CN113755447A - 293A cell strain for producing adenovirus and preparation and application thereof - Google Patents

Abstract

The invention relates to the technical field of cell engineering, in particular to a 293A cell strain for producing adenovirus and preparation and application thereof. The cell is obtained by inserting an exogenous fragment containing LoxM2, HPRT intron and Hygro cassette into the position of Ad5 gene in 293A cell genome. The replicative adenovirus genome generated by the cell of the invention can not form complete adenovirus particles because the genome exceeds the packaging limit of the adenovirus particles, thereby solving the problems of RCA and HDEP pollution in the adenovirus production process.

Description

293A cell strain for producing adenovirus and preparation and application thereof

Technical Field

The invention relates to the technical field of cell engineering, in particular to a 293A cell strain for producing adenovirus and preparation and application thereof.

Background

Replication-defective adenoviruses have wide applications in gene medicine, vaccine preparation, oncolytic adenoviruses and the like.

Most of the replication-defective adenoviruses currently used are serotype 5 adenoviruses in which the E1 region and the E3 region are deleted. Since the E1 region is an essential region for adenovirus replication, replication-defective adenoviruses must proliferate in a complementing cell line containing the E1 region. The most widely used replication-defective adenovirus packaging cell is 293, which comprises sequences of 1-4344 bp in Ad5 genome, including inverted repeat sequence (ITR) at 5' end, packaging signal, E1a, E1b gene and pIX coding sequence. However, Replication-deficient adenoviruses overlap with the adenovirus genome fragment in 293 cells, and when homologous recombination occurs in the cells, the Replication-deficient adenoviruses can be converted into Replication-competent adenoviruses (RCA) by recovering the E1 region. The probability of RCA appearance gradually increases with the number of passages. RCA is a pollutant in the replication-defective adenovirus amplification process, and has potential safety problem when being used in human body, so RCA detection is necessary for replication-defective adenovirus virus medicine used in clinic.

The approach to solve this problem is mainly to construct and modify cell lines in two directions: (1) deletion regions are added when constructing adenoviral vectors, such as deletion of multiple regions essential for replication, e.g., region E2 and/or region E4. Replication-defective adenoviruses deleted for multiple fragments need to be recombined for multiple times to regain replication capacity, so that the occurrence probability of RCA is reduced and the capacity of a vector is increased, but with the increase of deletion regions, the difficulty in constructing a stable cell line for replication is increased, and the virus titer may be reduced with the increase of the deletion regions; (2) the cell line is modified, namely the Ad5 DNA sequence in the 293 cell line is deleted from the homologous region of the adenovirus vector, the homologous sequence is reduced as much as possible, and the RCA is reduced or eliminated, for example, the cell line PER.C6 widely applied to adenovirus packaging at present, although the effect of preventing the RCA is achieved by greatly reducing the homologous part between the cell genome and the adenovirus vector. However, 177bp homologous portions still exist in the vector used for matching with PER.C. 6 cells and PER.C. 6 cells, and helper-dependent E1-containing particles (HDEP) are generated by unconventional homologous recombination with a very low probability. HDEP contains the E1 region, and in the presence of an adenoviral vector or a wild-type adenovirus, a replication-competent recombinant adenovirus product may be produced, with potential risk.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a 293A cell strain for producing adenovirus and preparation and application thereof, and can solve the problems of RCA and HDEP pollution in the production process of adenovirus.

In order to achieve the purpose, the invention adopts the technical scheme that: a cell obtained by inserting an exogenous fragment comprising LoxM2, HPRT intron and Hygro cassette at the position of Ad5 gene (NCBI number: AY339865.1 of Ad5 virus) in the genome of 293A cells is provided. In the presence of Cre enzyme, LoxM2 can be inserted into other genes in a targeted manner, so that convenience is brought to further modification of cell strains. HPRT intron is an intron of the human gene, which can also be replaced by other suitable introns. Hygro cassette is used to provide a selection marker during cell construction.

The replicative adenovirus genome generated by the cell of the invention cannot form complete adenovirus particles because the genome exceeds the packaging limit of the adenovirus particles, thereby solving the problem of RCA and HDEP pollution in the adenovirus production process.

As a preferred embodiment of the cell of the present invention, the nucleotide sequence of LoxM2 is shown in SEQ ID NO.1, the nucleotide sequence of HPRT intron is shown in SEQ ID NO.2, and the nucleotide sequence of Hygro cassette is shown in SEQ ID NO. 3.

As a preferred embodiment of the cell of the present invention, the nucleotide sequence of the exogenous fragment is shown in SEQ ID NO. 5.

As a preferred embodiment of the cell of the present invention, the insertion site is 3522bp of Ad5 gene. This insertion site is chosen in the present invention because it is located at the intron of the Ad 5E 1B gene, and insertion of the foreign gene does not affect the normal expression of the gene, but if recombination occurs, the foreign fragment will be contained in the viral genome such that the genome exceeds the packaging limit and cannot form a complete particle.

The invention also provides a construction method of the cell, which is characterized in that an exogenous fragment containing LoxM2, HPRT intron and Hygro cassette is inserted into the position of the Ad5 gene in the 293A cell genome.

As a preferred embodiment of the construction method, the construction method is to use a non-homology-dependent targeted integration method to make the genome of 293A cells and plasmids carrying the exogenous fragments form DNA double-strand breaks by using a CRISPR/Cas9 system, and insert the linearized exogenous fragments into targeted sites of the cell genome by using a non-homology end-linked DNA repair pathway, so as to form targeted integration.

Non-homology dependent targeted integration (or HITI) dsb (double strand break) is formed by using CRISPR/Cas9 system for both genome and plasmid carrying exogenous fragment, and the linearized exogenous fragment is inserted into the targeted site of the cell genome by using DNA repair pathway of Non-homologous end joining (NHEJ) to form targeted integration. When the method mediates the site-specific insertion of the exogenous fragment into the cell genome, compared with the terminal-dependent homologous recombination HDR (the homologous-directed repair), the method can simultaneously edit the divided cell and the non-divided cell, has higher editing efficiency and lower insertion and deletion probability, and has wide application prospect. While most of the current methods for constructing such cell lines insert fragments into cells according to HDR homologous recombination, such targeted insertion methods are inefficient.

A293A cell strain which does not generate RCA and HDEP in the process of producing the replication-defective adenovirus vector is constructed by using the HITI method and is named as VB-293A. This cell line inserted an approximately 9Kb foreign fragment comprising LoxM2, HPRT intron and Hygro cassette at a position of 3522bp of the 293A Ad5 genome. When homologous recombination occurs, the genome of the generated replicative adenovirus cannot form a complete adenovirus particle because the genome exceeds the packaging limit of the adenovirus particle, so that the problem of RCA and HDEP pollution in the adenovirus production process is solved.

As a preferred embodiment of the construction method of the present invention, the plasmid carrying the exogenous fragment is pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA 2.

The invention provides a method for packaging adenovirus by using the cell, which is to infect the cell by adenovirus with deletion of E1 gene.

The invention also provides the application of the cell in preparing adenovirus.

The invention has the beneficial effects that:

the 293A cell strain for producing the non-RCA adenovirus is characterized in that an exogenous segment of about 9Kb is inserted into an intron region of E1b, and when a defective adenovirus vector is recombined in a cell, a genome exceeding the packaging limit of adenovirus particles is generated, so that the cell strain can be used for producing the adenovirus vector, the RCA and HDEP can be prevented from being generated, and the safety of the adenovirus vector is improved.

Drawings

FIG. 1: plasmid map of pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA 2.

FIG. 2: plasmid map of pRP [ CRISPR ] -EGFP/Puro-hCas9-U6> {293A/Ad-3522nt }.

FIG. 3: sequence map of the engineered 293A near the target site of insertion (including the insertion sequence).

FIG. 4: plasmid map of pAV [ Exp ] -CMV > EGFP.

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

The sequence related by the invention is as follows:

SEQ ID NO. 1: the nucleotide sequence of LoxM 2;

SEQ ID NO. 2: an HPTR intron nucleotide sequence;

SEQ ID NO. 3: a hygro cassette nucleotide sequence;

SEQ ID NO. 4: a gRNA recognition sequence;

SEQ ID No. 5: exogenous fragments (the exogenous fragments are obtained by connecting 3 gene fragments in sequence and do not contain gRNA recognition sequences);

SEQ ID NO. 6: hCas9 nucleotide sequence;

SEQ ID NO. 7: a gRNA transcription cassette;

SEQ ID NO. 8: a Stuffer-LA-F (60) primer sequence;

SEQ ID NO. 9: a Stuffer-LA-R (60) primer sequence;

SEQ ID NO. 10: a Stuffer-RA-F (60) primer sequence;

SEQ ID NO. 11: a Stuffer-RA-R (60) primer sequence;

SEQ ID NO. 12: Hygro-F (60) primer sequence;

SEQ ID NO. 13: Hygro-R (60) primer sequence.

EXAMPLE 1 obtaining of exogenous fragments

Respectively synthesizing gene fragments LoxM2 (the nucleotide sequence is shown as SEQ ID NO. 1), HPRT intron (the nucleotide sequence is shown as SEQ ID NO. 2) and hygro cassette (the nucleotide sequence is shown as SEQ ID NO. 3), sequentially connecting the 3 gene fragments to obtain an exogenous fragment (the nucleotide sequence is shown as SEQ ID NO. 5), and respectively connecting gRNA recognition sequences (the nucleotide sequences are shown as SEQ ID NO. 4) to two ends of the exogenous fragment to obtain the exogenous fragment containing the gRNA recognition sequences.

EXAMPLE 2 construction of recombinant plasmid

(1) Construction of plasmid pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA2

The foreign fragment containing the recognition sequence of gRNA obtained in example 1 was cloned into the plasmid pDOwn-SapI-ccdB-Chl-SapI to obtain the recombinant plasmid pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA2, and the recombinant plasmid map is shown in FIG. 1.

(2) Construction of hCas9 and gRNA-carrying plasmid

Synthesizing a gene fragment hCas9 (nucleotide sequence is shown as SEQ ID NO. 6) and a gRNA transcription frame (nucleotide sequence is shown as SEQ ID NO. 7), cloning the gRNA sequence into a plasmid PX330-U6-BbsI-CBh-hSpCas9-CMV > EGFP/Puro to obtain a recombinant plasmid pRP [ CRISPR ] -EGFP/Puro-hCas9-U6> {293A/Ad-3522nt }, wherein the map of the recombinant plasmid is shown as figure 2.

EXAMPLE 3 construction of VB-293A cell line

By 3X 10⁶And (3) paving 6 pore plates on each cell, and ensuring that the confluency of the cells reaches 80-90% every other day. The two plasmids constructed in example 2 were co-transfected into 293A cells (thermo Fisher Cat.: R70507) using Lipofect amine 3000 transfection reagent. According to the principle of NHEJ, hCas9/gRNA will simultaneously recognize and cleave the target site of the gene and the two target sites of plasmid pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA 2. The linearized exogenous fragment is inserted into a targeted site of the cell genome, thereby forming targeted integration. The total amount of plasmid transfected was 2.5. mu.g, plasmid pRP [ CRISPR ]]-EGFP/Puro-hCas9-U6>{293A/Ad-3522nt }: 1.7 mu g; plasmid pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA 2: 0.8 mu g; lipofectamine 3000: 7.5. mu.L. The liquid is changed every other day.

And (3) passaging the cells after 48 hours of transfection in a 6-well plate, changing the culture medium by using 150 mu g of Hygro +2.5 mu g of puro + 10% FBS + DMEM drug sieve every other day, carrying out drug sieve for 7 days, and enriching the positive cells by using a Hygro and Pure double drug sieve. After drug screening, extracting cell genome and carrying out PCR amplification by using three pairs of primers (Stuffer-LA-F (60)/Stuffer-LA-R (60), Stuffer-RA-F (60)/Stuffer-RA-R (60) and Hygro-F (60)/Hygro-R (60)), wherein the sequences of the three pairs of primers are shown in Table 1, and the three pairs of primers can be respectively amplified to obtain the partial sequences of the left arm, the right arm and the insert fragment of the integration site. The PCR product was sent to sequencing for validation of the insert sequence. And if the PCR results of the three pairs of primers are positive and the sequencing result is correct, the exogenous fragment is inserted into the target site.

After digesting the cells with the correct sequencing results from the six-well plate, the cells were diluted to 20cells/mL with a gradient of 150. mu.g Hygro + 10% FBS + DMEM, plated in 2 96-well plates at 100. mu.L per well, and the wells with only one cell were observed and labeled under a microscope. After 20 days, cells were plated on 12-well plates after they had grown to capacity in 96-well plates. After the cells grow over a 12-well plate, the cells are transferred to a 6-well plate, the cells are taken and respectively subjected to PCR amplification by using three pairs of primers (Stuffer-LA-F (60)/Stuffer-LA-R (60), Stuffer-RA-F (60)/Stuffer-RA-R (60), Hygro-F (60)/Hygro-R (60)), PCR amplification products are sent to sequencing, and whether a cell monoclonal target site is inserted into an exogenous sequence or not is preliminarily verified. And (3) respectively sequencing and comparing the partial sequences of the left arm, the right arm and the inserted fragment of the integration site by three pairs of primers, further amplifying and culturing the cell monoclonal with correct sequencing, extracting a genome group to test the inserted exogenous sequence and the nearby E1b gene, and confirming that the inserted fragment and the nearby sequence have no mutation. The obtained monoclonal cell line with correct sequencing was designated VB-293A, and the sequence map of the cell near the inserted target site is shown in FIG. 3.

TABLE 1 primer names and sequences

Primer name	Sequence of
		Stuffer-LA-F(60)	GTGAGGCTACAGAGGAGGCTAGGAATCT
Stuffer-LA-R(60)	AAGGGTCGTCCGCAGGATTCAG
		Stuffer-RA-F(60)	GTCTGGACCGATGGCTGTGTAGAAGT
Stuffer-RA-R(60)	GAAATGGTGAGAGCCATCCACACAA
		Hygro-F(60)	CAAGACCTGCCTGAAACCGAACTG
Hygro-R(60)	AAGCATCAGCTCATCGAGAGCCTG

TABLE 2 sequencing primer names and sequences

Primer name	Sequence of
		HPRT-F1	TTGATTCTTTGAATCTGGGTCACC
HPRT-F2	gccgaggtggagatagatacg
		HPRT-F3	aaggtgctgaggtacgatgag
HPRT-F4	tctcttgttcatgtcctactgttc
		HPRT intron-F1	tttaagggcaggaatcaccg
HPRT intron-F2	gattctcccacttcagcctccc
		HPRT intron-F3	aagtaacttgcccaagattaccc
HPRT intron-R3	TCTTCTCTGCTGATCTGGTTGG
		HPRT intron-F4	ccaaccagatcagcagagaa
HPRT intron-F5	tgcccattccagaagtttg
		HPRT intro-F9	AGCAGTCCTTGTCACAGATGATG
HPRT intro-R1	GAAGTAAGTTGCCCACAGCCACAC
		HPRT intro-R4	CACAGTTTTGGAGGCTGGGAA
HPRT intro-R4R-R	AGCCTCTGAGCCCAGAAAGC
		hygro-R2	CTCGGCCCAAAGCATCAGC
Stuffer-RA-R2(60)	GCTTCCATCAAACGAGTTGGTGCT

Example 4 detection of the ability of VB-293A cells to Encapsulate

The detection of the virus-encapsulating capacity of the VB-293A cell comprises the following steps:

(1) after shaking overnight, the plasmid pAV [ Exp ] -CMV > EGFP (vector fruit der product No.: VB150925-10024) was extracted and the plasmid map was as shown in FIG. 4. The plasmid was digested with PacI and cut into two fragments: 2074bp, 33123bp, run glue to confirm complete digestion. The digested product was heated at 65 ℃ for 20min to inactivate the PacI enzyme.

(2) And (2) spreading a VB-293A monoclonal cell strain and a 293A cell strain (control) which are constructed in the example 3 on a 6-well plate respectively, and directly taking the enzyme-cutting inactivation product (containing Ad5 fragment) obtained in the step (1) to transfect the cells when the cells grow to 80% -90%. The DNA dose was 2.5. mu.g, and VB-293A and 293A cells were transfected with 7.5. mu.L of Lipofectamine 3000. The liquid is changed every other day, and the observation is continued for 10 days until the occurrence of comet-like spots. Liquid is changed after the comet-shaped spots appear until 90% of cells are diseased, and toxicity is collected. The cells and supernatant were collected and gently blown with a pipette to drop off the cells. Freeze-thaw was repeated three times at 37 deg.c/liquid nitrogen to lyse cells and release virus. Centrifuge at 2000g for 10min at 4 ℃. The virus-containing supernatants were aliquoted and frozen at-80 ℃.

(3) Recovering and expanding A549 cells, digesting the A549 cells, resuspending the A549 cells by using 10% FBS + DMEM, and diluting the A549 cells into 4 x 10 cells by using 270mL 10% FBS + DMEM after resuspension⁵cells/mL of cell suspension were plated in 12-well plates, 1mL per well, in a total of 21 plates in 12-well plates. The 12-well plate was placed in a 37 ℃ incubator overnight. Removing 12-well plate stock culture solution every other day, packaging and amplifying VB-293A monoclonal cell strain and 293A cell 3 × 10¹¹VP adenovirus was diluted in 120mL 10% FBS + DMEM and added to 12 well plates at 1mL per well. Each sample was infected separately in 10 12 well plates. One 12-well plate was set up and 10% FBS + DMEM was added as a negative control. The 12-well plate was incubated in an incubator at 37 ℃ for 14 days. Fluid replacement was performed on day 7, adding 1mL 10% FBS + DMEM per well. After 14 days, the number of wells showing CPE (cytopathic effect) in 10 12-well plates packed with the amplified adenovirus using VB-293A monoclonal cell line and 293A cells, respectively, was observed, while the negative control well plate showed no CPE.

TABLE 3 detection of the cytotoxic potency of VB-293A cells

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Yun boat Biotechnology (Guangzhou) Ltd

<120> 293A cell strain for producing adenovirus and preparation and application thereof

<130> 2021.9.23

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 34

<212> DNA

<213> Artificial sequence

<400> 1

ataacttcgt ataagaaacc atatacgaag ttat 34

<210> 2

<211> 6325

<212> DNA

<213> Artificial sequence

<400> 2

ggaaaagagg actgcgtgtg ggaagagaag gtggaaatgg cgttttggtt gacatgtgcc 60

gcctgcgagc gtgctgcggg gaggggccga gggcagattc gggaatgatg gcgcggggtg 120

ggggcgtggg ggctttctcg ggagaggccc ttccctggaa gtttggggtg cgatggtgag 180

gttctcgggg cacctctgga ggggcctcgg cacggaaagc gaccacctgg gagggcgtgt 240

ggggaccagg ttttgccttt agttttgcac acactgtagt tcatctttat ggagatgctc 300

atggcctcat tgaagcccca ctacagctct ggtagcggta accatgcgta tttgacacac 360

gaaggaacta gggaaaaggc attaggtcat ttcaagccga aattcacatg tgctagaatc 420

cagattccat gctgaccgat gccccaggat atagaaaatg agaatctggt ccttaccttc 480

aagaacattc ttaaccgtaa tcagcctctg gtatcttagc tccaccctca ctggtttttt 540

cttgtttgtt gaaccggcca agctgctggc ctccctcctc aaccgttctg atcatgcttg 600

ctaaaatagt caaaaccccg gccagttaaa tatgctttag cctgctttat tatgattatt 660

tttgttgttt tggcaatgac ctggttacct gttgtttctc ccactaaaac tttttaaggg 720

caggaatcac cgccgtaact ctagcactta gcacagtact tggcttgtaa gaggtcctcg 780

atgatggttt gttgaatgaa tacattaaat aattaaccac ttgaacccta agaaagaagc 840

gattctattt catattaggc attgtaatga cttaaggtaa agagcagtgc tattaacgga 900

gtctaactgg gaatccagct tgtttgggct atttactagt tgtgtggctg tgggcaactt 960

acttcacctc tctgggctta agtcatttta tgtatatctg aggtgctggc tacctcttgg 1020

agttattgag aggattataa gacagtctat gtgaatcagc aacccttgca tggcccctgg 1080

cggggaacag taataatagc catcatcatg tttacttaca tagtcctaat tagtcttcaa 1140

aacagccctg tagcaatggt atgattatta ccattttaca gatgaggaac ctttgaagcc 1200

tcagagaggc taacagacat accctaggtc atacagttat taagagaagg agctctgtct 1260

cgaacctagc tctctctctc tcgagtaata ccagttaaaa aataggctac aaataggtac 1320

tcaaaaaaat ggtagtggct gttgttttta ttcagttgct gaggaaaaaa tgttgatttt 1380

tcatctctaa acatcaactt acttaattct gccaatttct tttttttgag acagggtctc 1440

actctgtcac ctaggatgga gtgcagtggc acaatcactg ctcactgcag cctcgacttc 1500

ccgggctcgg gtgattctcc ccaggctcag gggattctcc cacttcagcc tcccaagtag 1560

ctgggactac aggtgcgcac caccatccct ggctaatatt tgtactttat tttatttatt 1620

tatttattta ttttttgaga tggagtttcg ctcttgttgc ccgggctgga gtacagtggc 1680

atgatctcgg ctcagtgcaa cctctgcctc ccgggttcaa gcgattctcc tacctcatcc 1740

ccctgagtag ctgggattac aggcgcctgc caccatgcct ggctaatttt ttgtattttt 1800

aatagagacg aggtttcacc atgttggcca ggctactctc gaactcctga tctcaggtga 1860

tccacccgcc ttggcctccc aaagtgctgg gattacaggc gtgagccact gcgcccggcc 1920

taatatttgt attttttgta gagatggtgt tttgccatgt tgtccaggct ggtcttgaac 1980

tcctgagctc aagcgatctg cccgcctctg cttcccaaag tgctgggatt acaggcatga 2040

gccaccgtgc ctggcctagg tagacgcttt tagctttggg gtgtgatgcc tgccccagta 2100

tatagtgaat ttaattattg ctagagctgg ctgtttgtta gttttctttg aacataagat 2160

actcattgtt tttagtttgc aaatccctct tcctttttaa aaaatttctt tcccttaaat 2220

tgtttgcatg ttagcaataa caaatgctta aatggtgcta tgtgctagat actcttctaa 2280

gccctgttat gtatattaac taatttttta aattacacaa atcagagagg ttaagtaact 2340

tgcccaagat tacccaacaa tactaggatt tgaacctaag tttgtctcac cccagattct 2400

gctcttaatc tctaaacttt taagttagta gtgacaatag taggtattta ttgaatactt 2460

aactatgttt taggcgttga agtaaatatt ttgcaggcat tatctaatgt aaacacccta 2520

aagttacata acaggtaccc tttaggtaaa taaacactag tatgaccttg gaggcacaga 2580

tagttgaagt aacttgccca atatcactta catgaaattg gccctcaaat gtgtctgata 2640

caacccatgc tgcttgtaac tatcgtttta aactgccagg gtaaacttgg acacacttga 2700

gctaagaaaa agcttttaga tttttgcaaa ttaatgtgaa agatatgctt tatgtggata 2760

taatatcttc taaatttcgg ggatggtagt cctagaaatg taatcctgcc ctagccgagc 2820

ttaccctgcc aataattttt tacagaattg gtaaaacgga gcaccttttt tttgtccttg 2880

gccacactgt tatcaacagg gtgtagattg acatcaatct gtaggtgtaa accagaatta 2940

ctctttgtga ccaccaggaa atagagcagt tcagttcagg ggtttctttc tgtgaattta 3000

gcactgtgac ctgcatacta caagtctact ttgttttcta tccattgttt gtatctgggt 3060

attgcaaaag gtaggaaaag gaccaaccag atcagcagag aagagttgcc ttggagtttt 3120

cttttagttt tctgcagttc attagatagt aactaggcca tgtcatttta ctcccttgta 3180

gtgaagatat gttgaagttg tactggtata ctcttctacc tttctgtaat tttatattgt 3240

gtagacttga taaaatttat gtgtcaatca ccaccattaa tatcaatatt gagcctcaat 3300

tcttattttt ctgcccagtg gctgccaaat tactaacatt tacaataatt cactactact 3360

aagataatct actagttcga tcacatactt caaattgtta tggaactact gtcttcagca 3420

ttgtgcttct gataactgat aagtataatt ttttttttgt ccagagtgaa catgtctatt 3480

cttccactgt acacactaat aaaaggaaaa attgtaatat tgggtaaatt catgtcctta 3540

cacatgtagt agttatgagc ccatgtccct agaatgagta ataatttatc cctcccttgg 3600

ttgaatagtc aagaatgctg attttaattc ttctaacagc tttatccctc agaagggaag 3660

gcaagcaagt tatatatgta gtttatttgt aagactgata tgaaattgga agatgaatct 3720

actattagct ttaattattt ttacatttag gaatattgca tcagtaactc ataattttgg 3780

ttttctgtta tcctgagtta acacaaatta tccaaggaga tggcggatca tctgctttga 3840

ggtgtttttt tttgagaatt ttaatgtatc tgaatataaa aggtaaaaat atgccaacta 3900

gcaatttctg cccattccag aagtttggaa atattactca ttactaggaa ttaaataaaa 3960

tatggtttat ctattgttat acctctttta attcacatag ctcattttta tcttttattt 4020

ttgtttgttt tttttgagat ggagtcttgc tctgtcacca ggcaggagtg cagtgatgca 4080

atctcggctc actctagcca ccgactccct ggttcaagcg attctcctgc ctgagccttc 4140

tgagtagctg ggattacagg caggcaccac cacgcccagc taatttttgt agagacagga 4200

tttcaccgtg ttggccagga tggtctccat ctcctgacct catgatctgc ctgcttcggc 4260

ctcccaaagt gctgggatta caggtgggag ccactacgcc tggcccacat agctcatttt 4320

tagactcact tccattaagt cttgtttgga cccacgaaca ttgtcttttt ttttttaaga 4380

tggagtttca cttttgttgc ccagactgta gtgcaatggt gcaatctcag ctcactgcaa 4440

tctctgcctc ctgggttcta gcaattctcc tgcctcagcc tcccgagtag ctggaattac 4500

aggcgcccgc caccacgccc agctaatttt tgtgttttta gtagagacgg ggtttcacca 4560

tgttgggcag gccaggggtg atccgcccac ctcagcctcc caaagtgctg ggattacagg 4620

tgtgagccac cgcatctggc caacatgtct tttttttttt tttccttttt aaccacaaag 4680

agacttaagc agtccttgtc acagatgatg aattgatgtt gcaagtattg tcttagcttg 4740

gattaatttt cttgcttact gtaattttag ataatatagc tttgtaatta gagattttat 4800

gtgtaaacca caaaaatgtt tacatgaagg ccattattac agatgtgacg tgcataatta 4860

ttagtaattt gtatgtttac atgggtcagt ctggcaaaaa attatgaagt tttaaaaatt 4920

aaaaaaaatt ataatgccag ttttactgga aagtaaaatt atttcagtaa tcgattatag 4980

caaaagtatt gattttcatt ccagacaaaa gtcagaatga aaggtaattt ctcaatactc 5040

tttcagatta ataaaagtac ctgtagcgat ttttatcatt cacaagtata tcacaagtaa 5100

gttagaattt gagaactgtg ttctagatct ctgaggagat gcagtcagat ttctgaactg 5160

tctcagcaaa tggtaagtaa cttagagcta gtaattaata acctgtcctt tgatttctga 5220

ttcagccaag aatggccata tttgggaaag gcagatctgg agagtaacca cgttttcatt 5280

catttaccac ttctaggccc ctccagagct ctcagatatt ttggggttga gcccttcccc 5340

aaagccatac aggacctttt ttttgtgatc tgttctagcc atttttatgt tgggtgcttg 5400

ttatggactg agcatttatg tcctcccaca ccccccccat accttttttg aagtcctaac 5460

ccccagtgtg atggtatttg gagacagggc ctttggaagg taattacagt tagaagaagt 5520

cgggagggtt gggcccaggt ctgattggat tagtgccctt atatgaaaag acaccaggac 5580

gggcgcagtg gctcacacct gtaatcccag cactttggga ggccaaggtg ggtggatcac 5640

gaggtcagga gtttgagacc agcctggcca atgtagtgaa acaccatctc tactaaaaat 5700

acaaaaatta gctgggtgtg gtagcgggct cctgtcatcc aagctactcg ggagggtgag 5760

gcatgagaat cacttgaacc cgggagttgg aggttgcagt gagcccagat tgtgccactg 5820

tactccagcc tgggtgacag agtgagactc tgtctcaaaa aagaaaaaaa aaaaaaaaga 5880

gacaccagag agcttgttag aagaggtcat gtgagcacac agttagaaga ccttcaagcc 5940

aaagaagagg cctgagattg aaacctacct tgcaggtacc ttaattttgg acttcccagc 6000

ctccaaaact gtgagaaata agtttctgtt aagtcactca gtctgtggta ttttgttatg 6060

gcagcctgag caggtagttg ttctttcaga aggtgttgat aataaccaca tgcaacacca 6120

agtcacaaat aataaaacag atgtaactta tattcataca gaaagttggg cactgccatt 6180

gccttgttgg tttacacggc tgtgctagtt cagtagcaga aaggtgctgg tctcctttac 6240

tcagtttaca atctaggcag tagaatgtaa tcactgcttt aaacttgata ctgcttaggg 6300

agagaatcat tggtgctggg taact 6325

<210> 3

<211> 1771

<212> DNA

<213> Artificial sequence

<400> 3

ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 60

ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc 120

ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc 180

ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa 240

gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta 300

ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg 360

ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga 420

aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc 480

tcctcttcct catctccggg cctttcgacc tcacgtggcc accatgaaaa agcctgaact 540

caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat 600

gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata 660

tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca 720

ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat ttagcgagag 780

cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac 840

cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga 900

tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac 960

atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat 1020

ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga 1080

ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac 1140

ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca 1200

atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac 1260

gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat 1320

gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc 1380

agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg 1440

tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc 1500

cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc agacatgata 1560

agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 1620

tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 1680

aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt 1740

taaagcaagt aaaacctcta caaatgtggt a 1771

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

cacacatttc agtacctcaa t 21

<210> 5

<211> 8794

<212> DNA

<213> Artificial sequence

<400> 5

catttcagta cctcaataaa cataacttcg tataagaaac catatacgaa gttattgcgc 60

gggacgtcct ttgtttacgt cccgtcggcg ctgaatcctg cggacgaccc ttctcggggt 120

cgcttgggac tctctcgtcc ccttctccgt ctgccgttcc gaccgaccac ggggcgcacc 180

tctctttacg cggactcccc gtctgtgcct tctcatctgc cggaccgtgt gcacttcgct 240

tcacctctgc acgtcgcatg gagaccaccg tgaacgccca ccaaatattg cccaaggtct 300

tacataagag gactcttgga ctctcagcaa tgtcaacgac cgaccttgag gcatacttca 360

aagactgttt gtttaaagac tgggaggagt tgggggagga gattaggtta aaggtctttg 420

tactaggagg ctgtaggcat aaattggtct gcgcaccagc accatgcaac tttttcacct 480

ctgcctaatc atctcttgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg 540

gctttggggc atggacatcg acccttataa agaatttgga gctactgtgg agttactctc 600

gtttttgcct tctgacttct ttccttcagt acgagatctt caagtttgta caaaaaagca 660

ggctggaaaa gaggactgcg tgtgggaaga gaaggtggaa atggcgtttt ggttgacatg 720

tgccgcctgc gagcgtgctg cggggagggg ccgagggcag attcgggaat gatggcgcgg 780

ggtgggggcg tgggggcttt ctcgggagag gcccttccct ggaagtttgg ggtgcgatgg 840

tgaggttctc ggggcacctc tggaggggcc tcggcacgga aagcgaccac ctgggagggc 900

gtgtggggac caggttttgc ctttagtttt gcacacactg tagttcatct ttatggagat 960

gctcatggcc tcattgaagc cccactacag ctctggtagc ggtaaccatg cgtatttgac 1020

acacgaagga actagggaaa aggcattagg tcatttcaag ccgaaattca catgtgctag 1080

aatccagatt ccatgctgac cgatgcccca ggatatagaa aatgagaatc tggtccttac 1140

cttcaagaac attcttaacc gtaatcagcc tctggtatct tagctccacc ctcactggtt 1200

ttttcttgtt tgttgaaccg gccaagctgc tggcctccct cctcaaccgt tctgatcatg 1260

cttgctaaaa tagtcaaaac cccggccagt taaatatgct ttagcctgct ttattatgat 1320

tatttttgtt gttttggcaa tgacctggtt acctgttgtt tctcccacta aaacttttta 1380

agggcaggaa tcaccgccgt aactctagca cttagcacag tacttggctt gtaagaggtc 1440

ctcgatgatg gtttgttgaa tgaatacatt aaataattaa ccacttgaac cctaagaaag 1500

aagcgattct atttcatatt aggcattgta atgacttaag gtaaagagca gtgctattaa 1560

cggagtctaa ctgggaatcc agcttgtttg ggctatttac tagttgtgtg gctgtgggca 1620

acttacttca cctctctggg cttaagtcat tttatgtata tctgaggtgc tggctacctc 1680

ttggagttat tgagaggatt ataagacagt ctatgtgaat cagcaaccct tgcatggccc 1740

ctggcgggga acagtaataa tagccatcat catgtttact tacatagtcc taattagtct 1800

tcaaaacagc cctgtagcaa tggtatgatt attaccattt tacagatgag gaacctttga 1860

agcctcagag aggctaacag acatacccta ggtcatacag ttattaagag aaggagctct 1920

gtctcgaacc tagctctctc tctctcgagt aataccagtt aaaaaatagg ctacaaatag 1980

gtactcaaaa aaatggtagt ggctgttgtt tttattcagt tgctgaggaa aaaatgttga 2040

tttttcatct ctaaacatca acttacttaa ttctgccaat ttcttttttt tgagacaggg 2100

tctcactctg tcacctagga tggagtgcag tggcacaatc actgctcact gcagcctcga 2160

cttcccgggc tcgggtgatt ctccccaggc tcaggggatt ctcccacttc agcctcccaa 2220

gtagctggga ctacaggtgc gcaccaccat ccctggctaa tatttgtact ttattttatt 2280

tatttattta tttatttttt gagatggagt ttcgctcttg ttgcccgggc tggagtacag 2340

tggcatgatc tcggctcagt gcaacctctg cctcccgggt tcaagcgatt ctcctacctc 2400

atccccctga gtagctggga ttacaggcgc ctgccaccat gcctggctaa ttttttgtat 2460

ttttaataga gacgaggttt caccatgttg gccaggctac tctcgaactc ctgatctcag 2520

gtgatccacc cgccttggcc tcccaaagtg ctgggattac aggcgtgagc cactgcgccc 2580

ggcctaatat ttgtattttt tgtagagatg gtgttttgcc atgttgtcca ggctggtctt 2640

gaactcctga gctcaagcga tctgcccgcc tctgcttccc aaagtgctgg gattacaggc 2700

atgagccacc gtgcctggcc taggtagacg cttttagctt tggggtgtga tgcctgcccc 2760

agtatatagt gaatttaatt attgctagag ctggctgttt gttagttttc tttgaacata 2820

agatactcat tgtttttagt ttgcaaatcc ctcttccttt ttaaaaaatt tctttccctt 2880

aaattgtttg catgttagca ataacaaatg cttaaatggt gctatgtgct agatactctt 2940

ctaagccctg ttatgtatat taactaattt tttaaattac acaaatcaga gaggttaagt 3000

aacttgccca agattaccca acaatactag gatttgaacc taagtttgtc tcaccccaga 3060

ttctgctctt aatctctaaa cttttaagtt agtagtgaca atagtaggta tttattgaat 3120

acttaactat gttttaggcg ttgaagtaaa tattttgcag gcattatcta atgtaaacac 3180

cctaaagtta cataacaggt accctttagg taaataaaca ctagtatgac cttggaggca 3240

cagatagttg aagtaacttg cccaatatca cttacatgaa attggccctc aaatgtgtct 3300

gatacaaccc atgctgcttg taactatcgt tttaaactgc cagggtaaac ttggacacac 3360

ttgagctaag aaaaagcttt tagatttttg caaattaatg tgaaagatat gctttatgtg 3420

gatataatat cttctaaatt tcggggatgg tagtcctaga aatgtaatcc tgccctagcc 3480

gagcttaccc tgccaataat tttttacaga attggtaaaa cggagcacct tttttttgtc 3540

cttggccaca ctgttatcaa cagggtgtag attgacatca atctgtaggt gtaaaccaga 3600

attactcttt gtgaccacca ggaaatagag cagttcagtt caggggtttc tttctgtgaa 3660

tttagcactg tgacctgcat actacaagtc tactttgttt tctatccatt gtttgtatct 3720

gggtattgca aaaggtagga aaaggaccaa ccagatcagc agagaagagt tgccttggag 3780

ttttctttta gttttctgca gttcattaga tagtaactag gccatgtcat tttactccct 3840

tgtagtgaag atatgttgaa gttgtactgg tatactcttc tacctttctg taattttata 3900

ttgtgtagac ttgataaaat ttatgtgtca atcaccacca ttaatatcaa tattgagcct 3960

caattcttat ttttctgccc agtggctgcc aaattactaa catttacaat aattcactac 4020

tactaagata atctactagt tcgatcacat acttcaaatt gttatggaac tactgtcttc 4080

agcattgtgc ttctgataac tgataagtat aatttttttt ttgtccagag tgaacatgtc 4140

tattcttcca ctgtacacac taataaaagg aaaaattgta atattgggta aattcatgtc 4200

cttacacatg tagtagttat gagcccatgt ccctagaatg agtaataatt tatccctccc 4260

ttggttgaat agtcaagaat gctgatttta attcttctaa cagctttatc cctcagaagg 4320

gaaggcaagc aagttatata tgtagtttat ttgtaagact gatatgaaat tggaagatga 4380

atctactatt agctttaatt atttttacat ttaggaatat tgcatcagta actcataatt 4440

ttggttttct gttatcctga gttaacacaa attatccaag gagatggcgg atcatctgct 4500

ttgaggtgtt tttttttgag aattttaatg tatctgaata taaaaggtaa aaatatgcca 4560

actagcaatt tctgcccatt ccagaagttt ggaaatatta ctcattacta ggaattaaat 4620

aaaatatggt ttatctattg ttatacctct tttaattcac atagctcatt tttatctttt 4680

atttttgttt gttttttttg agatggagtc ttgctctgtc accaggcagg agtgcagtga 4740

tgcaatctcg gctcactcta gccaccgact ccctggttca agcgattctc ctgcctgagc 4800

cttctgagta gctgggatta caggcaggca ccaccacgcc cagctaattt ttgtagagac 4860

aggatttcac cgtgttggcc aggatggtct ccatctcctg acctcatgat ctgcctgctt 4920

cggcctccca aagtgctggg attacaggtg ggagccacta cgcctggccc acatagctca 4980

tttttagact cacttccatt aagtcttgtt tggacccacg aacattgtct tttttttttt 5040

aagatggagt ttcacttttg ttgcccagac tgtagtgcaa tggtgcaatc tcagctcact 5100

gcaatctctg cctcctgggt tctagcaatt ctcctgcctc agcctcccga gtagctggaa 5160

ttacaggcgc ccgccaccac gcccagctaa tttttgtgtt tttagtagag acggggtttc 5220

accatgttgg gcaggccagg ggtgatccgc ccacctcagc ctcccaaagt gctgggatta 5280

caggtgtgag ccaccgcatc tggccaacat gtcttttttt tttttttcct ttttaaccac 5340

aaagagactt aagcagtcct tgtcacagat gatgaattga tgttgcaagt attgtcttag 5400

cttggattaa ttttcttgct tactgtaatt ttagataata tagctttgta attagagatt 5460

ttatgtgtaa accacaaaaa tgtttacatg aaggccatta ttacagatgt gacgtgcata 5520

attattagta atttgtatgt ttacatgggt cagtctggca aaaaattatg aagttttaaa 5580

aattaaaaaa aattataatg ccagttttac tggaaagtaa aattatttca gtaatcgatt 5640

atagcaaaag tattgatttt cattccagac aaaagtcaga atgaaaggta atttctcaat 5700

actctttcag attaataaaa gtacctgtag cgatttttat cattcacaag tatatcacaa 5760

gtaagttaga atttgagaac tgtgttctag atctctgagg agatgcagtc agatttctga 5820

actgtctcag caaatggtaa gtaacttaga gctagtaatt aataacctgt cctttgattt 5880

ctgattcagc caagaatggc catatttggg aaaggcagat ctggagagta accacgtttt 5940

cattcattta ccacttctag gcccctccag agctctcaga tattttgggg ttgagccctt 6000

ccccaaagcc atacaggacc ttttttttgt gatctgttct agccattttt atgttgggtg 6060

cttgttatgg actgagcatt tatgtcctcc cacacccccc ccataccttt tttgaagtcc 6120

taacccccag tgtgatggta tttggagaca gggcctttgg aaggtaatta cagttagaag 6180

aagtcgggag ggttgggccc aggtctgatt ggattagtgc ccttatatga aaagacacca 6240

ggacgggcgc agtggctcac acctgtaatc ccagcacttt gggaggccaa ggtgggtgga 6300

tcacgaggtc aggagtttga gaccagcctg gccaatgtag tgaaacacca tctctactaa 6360

aaatacaaaa attagctggg tgtggtagcg ggctcctgtc atccaagcta ctcgggaggg 6420

tgaggcatga gaatcacttg aacccgggag ttggaggttg cagtgagccc agattgtgcc 6480

actgtactcc agcctgggtg acagagtgag actctgtctc aaaaaagaaa aaaaaaaaaa 6540

aagagacacc agagagcttg ttagaagagg tcatgtgagc acacagttag aagaccttca 6600

agccaaagaa gaggcctgag attgaaacct accttgcagg taccttaatt ttggacttcc 6660

cagcctccaa aactgtgaga aataagtttc tgttaagtca ctcagtctgt ggtattttgt 6720

tatggcagcc tgagcaggta gttgttcttt cagaaggtgt tgataataac cacatgcaac 6780

accaagtcac aaataataaa acagatgtaa cttatattca tacagaaagt tgggcactgc 6840

cattgccttg ttggtttaca cggctgtgct agttcagtag cagaaaggtg ctggtctcct 6900

ttactcagtt tacaatctag gcagtagaat gtaatcactg ctttaaactt gatactgctt 6960

agggagagaa tcattggtgc tgggtaacta cccagctttc ttgtacaaag tggttctacc 7020

gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 7080

gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 7140

gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 7200

ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 7260

gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 7320

ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 7380

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 7440

ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 7500

cctcatctcc gggcctttcg acctcacgtg gccaccatga aaaagcctga actcaccgcg 7560

acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc 7620

tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg 7680

cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca 7740

tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aatttagcga gagcctgacc 7800

tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg 7860

cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc 7920

cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt 7980

gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac 8040

accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc 8100

cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat 8160

ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag 8220

gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac 8280

ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc 8340

attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg 8400

gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa 8460

atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt 8520

ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcagacatg ataagataca 8580

ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 8640

tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 8700

acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 8760

agtaaaacct ctacaaatgt ggtagtcgcc caca 8794

<210> 6

<211> 5320

<212> DNA

<213> Artificial sequence

<400> 6

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 120

gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 180

cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt 240

tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc 300

cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt 360

tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg 420

cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa 480

tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta 540

taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg 600

ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt 660

gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt 720

taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat 780

cacttttttt caggttggac cggtgccacc atggactata aggaccacga cggagactac 840

aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg 900

aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc 960

ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa 1020

ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg 1080

ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga 1140

tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc 1200

aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag 1260

aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag 1320

taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg 1380

cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag 1440

ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc 1500

tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc 1560

ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc 1620

gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac 1680

ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac 1740

gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg 1800

gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag 1860

atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac 1920

ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc 1980

ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag 2040

ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg 2100

aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc 2160

caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca 2220

ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac 2280

gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa 2340

accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc 2400

atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac 2460

agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc 2520

gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg 2580

ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa 2640

atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg 2700

ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa 2760

aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg 2820

atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg 2880

aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg 2940

gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga 3000

aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc 3060

caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc 3120

gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg 3180

ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag 3240

aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg 3300

ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg 3360

tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac 3420

cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc 3480

atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc 3540

tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg 3600

attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg 3660

gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg 3720

gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg 3780

gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag 3840

ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc 3900

gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc 3960

gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag 4020

gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc 4080

ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag 4140

atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa 4200

gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg 4260

cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac 4320

ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag 4380

ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga 4440

agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa 4500

aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag 4560

agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa 4620

tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat 4680

aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag 4740

cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg 4800

tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac 4860

ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc 4920

gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc 4980

atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg 5040

gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct 5100

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5160

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5220

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5280

agggggagga ttgggaagag aatagcaggc atgctgggga 5320

<210> 7

<211> 352

<212> DNA

<213> Artificial sequence

<400> 7

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg attgaggtac tgaaatgtgt gttttagagc tagaaatagc aagttaaaat 300

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt 352

<210> 8

<211> 26

<212> DNA

<213> Artificial sequence

<400> 8

gatctggaag gtgctgaggt acgatg 26

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<400> 9

aagggtcgtc cgcaggattc ag 22

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

cactcgtccg agggcaaagg a 21

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<400> 11

gcttccatca aacgagttgg tgct 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<400> 12

caagacctgc ctgaaaccga actg 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<400> 13

aagcatcagc tcatcgagag cctg 24

Claims (9)

1. A cell obtained by inserting an exogenous fragment comprising LoxM2, HPRT intron and Hygro cassette into the genome of 293A cells at the position of Ad5 gene.

2. The cell according to claim 1, wherein the nucleotide sequence of LoxM2 is shown in SEQ ID No.1, the nucleotide sequence of HPRT intron is shown in SEQ ID No.2, and the nucleotide sequence of Hygro cassette is shown in SEQ ID No. 3.

3. The cell of claim 2, wherein the exogenous fragment has the nucleotide sequence set forth in SEQ ID No. 5.

4. The cell of claim 1, wherein the insertion is at a position 3522bp of the Ad5 gene.

5. The method for constructing the cell according to any one of claims 1 to 4, wherein the method comprises inserting an exogenous fragment comprising LoxM2, HPRT intron and Hygro cassette at the position of Ad5 gene in 293A cell genome.

6. The construction method according to claim 5, wherein the construction method is to use a non-homology-dependent targeted integration method to make the genome of 293A cell and the plasmid carrying the exogenous fragment form DNA double-strand break by using CRISPR/Cas9 system, and insert the linearized exogenous fragment into the targeted site of the cell genome by using a non-homology end-linked DNA repair pathway, so as to form targeted integration.

7. The method of claim 6, wherein the plasmid carrying the exogenous fragment is pDOwn-HBV ployA-HPRT intron-mPGK-Hygro-SV40 pA-gRNA 2.

8. A method of packaging an adenovirus using a cell according to any one of claims 1 to 4, wherein said method comprises infecting said cell with an adenovirus lacking the E1 gene.

9. Use of a cell according to any one of claims 1 to 4 in the preparation of an adenovirus.

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