cassette that consists of a regulating region of the specific transcription of neuronal tissue joined operatively to a polynucleotide of interest.

2. An adeno-associated vector, according to claim 1, CHARACTERIZED in that the serotype of the AAV is selected from a group that includes AAV6, AAV7, AAV8 and AAV9.

3. An adeno-associated vector, according to claim 2, CHARACTERIZED in that the serotype of the AAV is AAV6.

4. An adeno-associated vector, according to claims 1, 2 and 3, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a promotor region selected from the group that includes Pgk1, Cam 2 and Thy 1, among others.

5. An adeno-associated vector, according to claim 4, CHARACTERIZED in that the promotor region selected from the group is Pgk1.

6. An adeno-associated vector, according to claims 1, 2 and 3, CHARACTERIZED in that it comprises a coding region for an immune response site selected from the group Ha, Flag, Gfp, His and Myc, among others.

7. An adeno-associated vector, according to claim 6, CHARACTERIZED in that it comprises the coding region for an immune response site is Ha.

8. An adeno-associated vector, according to claim 4, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a specific promotor region selected for neurons.

9. An adeno-associated vector, according to claim 8, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a promotor region selected from the group that comprises neuron specific Pgk1, Cam 2 and Thy1, among others.

10. An adeno-associated vector, according to claim 9, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a neuron specific promotor region Pgk1.

11. An adeno-associated vector, according to the previous claims, CHARACTERIZED in that the expression cassette comprises a regulatory post-transcriptional region.

12. An adeno-associated vector, according to claim 11, CHARACTERIZED in that the post-transcriptional regulatory region is the American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

13. An adeno-associated vector, according to claims 1 to 12, CHARACTERIZED in that the IRTs of the adeno-associated virus are IRTs derived from AAV6, AAV7, AAV8 and AAV9, preferably AAV6.

14. An adeno-associated vector, according to claims 1 to 13, CHARACTERIZED in that the target sequences to be transcribed comprise a XBP1s and XBP1u.

15. An adeno-associated vector, according to claim 14, CHARACTERIZED in that the target sequence to be transcribed is XBP1s.

16. An adeno-associated vector, according to claim 14, CHARACTERIZED in that the target sequence to be transcribed in XBP1u.

17. An adeno-associated vector, according to claims 1 to 16, CHARACTERIZED in that the polynucleotide of interest codifies proteins within the group that comprises XBP1s and XBP1u, which act systemically close to or with neuronal cells.

18. An adeno-associated vector, according to claim 16, CHARACTERIZED in that the polynucleotide of interest codifies the protein XBP1s.

19. An adeno-associated vector, according to claim 16, CHARACTERIZED in that the polynucleotide of interest codifies the protein XBP1u.

20. An adeno-associated vector, according to claim 17, CHARACTERIZED in that the polynucleotide of interest that acts systemically close to or with neuronal cells, is specific for cells in the hippocampus.

21. An adeno-associated vector, according to claim 20, CHARACTERIZED in that the polynucleotide of interest that acts systemically close to or with neuronal cells is specific for cells in the hippocampus, preferably in the CA3 area.

22. A pharmaceutical composition, CHARACTERIZED in that is comprises an adeno-associated vector described in the previous claims and a pharmaceutically acceptable excipient.

23. A pharmaceutical composition, according to claim 22, CHARACTERIZED in that it comprises a dose of the virus in a range between 10.sup.9 to 10.sup.13 copies of genome (CG) per ml of composition.

24. The use of a pharmaceutical composition, according to claim 22, CHARACTERIZED in that it is useful in the optimization of the memory and cognitive processes.

25. Use of an adeno-associated vector, according to claim 1, CHARACTERIZED in that it is useful in the optimization of the memory and cognitive processes in a mammal.

26. Use of an adeno-associated vector, according to claim 25, CHARACTERIZED in that that mammal is a human.

27. Use of an adeno-associated vector, according to claim 25, CHARACTERIZED in that the adeno-associated vector or the pharmaceutical composition is administrated systemically or locally.

28. Use of an adeno-associated vector, according to claim 25, CHARACTERIZED in that the adeno-associated vector or the pharmaceutical compositions require the expression of the polynucleotide of interest in the neuronal tissue.

29. Method of therapeutic application with an adeno-associated vector, according to claim 1, CHARACTERIZED in that it comprises: a. the contact of the neuronal cells with the adeno-associated virus described in claims 1 to 21; and b. expression of the virus in the neuronal cells.

30. Method of therapeutic application with an adeno-associated vector, according to claim 29, CHARACTERIZED in that the administration routes are subject to the virus passing the haemato-encephalic barrier and comprise the nasal route; by direct intraventricular and/or intrathecal injection, among others.

31. A polynucleotide CHARACTERIZED in that it comprises an expression cassette flanked by the ITRs of an adeno-associated virus, where that expression cassette consists of a promotor, a codifying region for immune response and a polynucleotide of interest.

32. A polynucleotide, according to claim 31, CHARACTERIZED in that the region of the promotor is selected from the group that includes Pgk 1, Cam 2 and Thy 1, among others.

33. A polynucleotide, according to claim 32, CHARACTERIZED in that the promotor region selected from the group is Pgk1.

34. A polynucleotide, according to claim 31, CHARACTERIZED in that it comprises a codifying region for an immune response site selected from the group Ha, Flag, Gfp, His and Myc, among others.

35. A polynucleotide, according to claim 34, CHARACTERIZED in that it comprises the codifying region for an immune response site that is Ha.

36. A polynucleotide, according to claim 31, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a promotor region selected specifically for neurons.

37. A polynucleotide, according to claim 36, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a promotor region selected from the neuron specific group that comprises Pgk 1, Cam 2 and Thy1, among others.

38. A polynucleotide, according to claim 37, CHARACTERIZED in that the regulating region of the specific transcription of neuronal tissue comprises a neuron specific promotor region Pgk1.

39. A polynucleotide, according to claims 31 to 38, CHARACTERIZED in that the expression cassette also comprises a post-transcriptional regulatory region.

40. A polynucleotide, according to claim 39, CHARACTERIZED in that the post-transcriptional regulatory region is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

41. A polynucleotide, according to claim 31, CHARACTERIZED in that the target sequences to be transcribed comprise the sequences XBP1s and XBP1u.

42. A polynucleotide, according to claim 41, CHARACTERIZED in that the target sequence to be transcribed is XBP1s.

43. A polynucleotide, according to claim 41, CHARACTERIZED in that the target sequence to be transcribed in XBP1u.

44. A polynucleotide, according to claims 31 to 43, CHARACTERIZED in that the polynucleotide of interest codifies proteins within the group that includes XBP1s and XBP1u, which act systemically close to or with neuronal cells.

45. A polynucleotide, according to claim 44, CHARACTERIZED in that the polynucleotide of interest codifies the protein XBP1s.

46. A polynucleotide, according to claim 44, CHARACTERIZED in that the polynucleotide of interest codifies the protein XBP1u.

47. A polynucleotide, according to claim 31, CHARACTERIZED in that the polynucleotide of interest that acts systemically close to or with neuronal cells, is specific for cells in the hippocampus.

48. A polynucleotide, according to claim 47, CHARACTERIZED in that the polynucleotide of interest that acts systemically close to or with neuronal cells, is specific for cells in the hippocampus, preferably in area CA3.

49. A plasmid, CHARACTERIZED in that it comprises the sequences of an adeno-associate, an expression cassette flanked by the ITRs of the adeno-associated virus, where the expression cassette comprises a promotor, a codifying region for immune response and a polynucleotide of interest, like the one deposited in the international agency of biological deposit, American Type Culture Collection (ATCC) with deposit number PTA-121708.

50. An adeno-associated virus, CHARACTERIZED in that is comprises the viral genome described in claims 31 to 48.

51. A method to obtain an adeno-associated viral vector CHARACTERIZED in that it comprises the steps of: a. provide a cell that comprises a polynucleotide according to any of the claims 31 to 48, with the proteins AAV Cap, with the proteins AAV Rep and the viral proteins on which AAV depends for its replication: b. maintain the cells under adequate conditions for the assembly of the AAV; and c. purify the adeno-associated viral vector produced by the cell.

52. A method, according to claim 51, CHARACTERIZED in that the AAV is dependent on the replication derived from the adenovirus.

53. A method, according to claims 49 and 51, CHARACTERIZED because the proteins Cap and Rep of the adeno-associated virus are derived from an AAV selected from the serotypes AAV6, AAV7, AAV8 and AAV9.

54. A method according to claim 53, CHARACTERIZED in that the proteins Cap and Rep of the adeno-associated virus are derived from the serotype AAV6.

Description

DESCRIPTION OF THE FIGURES

[0139] FIG. 1/17

[0140] In this figure, an evaluation is made of the levels of expression of several genes related with the memory measured in the hippocampus, such as XBP1.sup.f/f (n=4 mice) and XBP1.sup.Nes−/− (n=5 to 6 mice) using PCR in real time.

[0141] FIG. 2/17

[0142] This figure presents the levels of mRNA of the genes related with the memory, indicated in FIG. 3/17, measured in the amygdala using PCR in real time. In c, d and f the averages are shown and a statistical analysis was carried out using Student's t-test (*: p<0.05, **: p<0.01, *** p<0.001, ns: not significant). All the samples were standardized with the levels of β-actin.

[0143] FIG. 3/17

[0144] This figure presents the levels of BDNF and KIF17 protein that were analyzed by Western blot using extracts of hippocampus obtained from 6-month old animals for XBP1.sup.f/f and XBP1.sup.Nes−/−. The levels of β-actin or Hsp90 were used as load control. The average and the standard error are shown as the times of change in comparison with control animals (n=3). The bands were spliced from the same gel and their exposure to the film.

[0145] FIG. 4/17

[0146] This figure shows the levels of mRNA of the genes of the UPR indicated. These genes were measured in the dissected hippocampus of mice XBP1.sup.Nes−/− or of animals XBP1.sup.f/f using PCR in real time. The analysis was executed at 6 months of age (n=3 per group for Xbp1Δ and n=5 per group for Wfs1, Edem and Bip).

[0147] FIG. 5/17

[0148] This figure shows the alterations in the long-term memory and the long-term potentiation of XBP1 conditioned in knock-out mice. Here we see a bar graph where the XBP1.sup.Nes−/− mice are presented and the control of the same litter (XBP1.sup.f/f) of male mice where conditioning to contextual fear was tested in the test. The percentage of events of immobility during the test was calculated (XBP1.sup.f/f: n=4 and XBP1.sup.Nes−/−: n=6 per group). A statistical analysis was made using the Student's t-test (*: p<0.05).

[0149] FIG. 6/17

[0150] Presented here in parallel, in another bar graph, is the result obtained when the animals were trained and evaluated using the paradigm of memory flexibility. The analysis shows the average number of tests to find the criteria of four consecutive days (n=4 per group). A statistical analysis was carried out using the Student's t-test (***: P<0.001).

[0151] FIG. 7/17

[0152] This figure presents the electrophysiological records of the LTP carried out on hippocampal slices derived from XBP1.sup.Nes−/− or XBP1.sup.f/f of the same litter of control mice (n=7 per group). Representative records of the fEPSP are shown after three stimulation trains with 100 Hz in the collateral circuit of Schaffer-CA1. The statistical analysis was carried out using two-way ANOVA (***: p<0.001).

[0153] FIG. 8/17

[0154] This figure shows that the overexpression of XBP1s in the neurons improves the long-term memory. In this figure, a specific neuronal transgenic strain of XBP1s is presented, created using the promotor of the prion to induce the expression in the CNS (Tg.sup.XBP1s). In the left panel, we observe the levels of XBP1s in hippocampus analyzed by Western blot with the levels of β-actin as load monitor. In the right panel, the level of learning was evaluated comparing animals Tg.sup.XBP1s and the control litter, using the memory flexibility test. The number of tests to reach this criterion is presented (n=5 per group). The statistical analysis was carried out using a two-way ANOVA, followed by a Bonferroni post-test (*: p<0.05, ** p<0.01, *** p<0.001).

[0155] FIG. 9/17

[0156] The LTP was measured in hippocampus slices Tg.sup.XBP1s and in control animals by theta burst stimulation (n=7 per group). The records of the fEPSP are shown. A statistical analysis was made using a two-way ANOVA (***: p<0.001).

[0157] FIG. 10/17

[0158] This figure shows that the local expression of XBP1s in the hippocampus improves the long-term memory tests in three-month old mice that were injected with an adeno-associated virus (AAV) serotype 6 to deliver XBP1s-HA (AAV/XBP1s-HA) or the empty vector (AAV/MOCK) in the hippocampus using bilateral stereotaxis. Fourteen days after the injection, the animals were trained and evaluated in the memory flexibility test (n=6 per group). The statistical analysis was carried out via a two-way ANOVA followed by a Bonferroni post-test (*: p<0.05, **: p<0.01, ***: p<0.001).

[0159] FIG. 11/17

[0160] In this diagram, Xbp1s is presented in the left panel and Bdnf in the right panel. The mARN levels were measured by PCR in real time in the total of cADN obtained as of the hippocampus of wild-type mice injected with AAV/XBP1s or AAV/MOCK particles. The expression values were standardized with the levels of β-actin (n=6 per group). A statistical analysis was made using the Student's t-test (*: p<0.05).

[0161] FIG. 12/17

[0162] In these graphs, mice were evaluated that had been injected with particles of AAV, described in FIG. 10/17, in the hippocampus by means of two different virus titles (1×:1×10.sup.6 Tus, 10×: 1×10.sup.7 TUS) by means of bilateral cerebral stereotaxis. In the left panel, images representative of the immunohistochemistry of injected animals can be seen, where they are shown after the tincture with the anti-HA antibody where the arrowheads indicate the HA-positive neurons. The scale of the bar: 100 μm.

[0163] In the right-hand panel, the mice were trained for the oasis labyrinth test and the percentage of success in the task was measured over time (1× title: AAV/MOCK n=9; AAV/XBP1s-HA: n=10; 10× title: AAV/MOCK n=5; AAV/XBP1s-HA: n=5). A statistical analysis was carried out by means of a two-way ANOVA followed by a Bonferroni post-test (*: p<0.05, ***: p<0.001). The average and the standard error are presented in all the figures.

[0164] FIG. 13/17

[0165] This figure presents that the overexpression of XBP1s in the hippocampus of mice improves the performance in the Oasis test. The mice were injected by bilateral stereotaxis with the serotype 6 of the adeno-associated virus (AAV) to deliver XBP1s-HA (AAV/XBP1s-HA) or particles of an empty vector (AAV/MOCK) in the hippocampus of wild-type mice using the different titres of virus (1×: 1×10.sup.6 TUs, 10×: 1×10.sup.7 TUs). The mice were trained in the oasis labyrinth (FIG. 12/17, right-hand panel), and in the test the ratio of distances was measured (observed versus expected). The average and the standard error were presented in the figure. The statistical analysis was carried out using a two-way ANOVA followed by a Bonferroni post-test (*: p<0.05, **: p<0.01).

[0166] FIG. 14/17

[0167] This figure presents a work model where one sees an interaction between the virus AAV/XBP1s-HA and/or the AAV/XBP1u-HA virus and a cell from the hippocampus and how the mARN of Xbp1s and Xbp1u act on the group of genes (Ryr2, Ampa 3, Bdnf and Kif17) in the regulating of learning and the memory. Where the expression of XBP1 in the neurons of the hippocampus directly or indirectly (dotted lines) controls the expression of different genes implied in the establishment of the memory and other cognitive processes. The direct regulation of the genes of the cluster and the expression that is produced through the bonding of XBP1 to a bonding site UPRE B located in the proximal promoter region of Bdnf.

[0168] FIG. 15/17

[0169] This figure shows an outline of the AAV genome.

[0170] REP: Genes involved in the AAV replication mechanism.

[0171] VP: Genes involved in the formation and assembly of the capsid.

[0172] ITR: It is the equivalent of LTR, repeated terminal inverted sequence.

[0173] FIG. 16/17

[0174] This figure presents the vector of the AAV virus with the insert Xbp1s with the following specific description according to table VI.

TABLE-US-00003 TABLE VI Type Start Stop C Description LTR 1 141 /note = L-ITR promoter 150 >706 /note = PGK1 promoter frag 151 708 /note = 151 to 708 of #18 AAV-PGK1-MCS intron 721 1203 /note = beta-globin intron CDS 1219 1248 /note = /HA tag= CDS 1249 2364 /note = /xbp-1= frag <2368 2970 /note = 1 to 605 of WPRE frag <2378 >2967 /note = WPRE frag 2380 >2967 /note = 1094 to 1682 of WHV frag 2380 >2967 /note = 1 to 589 of WHV lentivirus CDS 2789 >2967 /codon_start = 1 /db_xref = PID: g336148 /note = X protein /translation = MAARLCCHLDSARDVLLLRPFGPQSSGPSFPRPAAGSAASSASSPSPSDESDLPLGR LPACFASASGPCCLVFTCADLRTMDSTVNFVSWHANRQLGMPSKDLWTPYIKDQLLTKWEEGSID PRLSIFVLGGCRHKCMRLL[Split] polyA_site 2976 3454 /note = hGH polyA LTR 3494 3634 /note = R-ITR insertion_seq 3726 4032 /note = f1 origin CDS 4551 5408 /note = Ampicillin resistance ORF insertion_seq 5559 6226 /note = pUC origin

[0175] FIG. 17/17

[0176] This figure presents the generation of the adenoviral plasmid (pAAV) for XBP1s.

[0177] (A) Description of the splitters used in the cloning of XBP1s with HA tag in the expression vector pAAV-PGK1-MCS. Splitters were designed that delimit the murine XBP1s sequence: the sense strand includes the sequence of the HA tag for the 5′ end (left panel) and the antisense strand includes the sequence of the HA tag for the 3′ end (right panel) indicated by the gray-colored box.

[0178] a) HA—sense strand

[0179] b) antisense strand

[0180] c) sense strand

[0181] d) HA—antisense strand

[0182] B) In the lower figure, the expression levels of HA were evaluated based on total extracts of proteins from HEK cells after 48 hours of transfection with the different constructs. The extracts of proteins were made in RIPA solution and 35 mg of proteins were analyzed using Western Blot in gels of acrylamide at 8%. The expression of HA was determined using a monoclonal primary antibody destined against HA (Dilution 1:1000, Covance, catalogue number MMS-101R) and the secondary antibody anti IgG of mice conjugated to peroxidase (dilution 1:3000). As load control, the expression of β-Actin was determined using a primary polyclonal antibody (dilution 1:1000, Santa Cruz, catalogue number sc-1616) and the secondary antibody anti-IgG of goat conjugated to peroxidase (dilution 1:3000).

EXAMPLE OF APPLICATION

[0183] Experimental Test 1

[0184] The transformed virus AAV/XBP1s-HA was applied locally in brains of wild mice to increase the expression of XBP1s and its activity. The selective expression in the hippocampus of adult mice was induced by bilateral stereotaxic injections of the AAV virus serotype 6 in order to free XBP1s and, on the other hand, a control vector AVV/MOCK, in the area of the hippocampal region CA1.

[0185] Two weeks after the injection, the rats were tested in memory flexibility tests. Results were observed similar to those observed in the Tg.sup.XBP1s animals, with the local expression of XBP1s in the hippocampus, resulting with an improved performance in the cognitive tasks (FIG. 10/17).

[0186] To see if the cognitive response is correlated with overexpression of any of the genes of the cluster (KIF 17, AMPA 3, BDNF, RYR3) related with the memory, the overexpression of the mARN of one of these genes in the hippocampus was evaluated. It was observed that a correlation exists between the increase of the mARN of the group of genes mentioned previously and the cognitive response (FIG. 11/17).

[0187] Experimental Test 2

[0188] To validate the results delivered in the experimental test 1, the virus was tested in another model of rodent using a cognitive test that evaluates the memory dependent on the hippocampus. Two different doses of AAV/XBP1s-HA (FIG. 12/17, right panel) were injected bilaterally in the CA3 region of the hippocampus of mice, and two weeks after the surgery their behavior was evaluated in the Oasis Maze (15) test.

[0189] The mice that express XBP1s-HA in the hippocampus (FIG. 12/17, left panel) present a significant increase in the percentage of successful attempts to find food hidden in the labyrinth. Moreover, these effects were dependent on the dose (FIG. 12/17, right panel and FIG. 13/17).

[0190] Material and Methods

[0191] Animals and Surgical Procedures:

[0192] For all the experiments, male mice, 3-6 months old, were used for XBP1.sup.Nes−/−, Tg.sup.XBP1s and mice of the wild type C57BL/6. The mice were maintained in a light-darkness cycle of 12:12 hours and they had free access to food and water, unless the experiment required it.

[0193] The guidelines established by the committee for the care and use of animals in the University of Chile, Chile were used for all the experiments in animals presented in this development.

[0194] Generation of Transgenic Mice XBP1s.

[0195] The cADN Xbp1s of mice was sub cloned in the Xho1 site of the vector.sup.2 MoPrP.Xho to control the expression under the promotor PrP and the microinjection of cells CBA-C57BL/6 derived from pronuclei of mice of fertilized oocytes. The genetic state of the mice was confirmed by PCR of genomic DNA from tails of mice (3 weeks) using the splitters for XBP1s:

TABLE-US-00004 Sense strand:  5′-ACACGCTTGGGAATGGACAC-3′ Antisense strand:  5′-CCATGGGAAGATGTTCTGGG-3′

[0196] Behavioral Tests

[0197] All the experiments were performed blindly, and different cohorts of animals were used for each behavioral test.

[0198] Conditioning of contextual fear: In the first days, the animals were allowed to become accustomed in the chamber (Med Associates, Burlington, Vt.) for 2 minutes and then they were presented with a base noise (80 dB) for 30 seconds. After an interval of 2 seconds, the animals were exposed to an electric shock of 0.5 mA, which is known as an unconditioned stimulus (US). This procedure was repeated five times. Twenty-four hours after the training, the animals are placed in the original chamber once again and the immobility events are evaluated for 5 minutes to determine the associations of the US with the context. The immobility events are automated with the Med Associates software (Burlington, Vt.) designed to determine 30 observations in five minutes. This experiment was carried out at Case Western Reserve University (CWUR) Rodent Behavior Core and was then repeated at the Harvard Neuro-discovery Center.

[0199] The flexibility of the Memory

[0200] The memory flexibility test was carried out according to the description of Chen et al (2000). The training is carried out up to ten tests per day, until the location of the platform is learned. Through a criterion a priori of three latent escapes of less than 20 seconds. After the finalization of the tests, the mouse was removed from the labyrinth, was dried and returned to his cubicle. The data related to the time spent in each quadrant of the pool was linked to a video tracking system of the water labyrinth (HVS Image, Hampton, United Kingdom).

[0201] Oasis Maze

[0202] A modified protocol of the Oasis Maze was used, that is a dry version equivalent to the water labyrinth in the requirement of hippocampal spatial navigation. The apparatus consisted of sand in an open field of 1.4 m in diameter, that is 50-cm above the floor with a 20-cm high wall, that is in an isolated room with constant distal visual signals. Twenty-one evenly spaced wells (4.5 cm in diameter, 2 cm high) were placed on the table and one of the wells is baited with 50 mg of food. Fourteen days after recovering from surgery, the task consisted in 15 tests of one minute each per session, one session per day, during four consecutive days. All the behavior of the animals was recorded on video, with the help of a video camera in zenithal position.

[0203] Startle Response

[0204] The mice were placed in a Plexiglas cylinder and were left at rest for five minutes. After the acclimatization, each subject was presented to 36 tests in one 9-minute test session. They are exposed to nine different levels of sound: 70, 74, 78, 82, 86, 90, 100, 110 and 120 (dB). Each stimulus was of 40 ms and was presented on four occasions with a pseudorandom purpose. The average interval between tests was of 15 s (it oscillated from 10 to 20 s). The startle response was registered during 65 ms (each measurement of the response of 1 ms) based on the appearance of the startle response. The maximum amplitude of the shock was registered during the 65 ms sampling window that was used as a dependent measure.

[0205] Rotarod

[0206] The mice were placed on a bar that rotates at 4 rpm during a minute of acclimation. The rod was accelerated at 0.1 rpm/s to 40.0 rpm. The test continued until all the mice fell off the rod. The latency in fallings and the rpm at the moment of the fall were registered for each mouse. Three tests were executed per mouse and an average calculated.

[0207] Hot Plate

[0208] The animal was placed on the plate at 55° C. The animal was observed until it showed a nociceptive response (for example, licking its rear legs, jumps or squeaks) or until the cut-off time is reached (30 seconds). The animal was removed and the latency of response was recorded. For the animals that do not respond before the cut-off time, the cut-off time was recorded.

[0209] Recognition of New Objects

[0210] The object recognition tests were carried out in the following manner. Twenty-four hours before the test the animals became accustomed to an open field for 15 minutes. The test wraps the presentation of two identical objects in an open field of 45×45 cm for ten minutes. The animals were allowed to explore freely and the frequency and duration of the explorations were quantified. One exploration was defined by direct visual contact at a distance of 1 cm or less, or a direct interaction with the object. After the training was concluded, the animals were placed again in their house-cage for one hour. The level of the object recognition memory was measured by switching one of the objects in the open field and allowing the animal out to explore the two objects for five minutes. The novel object was different but its exploratory index was similar. The relationship of the total novelty to the exploration of the object was used to determine the exploratory discrimination relationship. The locomotor activity was measured and the exploration of both objects during the training and test sessions to identify any object side/preference or general differences in the locomotor/exploratory activity.

[0211] Open Field

[0212] The locomotor activity and the observations of behavior related to anxiety were made while the mouse was in an “open field”. The open field consists of a 40-cm×40-cm box situated in a dimly lit room. The animals are placed in the open field and they are allowed to explore the enclosure freely for 15 minutes. During this period, the locomotor parameters such as the total distance of movement, speed, angular speed and the direction are measured to determine the basic locomotive activity and the presence of stereotypes.

[0213] Production of Adeno-Associated Vectors

[0214] The particles of the AAV serotype 6 (AAV2/6) virus were produced by the transfection of cells 293-AAV (Agilent Technologies, Santa Clara, Calif.) and they were purified in a gradient of iodixanol followed by affinity column chromatography. The number of particles of AAV that the genome contains in the suspension, as well as the infectivity of the vector's suspension in cells HEK293T were determined by means of TaqMan qPCR tests.

[0215] Preparation of the Adenoviral Plasmid (pAAV) for XBP1s.

[0216] For the development of this objective, the sequence of XBP1s murine was cloned in the adenoviral plasmid pAAV-PGK1-MCS, that expresses the transgene under the promotor PGK1. Due to the absence of antibodies that permit recognition of XBP1s in murine tissue, the sequence of the HA tag was included in the cloning strategy (FIG. 17/17 A), that will then allow us to identify the transduced cells and the expression of XBP1s (without excluding other epitope sequences such as Flag, Gfp, His and Myc, among others).

[0217] Therefore, we generate the amplification of XBP1s with the sequence of the HA tag at the terminus 5′ (left panel) and with the HA sequence in the terminus 3′ (right panel). The clones obtained were confirmed by means of DNA sequencing. In this way, we generated the constructs pAAV PGK HA-XBP1s that codify for the fusion protein XBP1s with the HA tag at the end of the amino terminal and pAAV PGK XBP1s-HA with the HA tag at the end of the carboxyl terminal. The empty adenoviral plasmid pAAV PGK was utilized as a control.

[0218] To confirm the expression of the constructs generated we transfected HEK cells with the different constructs, after 48 hours of transfection we executed the extraction of proteins that were evaluated by means of WB using an anti-HA antibody.

[0219] As can be seen in FIG. 17/17 B, we detected a band of the expected molecular weight for XBP1s (55 KDa) only in the cells transfected with the plasmid pAAV PGK HA-XBP1s and with pAAV PGK XBP1s-HA.

[0220] The cDNA XBP1s-HA that codifies C-terminal HA-labeled, the active form of XBP1 mice, was generated by amplifying by PCR of pCMVsport6-mXBP1s.

TABLE-US-00005 Sense strand 5′AGCTATCGATGAGATGATGGTGGTGGTGGCAGCGGCG3′; Anti-sense strand 5′ACGTAGATCTTTAGACGTAATCTGGAACATCGTATGGGTAGACACTAA TCAGCTGGGGGAAAA 3′

[0221] And they were sub cloned in the expression vector pAAV-pgk1-MCS that is derived from the plasmid pAAV-CMV-MCS (Clontech).

[0222] Stereotaxic Injections

[0223] The mice were anesthetized using ketamine/xylazine anesthesia (Ketamine: 100 mg/kg, xylazine: 10 mg/kg, Vetcom, Chile) and were placed in a stereotaxic with bars in the nose and ear for the mice (David Kopf Instruments, U.S.A.). Bilateral injections of AAV/XBP1s-HA, AAV/MOCK, AAV/BDNF-GFP or AAV/GFP were executed with the following concentrations: 1×10.sup.6 units of transduction/μl (TU) of AAV/XBP1s-HA and AAV/MOCK; 1×10.sup.9 viral genomes/μl (VG) were injected for AAV/BDNF-GFP and AAV/GFP. The expression of EGFP was controlled by PCR in real time after the injection of the AAV to corroborate that the efficiency of the transduction was the same as that obtained in these experiments for both constructions (data not shown). The injection of AAV was executed in a single point, the injection of 2 or 3 μl in the CA1 region of the hippocampus using a 5 μl Hamilton syringe (Hamilton, U.S.A.) in the following coordinates: AP: −0.194 cm μl/min and the needle is left in its place for 5 minutes before the retraction of the needle.

[0224] For the stereotaxic injections in the mice, the animals were anesthetized using the isoflurane inhalation anesthetic (halogenated ether 2-chlorine-2-difluoromethoxy-1,1,1-trifluor-ethane) and they were maintained at 1.0-2.0% of isoflurane in oxygen at 100% and they are placed in a stereotaxic frame with the nose and ear with bars for mice. Bilateral injections of AAV/XBP1s-HA or AAV/MOCK were executed with the following concentrations: 1×10.sup.6 TU (1×) or 1×10.sup.7 TU (10×). The AAVs injection was placed at a single point, the injection of 2 μl in the CA3 region of the hippocampus using a 5 μl Hamilton syringe (Hamilton, U.S.A.) in the following coordinates: AP: −0.33 cm, ML: 0.36 cm, DV: −0.33 cm (according to the Paxinos and Watson atlas, 1998). The injection was carried out at a speed of 05 μl/min and the needle was left in place for 5 minutes before it was retracted.

[0225] Preparation of Tissues for the Biochemical Analysis.

[0226] The mice were sacrificed by narcosis of CO.sub.2, the brains were removed, and the cortex, hippocampus, cerebellum and amygdala of both hemispheres were dissected rapidly in a plastic plate cooled with ice. The tissue was homogenized in 100 μl of phosphate buffered saline (PBS) (pH 7.4) supplemented with a mixture of protease inhibitors (Roche Applied Science, U.S.A.). The homogenized was divided to obtain mARN and the extraction of proteins was followed by standard purification and quantification protocols.

[0227] Extraction of ARN and PCR in Real Time.

[0228] The total ARN was isolated from the hippocampus, the amygdala, the cerebellum and the total brain. After the homogenization in PBS we have continued the ARN Trizol extraction protocol recommended by the manufacturer. The cDNA was synthesized with a kit of cDNA high capacity reverse transcription (Applied Biosystems). SYBR green and a System (Stratagene) Mx3005P QPCR were utilized for the quantitative RT-PCR. The relative amount of mARN was calculated by the comparative threshold cycle method with β-actin as control. The Primers of the sequences were obtained based on the PrimerBank (Table VII).

TABLE-US-00006 TABLE VII Sequence Target Forward Reverse Ttr 5′-TTGCCTCGCTGGACTGGTA-3′ 5′-TTACAGCCACGTCTACAGCAG-3′ Retn 5′-CTGTGTCATACGCCAAGAACA-3′ 5′-GGGGAGGTACAGGATGTGGAT-3′ GRia1 5′-GTCCGCCCTGAGAAATCCAG-3′ 5′-CTCGCCCTTGTCGTACCAC-3′ Gria2 5′-GCCGAGGCGAAACGAATGA-3′ 5′-CACTCTCGATGCCATATCGTTG-3′ Gria3 5′-ACCATCAGCATAGGTGGACTT-3′ 5′-ACGTGGTAGTTCAAATGGAAGG-3′ Gria4 5′-GGGAGGTGACTCCAAGGACA-3′ 5′-CCAGTCATGGATAACCTGGCT-3′ Myo5b 5′-CAGCAAGTGGTCAATGCACG-3′ 5′-TGGCGTAGTTGATACAAAACTGT-3′ Creb 5′-AGCCGGGTACTACCATTCTAC-3′ 5′-GCAGCTTGAACAACAACTTGG-3′ Bdnf 5′-CAGGTTCGAGAGGTCTGACGA-3′ 5′-CGCGTCCTTATGGTTTTCTTCG-3′ Camk11 5′-TGCCTGGTGTTGCTAACCC-3′ 5′-CCATTAACTGAACGCTGGAACT-3′ Ryr1 5′-CAGTTTTTGCGGACGGATGAT-3′ 5′-CACCGGCCTCCACAGTATTG-3′ Ryr2 5′-ATGCTTTAAGGCACAGCG-3′ 5′-CAGAGCCCGAATCATCCAGC-3′ Ryr3 5′-ACCAGCAGGAGCAAGTACG-3′ 5′-GGGGTCGTGTCAAAGTAGTCA-3′ Nr2a 5′-ACGTGACAGAACGCGAACTT-3′ 5′-TCAGTGCGGTTCATCAATAACG-3′ Nr2b 5′-GCCATGAACGAGACTGACCC-3′ 5′-GCTTCCTGGTCCGTGTCATC-3′ Pp2b/Caln 5′-AAATGAGGCCAGCTACCAAAC-3′ 5′-CCCGATTTGTCCAAGTCCAG-3′ Kif17 5′-GGGGCATCATTCCCAGAGC-3′ 5′-TTGTGTACCGTGTGCATGGAC-3′ Stx17 5′-TCAAAGTGGCAGGAATTGCAG-3′ 5′-AATTTTCCACCTGTGAAGCCTAA-3′ Kcnk1 5′-GAGGAGCTGCCTTATGAGGAC-3′ 5′-TCCCAATTCCAATTTCCCGAG-3′ Xpo4 5′-CCCCCAGAAGTGATCGCTC-3′ 5′-TGGTTTCCAAAATATGCCTGCAA-3′ Csnk2a 5′-AGGATAGCCAAGGTTCTGGGA-3′ 5′-CCATCGCTTACGGGAGTGTC-3′ Adb1 5′-GAACCCTGCAACTGTCGTC-3′ 5′-CCACGAGTAGGCCCATACC-3′ Pten 5′-TGGATTCGACTTAGACTTGACCT-3′ 5′-GCGGTGTCATAATGTCTCTCAG-3′ Map2k3 5′-GCCTCAGACCAAAGGAAAATCC-3′ 5′-GGTGTGGGGTTGGACACAG-3′ Ucqr10 5′-ATCCCTTCGCGCCTGTACT-3′ 5′-GTGCTCGTAGATCGCGTCT-3′ Nipsnap1 5′-CACGGCGGCTATTCACGAA-3′ 5′-GAACGGAACCAGCTTCCTTCA-3′ Xbp1Δ 5′-CCTGAGCCCCGGAGGAGAA-3′ 5′-CTCGAGCAGTCTGCGCTG-3′ Wfs1 5′-CCATCAACATGCTCCCGTTC-3′ 5′-GGGTAGGCCTCGCCAT-3′ Edem 5′-AACCCAATGGCCTGTCTGG-3′ 5′-AAGCCCTCTGGAACTTGCG-3′ Bip 5′-TCATCGGACGCACTTGGAA-3′ 5′-CAACCACCTTGAATGGCAAGA-3′ Actin 5′-CTCAGGAGGAGCAATGATCTTGAT-3′ 5′-TACCACCATGTACCCAGGCA-3′

[0229] Western Blot of Tissue.

[0230] The extraction of proteins based on the tissue of mice was carried out in RIPA Buffer (20 mM Tris pH 8.0, NaCl 150 mM, 0.1% of SDS, 0.5% deoxycholate, 0.5% of Triton X-100) that contains a mixture of inhibitors of the protease and a mixture of inhibitors of the phosphatase (Sigma, U.S.A.). An example of this quantification was executed with the BCA test kit (Pierce, U.S.A.). Total cellular extracts were separated by SDS-PAGE and were transferred to membranes of polyvinylidene difluoride. The following antibodies were used for the analysis of immunoblot: Hsp90 (1:3000 Santa Cruz), anti e1F2α phosphorylated, the total e1F2α and Hsp90 (1:1000, Cell Signaling), BDNF (1:1000, Alomone Labs), KIF17 (1:1000, Sigma), XBP1 (1:1000, Poly6195-BioLegend), β-actin and ATF4 (1:1000, Santa Cruz).

[0231] Preparation of the Tissue and the Histologic Analysis

[0232] The mice were anesthetized with the ketamine/xylazine mixture of anesthesia and they were fixed with paraformaldehyde at 4%. The mice were deeply anesthetized with 7% of chloral hydrate (350 mg/kg, ip) and they were fixed with paraformaldehyde at 4%. The brains were extracted, then fixed during the night at 4° C. in the same solution and subsequently placed at 30% of sucrose (Merck, U.S.A.) at 4° C. for 48 hours. The brains were frozen in an optimum compound for cutting coronal sections at an adequate temperature (Tissue Tek, U.S.A.): 25 μm for male mice and 50 μm for female mice that containing the hippocampus were cut in a cryostat (Leica, Germany) and then staining was executed in free-floating sections.

[0233] The immunostaining was executed via the universal kit plus ICQ LSAB (ABC Elite Kit, Vector Laboratories, U.S.A.). The sections were incubated with H.sub.2O.sub.2 at 3% in PBS for 30 minutes and were blocked for 2 hours with 0.5% of bovine serum albumin and 0.1% of Triton X-100. The sections were incubated during the night at 4° C. in a blocking solution with HA (1:800, Covance) as a primary antibody and they were washed three times with PBS and were incubated for one hour at room temperature with the biotinylated anti-mouse secondary antibody (1:1000). After rinsing three times, the sections were treated with avidin-biotin-peroxidase complex. The sections were developed using 3,3-diaminobenzidine for 5 minutes and they were visualized in an Olympus IX71 inverted microscope or in a DM5500 Leica for the digitalization of the complete sections.

[0234] Preparation and the Electrophysiology of the Slice of Hippocampus.

[0235] The slices of hippocampus were prepared according to standard procedures for mice aged 4-6 months. Transversal slices of 350 μm of the dorsal hippocampus were cut in artificial cold cephalorhachidian liquid (ACSF, in mM: 124 NaCl, 2,6 NaHCO3, 10 D-glucose, 2.69 KCl, 1.25 KH.sub.2PO.sub.4, CaCl.sub.2 2,5, 1.3 MgSO.sub.4, and 2,60 NaHPO.sub.4) using a vibratome (Leica VT 1000 s, Germany) and they were incubated in ACSF for more than one hour at room temperature. In all the experiments, picrotoxin was added (10 μM) to the ACSF perfusion means in order to suppress the inhibiting transmission GABAA. To evoke field excitatory postsynaptic potential (fEPSPs), Schaffer collateral fibers were activated for the bipolar cathodic stimulation, generated by a stimulator (Axon 700b, Molecular Devices, Sunnyvale, Calif.) connected to an isolation unit (Isoflex, AMPI, Jerusalem, Israel). To generate LTP, in mice XBP1.sup.Nes−/− that uses high frequency stimulation (HFS) that consists of three stimulus trains with an interval between trains of 20 s. Each train consisted of 100 Hz for 500 ms. In the Tg.sup.XBP1s mice we used the theta burst stimulation that consists of 5 stimulus trains with an interval between trains of 20 s. Each train consisted of 8 bursts at 5 Hz, each burst has 4 pulses at 100 Hz. The recordings were filtered at 2.0-3.0 kHz, sampling at 4.0 kHz using an A/D converter, and they are stored with a pClamp 10 computer (Molecular Devices).

[0236] Cultures and Neuronal Transfections.

[0237] The neuro2A cells and the HEK293T cells were obtained from the ATCC and were cultivated in a DMEM medium supplemented with 10% of bovine serum or 5%, respectively, and antibiotics (10000 U/ml of penicillin, 10 mg/ml of streptomycin) at 37° C. and 5% of CO.sub.2. The cortical neurons and those of the hippocampus were obtained on the 18.sup.th embryonic day described by Goslin and Banker (1991).

[0238] Statistics

[0239] The data is expressed as average and SEM. Depending on the experiments, the results were compared statistically using the Student's T test or the Mann-Whitney test, the two-way ANOVA followed by Holm-Sidack or Bonferroni as a post-hoc test or one-way Kruskal-Wallis ANOVA in ranges followed by the Dunn or Bonferroni Method as a post-hoc test.

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[0245] 6.—Rattiner, L. M., Davis, M. & Ressler, K. J. Differential regulation of brain-derived neurotrophic factor transcripts during the consolidation of fear learning. Learn Mem 11, 727-731, doi:10.1101/lm.83304 (2004).

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[0255] 16. Product Data Sheet Paav-MCS Expression vector, Catalog Number: VPK-410.

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TABLE-US-00007 TABLE II Characteristics and sequence of the plasmid pAAV-MCS alone.    1-130: left ITR  139-801: CMV Promotor  809-1301: Intron of human β-globin 1308-1383: MCS 1384-1862: PolyA 1902-2042: right ITR 2959-3819: Ampicillin resistance 5′ cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcg tcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagag agggagtggccaactccatcactaggggttcctgcggccgcacgcgtgga gctagttattaatagtaatcaattacggggtcattagttcatagcccata tatggagttccgcgttacataacttacggtaaatggcccgcctggctgac cgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccata gtaacgtcaatagggactttccattgacgtcaatgggtggagtatttacg gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgc cccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccag tacatgaccttatgggactttcctacttggcagtacatctacgtattagt catcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg gatagcggtttgactcacggggatttccaagtctccaccccattgacgtc aatgggagtttgttttgcaccaaaatcaacgggactttccaaaatgtcgt aacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtggga ggtctatataagcagagctcgatagtgaaccgtcagatcgcctggagacg ccatccacgctgattgacctccatagaagacaccgggaccgatccagcct ccgcggattcgaatcccggccgggaacggtgcattggaacgcggattccc cgtgccaagagtgacgtaagtaccgcctatagagtctataggcccacaaa aaatgctttcttcttttaatatacttttttgtttatcttatttctaatac tttccctaatctctttcatcagggcaataatgatacaatgtatcatgcct attgcaccattctaaagaataacagtgataatactgggttaaggcaatag caatatactgcatataaatatactgcatataaattgtaactgatgtaaga ggatcatattgctaatagcagctacaatccagctaccattctgcttttat tttatggttgggataaggctggattattctgagtccaagctaggcccttt tgctaatcatgttcatacctcttatcttcctcccacagctcctgggcaac gtgctggtctgtgtgctggcccatcactttggcaaagaattgggattcga acatcgattgaattccccggggatcctctagagtcgacctgcagaagctt gcctcgagcagcgctgctcgagagatctacgggtggcatccctgtgaccc ctccccagtgcctctcctggccctggaagttgccactccagtgcccacca gccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtcc ttctataatattatggggtggaggggggtggtatggagcaaggggcaagt tgggaagacaacctgtagggcctgcggggtctattgggaaccaagctgga gtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaa gcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcat gaccaggctcagctaatttttgtttttttggtagagacggggtttcacca tattggccaggctggtctccaactcctaatctcaggtgatctacccacct tggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccc tgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcagga acccctagtgatggagttggccactccctctctgcgcgctcgctcgctca ctgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg gcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcg gtattactccttacgcatctgtgcggtatttcacaccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggt ggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctc catcgattcaccatccatctcgccacgacgccggcatccccgtcaagctc taaatcgggggctccattagggaccgatttagtgctttacggcacctcga ccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccct gatagacggtttttcgccctttgacgaggagtccacgttattaatagtgg actatgaccaaactggaacaacactcaaccctatctcgggctattcattg atttataagggattttgccgatttcggcctattggttaaaaaatgagctg atttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaat tttatggtgcactctcagtacaatctgctctgatgccgcatagttaagcc agccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctg ctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcc tcgtgatacgcctatttttataggttaatgtcatgataataatggtttct tagacgtcaggtggcactatcggggaaatgtgcgcggaacccctatttga tattatctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacat ttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttat gctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagagggt gcacgagtgggttacatcgaactggatctcaacagcggtaagatcatgag agattcgccccgaagaacgattccaatgatgagcacattaaagactgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcg ccgcatacactattctcagaatgacttggttgagtactcaccagtcacag aaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgcc ataaccatgagtgataacactgcggccaacttacactgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaac tcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacg agcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactg gatggaggcggataaagttgcaggaccacttctgcgctcggcccttccgg ctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagt tatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaa aaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctt aacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaat actgtccttctagtgtagccgtagttaggccaccacttcaagaactctgt agcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagc tatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggg aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgc tcacatgt 3′

TABLE-US-00008 TABLE III Restriction map of AAV-PGK1-HA-Xbp-1-WPRE PGK1 189 to 528 nt Tag HA 1219 to 1248 nt XBP1 1249 to 2367 nt WPRE 2378 to 2967 nt CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC <100 GGACGTCCGTCGACGCGCGAGCGAGCGAGTGACTCCGGCGGGCCCGTTTCGGGCCCGCAGCCCGCTGGAAACCAGCGGGCCGGAGTCACTCGCTCGCTCG          10        20        30       40         50        60        70        80        90 GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGCGATCACGAGACTAGCCTCGACGATGGTCGAGTACCGGGTAGGGGA <200 CGCGTCTCTCCCTCACCGGTTGAGGTAGTGATCCCCAAGGACGCCGGCGTGCGCGCTAGTGCTCTGATCGGAGCTGCTACCAGCTCATGGCCCATCCCCT         110       120       130      140        150       160       170       180       190 GGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCC <300 CCGCGAAAAGGGTTCCGTCAGACCTCGTACGCGAAATCGTCGGGGCGACCCGTGAACCGCGATGTGTTCACCGGAGACCGGAGCGTGTGTAAGGTGTAGG         210       220       230      240        250       260       270       280       290                                                             <PGK Prom                                                             | ACCGGTAGGCGCGAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGT <400 TGGCCATCCGCGGTTGGCCGAGGCAAGAAACCACCGGGGAAGCGCGGTGGAAGATGAGGAGGGGATCAGTCCTTCAAGGGGGGGCGCGGCGTCGAGCGCA         310       320       330      340        350       360       370       380       390 CGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCAGCGGTGAGCAAATGGAAGCGGGTAGGCGTTTGGGGAGCGG <500 GCACGTCCTGCAGTGTTTACCTTCATCGTGCAGAGTGATCAGAGCACGTGTACCTGTCGTGGGGACTCGTTACTCTTCGCCCATCCGGAAACCCCGTCGC         410       420       430      440        450       460       470       480       490 CCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCC <600 GGTTATCGTCGAAACGAGGAAGCGAAAGACCCGAGTCTCCGACCCTTCCCCACCCAGGCCCCCGCCCGAGTCCCCGCCCGAGTCCCCGCCCGCCCGCGGG         510       520       530      540        550       560       570       580       590 CGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCTTTCGACCTCTAGCGG <700 GCTTCCAGGAGGCCTCCGGGCCGTAAGACGTGCGAAGTTTTCGCGTGCAGACGGCGGCGACAAGAGGAGAAGGAGTAGAGGCCCGGAAAGCTGGAGATCG         610       620       630      640        650       660       670       680       690 GGATCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAA <800 CCTAGCCTAAGCTTAGGGCCGGCCCTTGCCACGTAACCTTGCGCCTAAGGGGCACGGTTCTCACTGCATTCATGGCGGATATCTCAGATATCCGGGTGTT         710       720       730      740        750       760       770       780       790 AAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGC <900 TTTTACGAAAGAAGAAAATTATATGAAAAAACAAATAGAATAAAGATTATGAAAGGGATTAGAGAAAGAAAGTCCCGTTATTACTATGTTACATAGTACG         810       820       830      840        850       860       870       880       890 CTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGAT <1000 GAGAAACGTGGTAAGATTTCTTATTGTCACTATTAAAGACCCAATTCCGTTATCGTTATAAAGACGTATATTTATAAAGACGTATATTTAACATTGACTA         910       920       930      940        950       960       970       980       990 GTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAG <1100 CATTCTCCAAAGTATAACGATTATCGTCGATGTTAGGTCGATGGTAAGACGAAAATAAAATACCAACCCTATTCCGACCTAATAAGACTCAGGTTCGATC        1010      1020      1030     1040       1050      1060      1070      1080      1090 GCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGG <1200 CGGGAAAACGATTAGTACAAGTATGGAGAATAGAAGGAGGGTGTCGAGGACCCGTTGCACGACCAGACACACGACCGGGTAGTGAAACCGTTTCTTAACC        1110      1120      1130     1140       1150      1160      1170      1180      1190                                  HA tag                                  | GATTCGAACATCGATGAGATGTACCCATACGATGTTCCAGATTACGCAATGGTGGTGGTGGCAGCGGCGCCGAGCGCGGCCACGGCGGCCCCCAAAGTGC <1300 CTAAGCTTGTAGCTACTCTACATGGGTATGCTACAAGGTCTAATGCGTTACCACCACCACCGTCGCCGCGGCTCGCGCCGGTGCCGCCGGGGGTTTCACG        1210      1220      1230     1240       1250      1260      1170      1280      1290 TACTCTTATCTGGCCAGCCCGCCTCCGGCGGCCGGGCGCTGCCGCTCATGGTACCCGGTCCGCGGGCAGCAGGGTCGGAGGCGAGCGGGACACCGCAGGC <1400 ATGAGAATAGACCGGTCGGGCGGAGGCCGCCGGCCCGCGACGGCGAGTACCATGGGCCAGGCGCCCGTCGTCCCAGCCTCCGCTCGCCCTGTGGCGTCCG        1310      1320      1330     1340       1350      1360      1370      1380      1390 TCGCAAGCGGCAGCGGCTCACGCACCTGAGCCCGGAGGAGAAAGCGCTGCGGAGGAAACTGAAAAACAGAGTAGCAGCGCAGACTGCTCGAGATAGAAAG <1500 AGCGTTCGCCGTCGCCGAGTGCGTGGACTCGGGCCTCCTCTTTCGCGACGCCTCCTTTGACTTTTTGTCTCATCGTCGCGTCTGACGAGCTCTATCTTTC        1410      1420      1430     1440       1450      1460      1470      1480      1490 AAAGCCCGGATGAGCGAGCTGGAGCAGCAAGTGGTGGATTTGGAAGAAGAGAACCACAAACTCCAGCTAGAAAATCAGCTTTTACGGGAGAAAACTCACG <1600 TTTCGGGCCTACTCGCTCGACCTCGTCGTTCACCACCTAAACCTTCTTCTCTTGGTGTTTGAGGTCGATCTTTTAGTCGAAAATGCCCTCTTTTGAGTGC        1510      1520      1530     1540       1550      1560      1570      1580      1590 GCCTTGTGGTTGAGAACCAGGAGTTAAGAACACGCTTGGGAATGGACACGCTGGATCCTGACGAGGTTCCAGAGGTGGAGGCCAAGGGGAGTGGAGTAAG <1700 CGGAACACCAACTCTTGGTCCTCAATTCTTGTGCGAACCCTTACCTGTGCGACCTAGGACTGCTCCAAGGTCTCCACCTCCGGTTCCCCTCACCTCATTC        1610      1620      1630     1640       1650      1660      1670      1680      1690 GCTGGTGGCCGGGTCTGCTGAGTCCGCAGCAGGTGCAGGCCCAGTTGTCACCTCCCCAGAACATCTTCCCATGGACTCTGACACTGTTGCCTCTTCAGAT <1800 CGACCACCGGCCCAGACGACTCAGGCGTCGTCCACGTCCGGGTCAACAGTGGAGGGGTCTTGTAGAAGGGTACCTGAGACTGTGACAACGGAGAAGTCTA        1710      1720      1730     170        1750      1760      1770      1780      1790 TCTGAGTCTGATATCCTTTTGGGCATTCTGGACAAGTTGGACCCTGTCATGTTTTTCAAATGTCCTTCCCCAGAGTCTGCTAGTCTGGAGGAACTCCCAG <1900 AGACTCAGACTATAGGAAAACCCGTAAGACCTGTTCAACCTGGGACAGTACAAAAAGTTTACAGGAAGGGGTCTCAGACGATCAGACCTCCTTGAGGGTC        1810      1820      1830     1840       1850      1860      1870      1880      1890 AGGTCTACCCAGAAGGACCTAGTTCCTTACCAGCCTCCCTTTCTCTGTCAGTGGGGACCTCATCAGCCAAGCTGGAAGCCATTAATGAACTCATTCGTTT <2000 TCCAGATGGGTCTTCCTGGATCAAGGAATGGTCGGAGGGAAAGAGACAGTCACCCCTGGAGTAGTCGGTTCGACCTTCGGTAATTACTTGAGTAAGCAAA        1910      1920      1930     1940       1950      1960      1970      1980      1990 TGACCATGTATACACCAAGCCTCTAGTTTTAGAGATCCCCTCTGAGACAGAGAGTCAAACTAACGTGGTAGTGAAAATTGAGGAAGCACCTCTAAGCTCT <2100 ACTGGTACATATGTGGTTCGGAGATCAAAATCTCTAGGGGAGACTCTGTCTCTCAGTTTGATTGCACCATCACTTTTAACTCCTTCGTGGAGATTCCGAG        2010      2020      2030     2040       2050      2060      2070      2080      2090 TCAGAAGAGGATCACCCTGAATTCATTGTCTCAGTGAAGAAAGAGCCTTTGGAAGATGACTTCATCCCAGAGCTGGGCATCTCAAACCTGCTTTCATCCA <2200 AGTCTTCTCCTAGTGGGACTTAAGTAACAGAGTCACTTCTTTCTCGGAAACCTTCTACTGAAGTAGGGTCTCGACCCGTAGAGTTTGGACGAAAGTAGGT        2110      2120      2130     2140       2150      2160      2170      2180      2190 GCCATTGTCTGAGACCACCTTCTTGCCTGCTGGACGCTCACAGTGACTGTGGATATGAGGGCTCCCCTTCTCCCTTCAGTGACATGTCTTCTCCACTTGG <2300 CGGTAACAGACTCTGGTGGAAGAACGGACGACCTGCGAGTGTCACTGACACCTATACTCCCGAGGGGAAGAGGGAAGTCACTGTACAGAAGAGGTGAACC        2210      2220      2230     2240       2250      2260      2270      2280      2290 TACAGACCACTCCTGGGAGGATACTTTTGCCAATGAACTTTTCCCCCAGCTGATTAGTGTCTAAAGATCTATTCCGATAATCAACCTCTGGATTACAAAA <2400 ATGTCTGGTGAGGACCCTCCTATGAAAACGGTTACTTGAAAAGGGGGTCGACTAATCACAGATTTCTAGATAAGGCTATTAGTTGGAGACCTAATGTTTT        2310      2320      2330     2340       2350      2360      2370      2380      2390 TTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTAT <2500 AAACACTTTCTAACTGACCATAAGAATTGATACAACGAGGAAAATGCGATACACCTATGCGACGAAATTACGGAAACATAGTACGATAACGAAGGGCATA        2410      2420      2430     2440       2450      2460      2470      2480      2490 GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTT <2600 CCGAAAGTAAAAGAGGAGGAACATATTTAGGACCAACGACAGAGAAATACTCCTCAACACCGGGCAACAGTCCGTTGCACCGCACCACACGTGACACAAA        2510      2520      2530     2540       2550      2560      2570      2580      2590 GCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCG <2700 CGACTGCGTTGGGGGTGACCAACCCCGTAACGGTGGTGGACAGTCGAGGAAAGGCCCTGAAAAGCGAAAGGGGAGGGATAACGGTGCCGCCTTGAGTAGC        2610      2620      2630     2640       2650      2660      2670      2680      2690 CCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGC <2800 GGCGGACGGAACGGGCGACGACCTGTCCCCGAGCCGACAACCCGTGACTGTTAAGGCACCACAACAGCCCCTTCGACTGCAGGAAAGGTACCGACGAGCG        2710      2720      2730     2740       2750      2760      2770      2780      2790                                                       <FactorXa site                                                       | CTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTG <2900 GACACAACGGTGGACCTAAGACGCGCCCTGCAGGAAGACGATGCAGGGAAGCCGGGAGTTAGGTCGCCTGGAAGGAAGGGCGCCGGACGACGGCCGAGAC        2810      2820      2830     2840       2850      2860      2870      2880      2890 CGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATGGATCTACGGGTGGCATCCCTGTGACCCCTC <3000 GCCGGAGAAGGCGCAGAAGCGGAAGCGGGAGTCTGCTCAGCCTAGAGGGAAACCCGGCGGAGGGGCGTACCTAGATGCCCACCGTAGGGACACTGGGAGG        2910      2920      2930     2940       2950      2960      2970      2980      2990 CCCAGTGCCTCTCCTGGGGCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTGTGACTAGGTGTGGTTC <3100 GGGTCAGGGAGAGGACCCGGACCTTCAACGGTGAGGTCACGGGTGCTCGCAACAGCATTATTTTAATTCAACGTAGTAAAACAGACTGATCCACAGGAAG        3010      3020      3030     3040       3050      3060      3070      3080      3090 TATAATATTATGGGGTGGACGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAAGCTGTAGGGCCTGCGGGGTCTATTGCGAACCAAGCTGGAGTG <3200 ATATTATAATACCCCACCTCCCCCCACCATACCTCGTTCCCCGTTCAACCCTTCTGTTGGACATCGGGGACCCCCCAGATAACCCTTGGTTCGACCTCAC        3110      3120      3130     3140       3150      3160      3170      3180      3190                 >AGH poly signsl                 | CAGTGCCACAATCTTGGCTCAGTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTGCGGAGTTGTTGGGATTCCAGCCATGCATGAC <3300 GTCACCGTGTTAGAACCGAGTGGCGTTAGAGGCGGAGGACCCAAGTTCGCTAAGAGGACGAGTCGGAGGGCTCAACAACCGTAAGGTCGTACGTACTGAA        3210      3220      3230     3240       3250      3260      3270      3280      3290 CAGGGTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCGTAATCTCAGGTGATCTACCGACCTTTG <3400 GTCCGAGTCGATTAAAAACAAAAAAACCATCTCTGCCCCAAAGTGGTATAACCGGTCCGACCAGAGGTTGAGGATTAGAGTCCACTAGATGGGTGGAACC        3310      3320      3330     3340       3350      3360      3370      3380      3390 CCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTGCCTGTCCTTCTGATTTTGTAGGTAACCACGTGCGGACCGAGCGGCCGCAGGAACC <3500 GGACGGTTTAACGACCCTAATGTCCGCACTTCGTGACGAGGGAAGGGACATGAACACTAAAACATCCATTGGTGCACGCCTGGCTCGCCGGCGTCCTTGG        3410      3420      3430     3440       3450      3460      3470      3480      3490 CCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCGGCCC <3600 GGATCACTACCTCAACCGGTGAGGGAGAGACGCGCGAGCGAGCGAGTGACTCCGGCCCGCTGGTTTCCAGCGGGCTGCGGGCCCAAACGGGCCCGCCGGG        3510      3520      3530     3540       3550      3560      3570      3580      3590 TCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCA <3700 AGTCACTCGCTCGCTCGCGCGTCGACGGACGTCCCCGCGGACTACGCCATAAAAGAGGAATGCGTAGACACGCCATAAAGTGTGGCGTATGCAGTTTCGT        3610      3620      3630     3640       3650      3660      3670      3680      3690 ACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCT <3800 TGGTATCATGCGCGGGACATCGCCGCGTAATTCGCGCCGCCCACACCACCAATGCGCGTCGCACTGGCGATGTGAACGGTCGCGGGATCGCGGGCGAGGA        3710      3720      3730     3740       3750      3760      3770      3780      3790 TTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCC <3900 AAGCGAAAGAAGGGAAGGAAAAGAGCGGTGCAAGCGGCCGAAAGGGGCAGTTCGAGATTTAGCCCCCGAGGGAAATCCCAAGGCTAAATCACGAAATGCG        3810      3820      3830     3840       3850      3860      3870      3880      3890                                    >M13 origin                                  <F1 ori                                  | | ACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT <4000 TGGAGCTGGGGTTTTTTGAACTAAACCCACTACCAAGTGCATCACCCGGTAGCGGGACTATCTGCAAAAAGCGCGAAACTGCAACCTCAGGTGCAAGAAA        3910      3920      3930     3940       3950      3960      3970      3980      3990 TAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCGTATTGGTTA <4100 ATTATCACCTGAGAACAAGGTTTGACCTTGTTGTGAGTTGGGATAGAGCCCGATAAGAAAACTAAATATTCCCTAAAACGGCTAAAGCCGGATAACCAAT        4010      4020      4030     4040       4050      4060      4070      4080      4090 AAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG <4200 TTTTTACTCGACTAAATTGTTTTTAAATTGCGCTTAAAATTGTTTTATAATTGCAAATGTTAAAATACCACGTGAGAGTCATGTTAGACGAGACTACGGC        4110      4120      4130     4140       4150      4160      4170      4180      4190 CATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTC <4300 GTATCAATTCGGTCGGGGCTGTGGGCGGTTGTGGGCGACTGCGCGGGACTGCCCGAACAGACGAGGGCCGTAGGCGAATGTCTGTTCGACACTGGCAGAG        4210      4220      4230     4240       4250      4260      4270      4280      4290 CGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATA <4400 GCCCTCGACGTACACAGTCTCCAAAAGTGGCAGTAGTGGCTTTGCGCGCTCTGCTTTCCCGGAGACACTATGCGGATAAAAATATCCAATTACAGTACTA        4310      4320      4330     4340       4350      4360      4370      4380      4390 ATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA <4500 TATTACCAAAGAATCTGCAGTCCACCGTGAAAAGCCCCTTTACACGCGCCTTGGGGATAAACAAATAAAAAGATTTATGTAAGTTTATAGATAGGCGAGT        4410      4420      4430     4440       4450      4460      4470      4480      4490 TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTGCGGACATT <4600 ACTCTGTTATTGGGACTATTTACGAAGTTATTATAACTTTTTCCTTCTCATACTCATAAGTTGTAAAGGCACAGCGGGAATAAGGGAAAAAACGCCGTAA        4510      4520      4530     4540       4550      4560      4570      4580      4590 TTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC <4700 AACGGAAGGACAAAAACGAGTGGGTCTTTGCGACCACTTTCATTTTCTACGACTTCTAGTCAACCCACGTGCTCACCCAATGTAGCTTGACCTAGAGTTG        4610      4620      4630     4640       4650      4660      4670      4680      4690 AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTCGCGCGGTATTATCCCGTATTTGACG <4800 TCGCCATTCTAGGAACTCTCAAAAGCGGGGCTTCTTGCAAAAGGTTACTACTCGTGAAAATTTCAAGACGATACACCGCGCCATAATAGGGCATAACTGG        4710      4720      4730     4740       4750      4760      4770      4780      4790 CCGGGCAAGGGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGATGGCATGACAAGT <4900 GGCCCGTTCTCGTTGAGCCAGCGCCGTATGTGATAAGAGTCTTACTGAACCAACTCATGAGTGGTCAGTGTCTTTTCGTAGAATGCCTACCGTACTGTCA        4810      4820      4830     4840       4850      4860      4870      4880      4890 AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCCGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG <5000 TTCTCTTAATACGTCACGACGGTATTGGTACTCACTATTGTGACGCGGGTTGAATGAAGACTGTTGCTAGCCTCCTGGCTTCCTCGATTGGCGAAAAAAC        4910      4920      4930     4940       4950      4960      4970      4980      4990                                                                            >AmpR                                                                            | CACAACATGGGCGATCATGTAACTCGCCTTGATCGTTGGGAACCGAGCTGAATGAACCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGTGCAA <5100 GTGTTGTACCCCCTAGTACATTGAGCGGAACTAGCAACCCTTGGCCTCGACTTACTTCGGTATGGTTTGCTCCTCGCAGTGTGGTGCTACGGACATCGTT        5010      5020      5030     5040       5050      5060      5070      5080      5090 TGGCAACAACGTTGCGCAAACTATTAACTCGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC <5200 ACCGTTGTTGCAACGCGTTTGATAATTGACCGCTTGATGAATGAGATCGAAGGGCCGTTGTTAATTATCTGACCTACCTCCGCCTATTTCAACGTGCTGG        5110      5120      5130     5140       5150      5160      5170      5180      5190 ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGGTGATAAATCTGGAGCCGGTGACCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGAT <5300 TGAAGACGCGAGCCGGGAAGGCCGACCGACCAAATAACGACTATTTAGACCTCGGCCACTCCCACCCAGAGCCCCATAGTAACGTCGTGACCGCGGTCTA        5210      5220      5230     5240       5250      5260      5270      5280      5290 GGTAAAGCCGTCCCGTATCGTACTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACGATCGCTGAGATAGGTGCCTCACTGATTA <5400 CCATTCGGGAGGGCATACCATCAATAGATGTGCTGCCCCTCAGTCCGTTGATACCTACTTGCTTTATCTGTCTAGCGACTCTATCCACGGAGTGACTAAT        5310      5320      5330     5340       5350      5360      5370      5380      5390 AGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA <5500 TCGTAACCATTGACAGTCTGGTTCAAATGAGTATATATGAAATCTAACTAAATTTTGAAGTAAAAATTAAATTTTCCTAGATCCACTTCTAGGAAAAACT        5410      5420      5430     5440       5450      5460      5470      5480      5490 TAATCTCATGACCAAAATCCCTTAACTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTTCTG <5600 ATTAGAGTACTGGTTTTAGGGAATTGCACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTATTTCCTAGAAGAACTCTAGGAAAAAAAAGAC        5510      5520      5530     5540       5550      5560      5570      5580      5590 CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGATCAAGAGCTACCAACTCTTTTTCCCGAAGGTAACTGGCT <5700 GCGCATTAGACGACGAACGTTTGTTTTTTTGGTGGCGATGGTCGCCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGCTTCCATTGACCGA        5610      5620      5630     5640       5650      5660      5670      5680      5690 TCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT <5800 AGTCGTCTCGCGTCTATGGTTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTTCTTGAGACATCGTGGCGGATGTATGGAGCGAGCGA        5710      5720      5730     5740       5750      5760      5770      5780      5790                                                                          >ColE1 origin                                                                          | AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATTGTTACCGGATAAGGCGCAGCGGTCGGGCTGA <5900        5810      5820      5830     5840       5850      5860      5870      5880      5890 ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACGTACAGCGTGAGCTATGAGAAAGCGCCACCTTCCCGAAGA <6000 TGCCCCCCAAGCACGTGTGTCGGGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGATACTCTTTCGCGGTGCGAAGGGCTTC        5910      5920      5930     5940       5950      5960      5970      5980      5990 GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT <6100 CCTCTTTCCGCCTGTCCATAGGCCATTCGCGGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTTGCGGACCATAGAAAATATCAGGAC        6010      6020      6030     6040       6050      6060      6070      6080      6090 CGGGTTTCGGCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC <6200 GCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAG        6110      6120      6130     6140       6150      6160      6170      6180      6190 CTGGCCTTTTGCTGGCCTTTTGCTCACATGT <6231 GACCGGAAAACGACCGGAAAACGAGTGTACA        6210      6220      6230 Features: ColE1: [5560:6183] F1 ori: [4154:3714] M13 origin [3709:4161] AmpR: [4749:5408] hGE polyA signal: [2977:3257] mPGK Prom: [189:528] Amp prom: [4481:4509] BA tag:[1222:1248] FactorXa Site: [2861:2850]

TABLE-US-00009 TABLE IV Xbp1s (Mouse) ORIGIN    1 ctagggtaaa accgtgagac tcggtctgga aatctggcct gagaggacag cctggcaatc   61 ctcagccggg gtggggacgt ctgccgaaga tccttggact ccagcaacca gtggtcgcca  121 ccgtccatcc accctaaggc ccagtttgca cggcggagaa cagctgtgca gccacgctgg  181 acactcaccc cgcccgagtt gagcccgccc ccgggactac aggaccaata agtgatgaat  241 atacccgcgc gtcacggagc accggccaat cgcggacggc cacgacccta gaaaggctgg  301 gcgcggcagg aggccacggg gcggtggcgg cgctggcgta gacgtttcct ggctatggtg  361 gtggtggcag cggcgccgag cgcggccacg gcggccccca aagtgctact cttatctggc  421 cagcccgcct ccggcggccg ggcgctgccg ctcatggtac ccggtccgcg ggcagcaggg  481 tcggaggcga gcgggacacc gcaggctcgc aagcggcagc ggctcacgca cctgagcccg  541 gaggagaaag cgctgcggag gaaactgaaa aacagagtag cagcgcagac tgctcgagat  601 agaaagaaag cccggatgag cgagctggag cagcaagtgg tggatttgga agaagagaac  661 cacaaactcc agctagaaaa tcagctttta cgggagaaaa ctcacggcct tgtggttgag  721 aaccaggagt taagaacacg cttgggaatg gacacgctgg atcctgacga ggttccagag  781 gtggaggcca aggggagtgg agtaaggctg gtggccgggt ctgctgagtc cgcagcaggt  841 gcaggcccag ttgtcacctc cccagaacat cttcccatgg actctgacac tgttgcctct  901 tcagattctg agtctgatat ccttttgggc attctggaca agttggaccc tgtcatgttt  961 ttcaaatgtc cttccccaga gtctgctagt ctggaggaac tcccagaggt ctacccagaa 1021 ggacctagtt ccttaccagc ctccctttct ctgtcagtgg ggacctcatc agccaagctg 1081 gaagccatta atgaactcat tcgttttgac catgtataca ccaagcctct agttttagag 1141 atcccctctg agacagagag tcaaactaac gtggtagtga aaattgagga agcacctcta 1201 agctcttcag aagaggatca ccctgaattc attgtctcag tgaagaaaga gcctttggaa 1261 gatgacttca tcccagagct gggcatctca aacctgcttt catccagcca ttgtctgaga 1321 ccaccttctt gcctgctgga cgctcacagt gactgtggat atgagggctc cccttctccc 1381 ttcagtgaca tgtcttctcc acttggtaca gaccactcct gggaggatac ttttgccaat 1441 gaacttttcc cccagctgat tagtgtctaa agagccacat aacactgggc ccctttccct 1501 gaccatcaca ttgcctagag gatagcatag gcctgtctct ttcgttaaaa gccaaagtag 1561 aggctgtctg gccttagaag aattcctcta aagtatttca aatctcatag atgacttcca 1621 agtattgtcg tttgacactc agctgtctaa ggtattcaaa ggtattccag tactacagct 1681 tttgagattc tagtttatct taaaggtggt agtatactct aaatcgcagg gagggtcatt 1741 tgacagtttt ttcccagcct ggcttcaaac tatgtagccg aggctaggca gaaacttctg 1801 accctcttga ccccacctcc caagtgctgg gcttcaccag gtgtgcacct ccacacctgc 1861 ccccccgaca tgtcaggtgg acatgggatt catgaatggc ccttagcatt tctttctcca 1921 ctctctgctt cccaggtttc gtaacctgag ggggcttgtt ttcccttatg tgcattttaa 1981 atgaagatca agaatctttg taaaatgatg aaaatttact atgtaaatgc ttgatggatc 2041 ttcttgctag tgtagcttct agaaggtgct ttctccattt atttaaaact acccttgcaa 2101 ttaaaaaaaa agcaacacag cgtcctgttc tgtgatttct agggctgttg taatttctct 2161 ttattgttgg ctaaaggagt aatttatcca actaaagtga gcataccact ttttaaagtc 2221 aaaaaaaaaa aaaaaaaa

TABLE-US-00010 TABLE V Xbp1u (Mouse) ORIGIN    1 ctagggtaaa accgtgagac tcggtctgga aatctggcct gagaggacag cctggcaatc   61 ctcagccggg gtggggacgt ctgccgaaga tccttggact ccagcaacca gtggtcgcca  121 ccgtccatcc accctaaggc ccagtttgca cggcggagaa cagctgtgca gccacgctgg  181 acactcaccc cgcccgagtt gagcccgccc ccgggactac aggaccaata agtgatgaat  241 atacccgcgc gtcacggagc accggccaat cgcggacggc cacgacccta gaaaggctgg  301 gcgcggcagg aggccacggg gcggtggcgg cgctggcgta gacgtttcct ggctatggtg  361 gtggtggcag cggcgccgag cgcggccacg gcggccccca aagtgctact cttatctggc  421 cagcccgcct ccggcggccg ggcgctgccg ctcatggtac ccggtccgcg ggcagcaggg  481 tcggaggcga gcgggacacc gcaggctcgc aagcggcagc ggctcacgca cctgagcccg  541 gaggagaaag cgctgcggag gaaactgaaa aacagagtag cagcgcagac tgctcgagat  601 agaaagaaag cccggatgag cgagctggag cagcaagtgg tggatttgga agaagagaac  661 cacaaactcc agctagaaaa tcagctttta cgggagaaaa ctcacggcct tgtggttgag  721 aaccaggagt taagaacacg cttgggaatg gacacgctgg atcctgacga ggttccagag  781 gtggaggcca aggggagtgg agtaaggctg gtggccgggt ctgctgagtc cgcagcactc  841 agactatgtg cacctctgca gcaggtgcag gcccagttgt cacctcccca gaacatcttc  901 ccatggactc tgacactgtt gcctcttcag attctgagtc tgatatcctt ttgggcattc  961 tggacaagtt ggaccctgtc atgtttttca aatgtccttc cccagagtct gctagtctgg 1021 aggaactccc agaggtctac ccagaaggac ctagttcctt accagcctcc ctttctctgt 1081 cagtggggac ctcatcagcc aagctggaag ccattaatga actcattcgt tttgaccatg 1141 tatacaccaa gcctctagtt ttagagatcc cctctgagac agagagtcaa actaacgtgg 1201 tagtgaaaat tgaggaagca cctctaagct cttcagaaga ggatcaccct gaattcattg 1261 tctcagtgaa gaaagagcct ttggaagatg acttcatccc agagctgggc atctcaaacc 1321 tgctttcatc cagccattgt ctgagaccac cttcttgcct gctggacgct cacagtgact 1381 gtggatatga gggctcccct tctcccttca gtgacatgtc ttctccactt ggtacagacc 1441 actcctggga ggatactttt gccaatgaac ttttccccca gctgattagt gtctaaagag 1501 ccacataaca ctgggcccct ttccctgacc atcacattgc ctagaggata gcataggcct 1561 gtctctttcg ttaaaagcca aagtagaggc tgtctggcct tagaagaatt cctctaaagt 1621 atttcaaatc tcatagatga cttccaagta ttgtcgtttg acactcagct gtctaaggta 1681 ttcaaaggta ttccagtact acagcttttg agattctagt ttatcttaaa ggtggtagta 1741 tactctaaat cgcagggagg gtcatttgac agttttttcc cagcctggct tcaaactatg 1801 tagccgaggc taggcagaaa cttctgaccc tcttgacccc acctcccaag tgctgggctt 1861 caccaggtgt gcacctccac acctgccccc ccgacatgtc aggtggacat gggattcatg 1921 aatggccctt agcatttctt tctccactct ctgcttccca ggtttcgtaa cctgaggggg 1981 cttgttttcc cttatgtgca ttttaaatga agatcaagaa tctttgtaaa atgatgaaaa 2041 tttactatgt aaatgcttga tggatcttct tgctagtgta gcttctagaa ggtgctttct 2101 ccatttattt aaaactaccc ttgcaattaa aaaaaaagca acacagcgtc ctgttctgtg 2161 atttctaggg ctgttgtaat ttctctttat tgttggctaa aggagtaatt tatccaacta 2221 aagtgagcat accacttttt aaagtcaaaa aaaaaaaaaa aaaa

TABLE-US-00011 TABLE VIII Xbp1s (Human) ORIGIN    1 ggcgctgggc ggctgcggcg cgcggtgcgc ggtgcgtagt ctggagctat ggtggtggtg   61 gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc  121 gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga  181 ggggccagcc cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc  241 acgcacctga gccccgagga gaaggcgctg aggaggaaac tgaaaaacag agtagcagct  301 cagactgcca gagatcgaaa gaaggctcga atgagtgagc tggaacagca agtggtagat  361 ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat  421 ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct  481 gaagaggagg cggaagccaa ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc  541 gcagcaggtg caggcccagt tgtcacccct ccagaacatc tccccatgga ttctggcggt  601 attgactctt cagattcaga gtctgatatc ctgttgggca ttctggacaa cttggaccca  661 gtcatgttct tcaaatgccc ttccccagag cctgccagcc tggaggagct cccagaggtc  721 tacccagaag gacccagttc cttaccagcc tccctttctc tgtcagtggg gacgtcatca  781 gccaagctgg aagccattaa tgaactaatt cgttttgacc acatatatac caagccccta  841 gtcttagaga taccctctga gacagagagc caagctaatg tggtagtgaa aatcgaggaa  901 gcacctctca gcccctcaga gaatgatcac cctgaattca ttgtctcagt gaaggaagaa  961 cctgtagaag atgacctcgt tccggagctg ggtatctcaa atctgctttc atccagccac 1021 tgcccaaagc catcttcctg cctactggat gcttacagtg actgtggata cgggggttcc 1081 ctttccccat tcagtgacat gtcctctctg cttggtgtaa accattcttg ggaggacact 1141 tttgccaatg aactctttcc ccagctgatt agtgtctaag gaatgatcca atactgttgc 1201 ccttttcctt gactattaca ctgcctggag gatagcagag aagcctgtct gtacttcatt 1261 caaaaagcca aaatagagag tatacagtcc tagagaattc ctctatttgt tcagatctca 1321 tagatgaccc ccaggtattg tcttttgaca tccagcagtc caaggtattg agacatatta 1381 ctggaagtaa gaaatattac tataattgag aactacagct tttaagattg tacttttatc 1441 ttaaaagggt ggtagttttc cctaaaatac ttattatgta agggtcatta gacaaatgtc 1501 ttgaagtaga catggaattt atgaatggtt ctttatcatt tctcttcccc ctttttggca 1561 tcctggcttg cctccagttt taggtccttt agtttgcttc tgtaagcaac gggaacacct 1621 gctgaggggg ctctttccct catgtatact tcaagtaaga tcaagaatct tttgtgaaat 1681 tatagaaatt tactatgtaa atgcttgatg gaattttttc ctgctagtgt agcttctgaa 1741 aggtgctttc tccatttatt taaaactacc catgcaatta aaaggtacaa tgcaaaaaaa 1801 aaaaaaaaaa

TABLE-US-00012 TABLE IX Xbp1u (Human) ORIGIN    1 ggcgctgggc ggctgcggcg cgcggtgcgc ggtgcgtagt ctggagctat ggtggtggtg   61 gcagccgcgc cgaacccggc cgacgggacc cctaaagttc tgcttctgtc ggggcagccc  121 gcctccgccg ccggagcccc ggccggccag gccctgccgc tcatggtgcc agcccagaga  181 ggggccagcc cggaggcagc gagcgggggg ctgccccagg cgcgcaagcg acagcgcctc  241 acgcacctga gccccgagga gaaggcgctg aggaggaaac tgaaaaacag agtagcagct  301 cagactgcca gagatcgaaa gaaggctcga atgagtgagc tggaacagca agtggtagat  361 ttagaagaag agaaccaaaa acttttgcta gaaaatcagc ttttacgaga gaaaactcat  421 ggccttgtag ttgagaacca ggagttaaga cagcgcttgg ggatggatgc cctggttgct  481 gaagaggagg cggaagccaa ggggaatgaa gtgaggccag tggccgggtc tgctgagtcc  541 gcagcactca gactacgtgc acctctgcag caggtgcagg cccagttgtc acccctccag  601 aacatctccc catggattct ggcggtattg actcttcaga ttcagagtct gatatcctgt  661 tgggcattct ggacaacttg gacccagtca tgttcttcaa atgcccttcc ccagagcctg  721 ccagcctgga ggagctccca gaggtctacc cagaaggacc cagttcctta ccagcctccc  781 tttctctgtc agtggggacg tcatcagcca agctggaagc cattaatgaa ctaattcgtt  841 ttgaccacat atataccaag cccctagtct tagagatacc ctctgagaca gagagccaag  901 ctaatgtggt agtgaaaatc gaggaagcac ctctcagccc ctcagagaat gatcaccctg  961 aattcattgt ctcagtgaag gaagaacctg tagaagatga cctcgttccg gagctgggta 1021 tctcaaatct gctttcatcc agccactgcc caaagccatc ttcctgccta ctggatgctt 1081 acagtgactg tggatacggg ggttcccttt ccccattcag tgacatgtcc tctctgcttg 1141 gtgtaaacca ttcttgggag gacacttttg ccaatgaact ctttccccag ctgattagtg 1201 tctaaggaat gatccaatac tgttgccctt ttccttgact attacactgc ctggaggata 1261 gcagagaagc ctgtctgtac ttcattcaaa aagccaaaat agagagtata cagtcctaga 1321 gaattcctct atttgttcag atctcataga tgacccccag gtattgtctt ttgacatcca 1381 gcagtccaag gtattgagac atattactgg aagtaagaaa tattactata attgagaact 1441 acagctttta agattgtact tttatcttaa aagggtggta gttttcccta aaatacttat 1501 tatgtaaggg tcattagaca aatgtcttga agtagacatg gaatttatga atggttcttt 1561 atcatttctc ttcccccttt ttggcatcct ggcttgcctc cagttttagg tcctttagtt 1621 tgcttctgta agcaacggga acacctgctg agggggctct ttccctcatg tatacttcaa 1681 gtaagatcaa gaatcttttg tgaaattata gaaatttact atgtaaatgc ttgatggaat 1741 tttttcctgc tagtgtagct tctgaaaggt gctttctcca tttatttaaa actacccatg 1801 caattaaaag gtacaatgca

TABLE-US-00013 TABLE X Restriction map of AAV-PGK1-HA-Xbp-1(human)-WPRE- ggcggatccaattgcctaggcccaagggcgaattgtcacgactccacccc tccaggaacccctagtgatggagttggccactccctctctgcgcgctcg ctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttg gtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcccag atctgatatcatcgatgaattcaagcttcagctgctcgagttctatagtg tcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagc cgcgttctaacgacaatatgtccatatggtgcactctcagtacaatctgc tctgatgccgcatagttaagccagccccgacacccgccaacacccgctga cgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccg aaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaa tgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaa atgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatg tatccgctcatgagacaataaccctgataaatgcttcaataatattgaaa aaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttt ttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaa gtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaact ggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgtt ttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcc cgtattgacgccgggcaagagcaactcggtcgccgcatacactattctca gaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaac cgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttggg aaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactact tactctagcttcccggcaacaattaatagactggatggaggcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgct gataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcact ggggccagatggtaagccctcccgtatcgtagttatctacacgacgggga gtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcc tcactgattaagcattggtaactgtcagaccaagtttactcatatatact ttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaaga tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctac cagcggtggtttgtttgccggatcaagagctaccaactctttttccgaag gtaactggcttcagcagagcgcagataccaaatactgtccttctagtgta gccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcagacgatagttaccggataaggcgcagcgg tcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac ctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgg aacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttt atagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctt tttacggttcctggccttttgctggccttttgccacatgttctttcctgc gttatcccctgattctgtggataaccgtattaccgcctttgagtgagctg ataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgag gaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcaggttaacctggcttatcgaaattaatacgactcact atagggagaccggcagatctgtccctctctgcgcgctcgctcgctcactg aggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcc tcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactag gggttccttgtagttaatgattaacccgccatgctacttatctacaattc gcccttcggacgcgtggcttcgaactaggcaattgcatgaagaatctgct tagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgc gttgacattgattattgactagttattaatagtaatcaattacggggtca ttagttcatagcccatatatggagttccgcgttacataacttacggtaaa tggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataa tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaa tgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgta tcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg cctggcattatgcccagtacatgaccttatgggactttcctacttggcag tacatctacgtattagtcatcgctattaccatggtgatgcggttttggca gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc tccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgg gactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcgg taggcgtgtacggtgggaggtctatataagcagagctctctggctaacta gagaacccactgcttactggcttatcgaaattaatacgactcactatagg gagacccaagctggctagcgtttaaacttaagcttcctggctatggtggt ggtggcagccgcgccgaacccggccgacgggacccctaaagttctgcttc tgtcggggcagcccgcctccgccgccggagccccggccggccaggccctg ccgctcatggtgccagcccagagaggggccagcccggaggcagcgagcgg ggggctgccccaggcgcgcaagcgacagcgcctcacgcacctgagccccg aggagaaggcgctgaggaggaaactgaaaaacagagtagcagctcagact gccagagatcgaaagaaggctcgaatgagtgagctggaacagcaagtggt agatttagaagaagagaaccaaaaacttttgctagaaaatcagcttttac gagagaaaactcatggccttgtagttgagaaccaggagttaagacagcgc ttggggatggatgccctggttgctgaagaggaggcggaagccaaggggaa tgaagtgaggccagtggccgggtctgctgagtccgcagcaggtgcaggcc cagttgtcacccctccagaacatctccccatggattctggcggtattgac tcttcagattcagagtctgatatcctgttgggcattctggacaacttgga cccagtcatgttcttcaaatgcccttccccagagcctgccagcctggagg agctcccagaggtctacccagaaggacccagttccttaccagcctccctt tctctgtcagtggggacgtcatcagccaagctggaagccattaatgaact aattcgttttgaccacatatataccaagcccctagtcttagagataccct ctgagacagagagccaagctaatgtggtagtgaaaatcgaggaagcacct ctcagcccctcagagaatgatcaccctgaattcattgtctcagtgaagga agaacctgtagaagatgacctcgttccggagctgggtatctcaaatctgc tttcatccagccactgcccaaagccatcttcctgcctactggatgcttac agtgactgtggatacgggggttccctttccccattcagtgacatgtcctc tctgcttggtgtaaaccattcttgggaggacacttttgccaatgaactct ttccccagctgattagtgtctacccatacgatgttccagattacgcaatg taaagagccacataacactgggcccctttccctgaccatcacattgccta gaggatagcataggcctgaagggcgaattccagcacactggcggccgtta ctagagggcccgtttaaacccgctgatcacctcgactgtgccttctagtt gccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgca ttgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca gcaagggggaggattgggaagacaatagcaggcatgcctgcagcggtccg gtcgactctagaggatccgaaaaaacctcccacacctccccctgaacctg aaacataaaatgaatgcaattgttgttgttaacttgtttattgcagctta taatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat ttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatct tatcatgtctggatccccgcggtggcggccgcactagtcccgggttaatt aagctagcagatcttgatcacctaggcgtacgatttggccgctttacatg gtggcgaccggggatcctctagtaccaagctaattcctcacgacacctga aatggaagaaaaaaactttgaaccactgtctgaggcttgagaatgaacca agatccaaactcaaaaagggcaaattccaaggagaattacatcaagtgcc aagctggcctaacttcagtctccacccactcagtgtggggaaactccatc gcataaaacccctccccccaacctaaagacgacgtactccaaaagctcga gaactaatcgaggtgcctggacggcgcccggtactccgtggagtcacatg aagcgacggctgaggacggaaaggcccttttcctttgtgtgggtgactca cccgcccgctctcccgagcgccgcgtcctccattttgagctccctgcagc agggccgggaagcggccatctttccgctcacgcaactggtgccgaccggg ccagccttgccgcccagggcggggcgatacacggcggcgcgaggccaggc accagagcaggccggccagcttgagactacccccgtccgattctcggtgg ccgcgctcgcaggccccgcctcgccgaacatgtgcgctgggacgcacggg ccccgtcgccgcccgcggccccaaaaaccgaaataccagtgtgcagatct tggcccgcatttacaagactatcttgccagaaaaaaagcgtcgcagcagg tcatcaaaaattttaaatggctagagacttatcgaaagcagcgagacagg cgcgaaggtgccaccagattcgcacgcggcggccccagcgcccaggccag gcctcaactcaagcacgaggcgaaggggctccttaagcgcaaggcctcga actctcccacccacttccaacccgaagctcgggatcaagaatcacgtact gcagccaggtggaagtaattcaaggcacgcaagggccataacccgtaaag aggccaggcccgcgggaaccacacacggcacttacctgtgttctggcggc aaacccgttgcgaaaaagaacgttcacggcgactactgcacttatatacg gttctcccccaccctcgggaaaaaggcggagccagtacacgacatcactt tcccagtttaccccgcgccaccttctctaggcaccgg Features: XBP1s: [3257:5917-CW] cdd XBP1s: [3593:4720-CW] HA: [4721:4750-CW] L-ITR: [89:196-CW] R-ITR: [2629:2736-CW] T7: [2587:2606-CW] T7: [2532:3551-CW] SP6: [259:242-CCW] ColE1 origin: [1719:2347-CW] Amp: [908:1567-CW] SV40 late polyA: [5311:5120-CCW] Amp prom: [640:668-CW] HA tag: [4721:4747-CW]