CN108753727A - A kind of GPCR targeted drugs screening system and its structure and application - Google Patents
A kind of GPCR targeted drugs screening system and its structure and application Download PDFInfo
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Abstract
The present invention provides a kind of GPCR targeted drugs screening system and its structure and applications.The GPCR targeted drug screening systems, which is characterized in that including：β-arrestin are connected to the PB- β-arrestin-TEV Nia of composition with tobacco etch virus protease；And Tet response elements are connected to the PB-TRE-GFP of composition with GFP.The GPCR targeted drug screening system PiggyBac-Tango, compared to previous medicament sifting motion system, it is embodied the advantages of include：(1) high stability (constituent element being inserted into genome using PiggyBac, can express steadily in the long term)；(2) highly sensitive (exogenous origin gene integrator form is multicopy, and detection signal is amplified)；(3) signal-to-noise ratio is high；(4) inexpensive (not needing detection reagent)；(5) In vivo detection (cytotoxicity that ligand to be measured can be reacted).PiggyBac-Tango systems are the effective tools of GPCR targeted drugs research and development.
Description
Technical field
The present invention relates to a kind of GPCR targeted drugs screening system and its structure and applications.
Background technology
G protein coupled receptor (G protein-coupled receptors, GPCRs) family is most huge in the mankind
One of memebrane protein family and the most successful drug targets of current research, existing 34% or so marketed drug is with GPCR so far
For target spot1-3.According to statistics, during 2011 to 2015 years, the drug sales volume for targeting GPCR accounts for the 27% of world market, and sales volume reaches
To 890,000,000,000 U.S. dollars2,4.Although GPCR accountings in all druggability target spots are maximum, there is 3/4ths GPCR member still
It is not developed to drug target；Only about 80 kinds of GPCR molecules have corresponding small-molecule drug at present5,6.Therefore, it is based on weight
The GPCR ligands research and development demand that big disease is oriented to is very urgent.It is exactly to innovate that GPCR targeted drugs, which screen one of facing challenges,
The structure of high-flux medicaments sifting system.Quasi- research and development structure is high-throughput, general, high stability for we, is based on active somatic cell
GPCRs targeted drug screening systems.
The discoveries of GPCR Novel Ligands play the role of during new drug development with identification it is key, it is high for GPCR ligands
The method of flux screening should have the characteristics such as stabilization, on-radiation, low cost, general, easy to operate7.It is traditional common
Method is all based on the functional analysis of G-protein dependence mostly.But such methods are limited by G-protein type, are not to be suitable for
All GPCR greatly limit screening range, and detection method is cumbersome, and cost is higher8.B.L.Roth et al. develops entitled
PRESTO-Tango GPCR targeted drug screening systems, this method are the recruitments based on β-arrestin, and principle is when excitement
Type ligand L is combined with GPCR, activates corresponding GPCR, you can the C-terminal of recruitment β-arrestin albumen to receptor, and β-
The TEV that arrestin is connected below shears enzyme, can be recruited simultaneously, and then shears downstream TEV with GPCR carriers
Point, release tTA enter core, the TRE promoters that activation downstream tTA is relied on, and the reporter gene in the promoter downstream connection passes through inspection
Reporter gene is surveyed, situation is activated to react GPCR9.The advantage of this kind of method is to be suitable for all GPCR, and sensitivity
Height, detection signal is strong, shows that the method for the screening of the GPCR targeted drugs based on Tango systems is more expected to become innovation drug
Powerful.
It is worth noting that the structure of traditional GPCR-Tango systems is completed by the method for transient transfection, it cannot
Exist in the genome of cell for a long time, can be lost after a period of time, there are unstability for system；Medicine is limited in this way
The timeliness and reliability of object screening.Therefore, there is an urgent need for new technological means to build the screening system expressed steadily in the long term.
Transposons (transponson) is also referred to as transposable element, refer to can be transferred to from a chromosomal foci it is another
The interspersed repetitive sequence in site.As effective transgenosis tool, foreign gene can be imported host genome etc. by transposons
Gene code operates10.PiggyBac (PB) transposons belongs to one kind of DNA transposons, is a kind of moveable genetic elements, and
Swivel base, i.e. transposase (PBase) cutting PB transposons both ends inverted repeats are carried out by " shear-paste " swivel base pattern
The TTAA sequences of the end (inverted terminal repeat, ITR), transposons are scaled off from original gene seat, then
It is inserted on the new sites TTAA of genome.B transposon systems are as a kind of new gene transfer means, due to living with high swivel base
Property, swivel base it is safe and stable with it is seamless remove etc. characteristics and be concerned, in molecular medicine research have preferably apply before
Scape11,12.After present PiggyBac systems have been successfully used to genome research, gene therapy, stem cell induction and induction
The research fields such as differentiation, are the powerfuls of gene code13-15。
Due to existing Tango systems have the shortcomings that it is unstable, of high cost, cumbersome；And what it was finally detected
System is not based on active somatic cell, cannot directly react the cytotoxicity of ligand to be measured；It is clear that this allow for it cannot
As long-term and widely used screening system.Therefore the high throughput, high stability of structure GPCR targeted drugs, Vivo Studies on Screening system
It is imperative to unite.
Bibliography
1 Santos, R.et al.A comprehensive map of molecular drug targets.Nat
Rev Drug Discov 16,19-34, doi：10.1038/nrd.2016.230(2017).
2 Hauser, A.S., Attwood, M.M., Rask-Andersen, M., Schioth, H.B.&Gloriam,
D.E.Trends in GPCR drug discovery：New agents, targets and indications.Nat Rev
Drug Discov 16,829-842, doi：10.1038/nrd.2017.178(2017).
3 Rask-Andersen, M., Masuram, S.&Schioth, H.B.The druggable genome：
Evaluation of drug targets in clinical trials suggests major shifts in
Molecular class and indication.Annu Rev Pharmacol Toxicol 54,9-26, doi：
10.1146/annurev-pharmtox-011613-135943(2014).
4 The IDG Knowledge Management Center.Unexplored opportunities in the
druggable human genome.Nat.Rev.Drug Disc.http：//www.nature.com/nrd/posters/
druggablegenome/nrd_druggablegenome.pdf(2016).
5 Hutchings, C.J., Koglin, M., Olson, W.C.&Marshall, F.H.OPPortunities for
therapeutic antibodies directed at G-protein-coupled receptors.Nat Rev Drug
Discov 16,661, doi：10.1038/nrd.2017.173(2017).
6 Wilkinson, T.C.Discovery of functional monoclonal antibodies
Targeting G-protein-coupled receptors and ion channels.Biochem Soc Trans 44,
831-837, doi：10.1042/BST20160028(2016).
7 Harrison, C.&Traynor, J.R.The [35S] GTPgammaS binding assay：approaches
And applications in pharmacology.Life Sci 74,489-508 (2003)
8 Weber, M.et al.A 1536-well cAMP assay for Gs-and Gi-coupled
receptors using enzyme fragmentation complementation.Assay Drug Dev Technol
2,39-49, doi：10.1089/154065804322966306(2004).
9 Kroeze, W.K.et al.PRESTO-Tango as an open-source resource for
Interrogation of the druggable human GPCRome.Nat Struct Mol Biol 22,362-369,
doi：10.1038/nsmb.3014(2015).
10 Smit, A.F.Interspersed repeats and other mementos of transposable
Elements in mammalian genomes.Curr Opin Genet Dev 9,657-663 (1999)
11 Kim, A.&Pyykko, I.Size matters：vetsatile use of PiggyBac transposons
As a genetic manipulation tool.Mol Cell Biochem 354,301-309, doi：10.1007/
s11010-011-0832-3(2011).
12 Wilson, M.H., Coates, C.J.&George, A.L., Jr.PiggyBac transposon-
Mediated gene transfer in human cells.Mol Ther 15,139-145, doi：10.1038/
sj.mt.6300028(2007).
13 Rad, R.et al.PiggyBac transposon mutagenesis：a tool for cancer gene
Discovery in mice.Science 330,1104-1107, doi：10.1126/science.1193004(2010).
14 Kettlun, C., Galvan, D.L., George, A.L., Jr., Kaja, A.&Wilson,
M.H.Manipulating piggyBac transposon chromosomal integration site selection
In human cells.Mol Ther 19,1636-1644, doi：10.1038/mt.2011.129(2011).
15 Woltjen, K.et al.piggyBac transposition reprograms fibroblasts to
Induced pluripotent stem cells.Nature 458,766-770, doi：10.1038/nature07863
(2009).
Invention content
The object of the present invention is to provide build a kind of new GPCR targeted drugs screening system and its structure and application.
In order to achieve the above object, the present invention provides a kind of GPCR targeted drugs screening system (Piggybac-
Tango), which is characterized in that including：By β-arrestin and tobacco etch virus protease (TEV protease) connection composition
PB- β-arrestin-TEV Nia；And Tet response elements are connected to the PB-TRE-GFP of composition with GFP.
Preferably, PB- β-the arrestin-TEV Nia and PB-TRE-GFP are inserted using Piggybac as carrier
The target gene entered is multicopy, improves signal-to-noise ratio.
Preferably, PB- β-the arrestin-TEV Nia and PB-TRE-GFP are inserted using Piggybac as carrier
The form of the target gene entered is to stablize to be inserted into, and has stability.
Preferably, PB- β-arrestin-TEV Nia and the PB-TRE-GFP sequences be respectively SEQ ID NO.2 and
SEQ ID NO.5。
Preferably, the reporter gene of the system is GFP, result visualization.
Preferably, the GPCR targeted drug screening systems further include ADORA1 receptor plasmids.
Preferably, the GPCR targeted drugs screening system (Piggybac-Tango) further includes that GPCR is lost by tobacco
The GPCR-TEV-tTA of line virus protease (TEV protease) cleavage site and tetracycline trans-acting factor tTA compositions.
The present invention also provides the construction method for the cell line that can express Piggybac-Tango systems, feature exists
In, including：Using PiggyBac transposons as carrier, PB- β-arrestin-TEV Nia and PB-TRE-GFP are imported into cell strain
It in genome and is integrated, obtains the cell line that can express Piggybac-Tango systems.
The present invention also provides above-mentioned Piggybac-Tango systems or Piggybac-Tango systems can be expressed
Application of the cell line in the screening of GPCR targeted drugs.
The present invention strategy be：Exogenous gene high-efficient is imported in cell strain genome using Piggybac transposons, it is whole
It closes efficient and can express steadily in the long term.Integration form transposon-mediated Piggybac is multicopy, is also greatly enhanced in this way
Detection signal, improves sensitivity and signal-to-noise ratio.Reporter gene is GFP, and result visualization is easy to operate, save experiment at
This, and detected on active somatic cell, the cytotoxicity of ligand to be measured can be reacted.
The GPCR targeted drug screening system PiggyBac-Tango of the present invention, it is characterized in that high stability, multicopy shape
Formula and based on living cells screen.The GPCR targeted drugs screening system (Piggybac-Tango) is with PiggyBac transposons
It mediates, exogenous gene high-efficient is imported into cell strain genome, integration efficiency is high and can express steadily in the long term.PiggyBac transposons
The exogenous origin gene integrator of mediation is multicopy form, and amplification detection signal improves signal-to-noise ratio.Reporter gene is GFP, as a result visually
Change, saves experimental implementation and cost.Medicament sifting motion system detects for living cells, can react the cytotoxicity of ligand to be measured.
The present invention is directed to the limitation of previous system, and the characteristics of GPCR targeted drug screening systems of structure embody includes：
(1) high stability (constituent element is inserted into genome, can be expressed steadily in the long term), (2) highly sensitive (exogenous origin gene integrator form
Multicopy, detection signal is amplified), (3) signal-to-noise ratio is high, (4) inexpensive (not needing detection reagent), (5) In vivo detection (can
To react the cytotoxicity of ligand to be measured).
The experimental results showed that Piggybac-Tango systems of the invention have the advantage that：
(1) Piggybac-Tango systems have high stability.
(2) Piggybac-Tango system detectios signal is amplified, and improves sensitivity and signal-to-noise ratio.
(3) Piggybac-Tango system results visualize, and detecting step is easy, and experimental cost is low.
(4) Piggybac-Tango systems detect on active somatic cell, can react the cytotoxicity of ligand to be measured.
Description of the drawings
Fig. 1 is Piggybac-Tango system model figures；
Fig. 2 is Piggybac-Tango system element schematic diagrames；
Fig. 3 is element 2 and 3 qualification result figures；
(A) it is that element 2 identifies agarose gel electrophoresis figure and sequencing result figure；
(B) it is that element 3 identifies agarose gel electrophoresis figure and sequencing result figure；
Fig. 4 is the agarose gel electrophoresis figure and sequencing result that Piggybac-Tango systems are expressed in 293T cytotostatics
Figure；
Fig. 5 is Piggybac-Tango system verification result figures；
Fig. 6 is the agarose gel electrophoresis figure that Piggybac-Tango systems stablize expression in 4 monoclonal cell systems；
Fig. 7 is Piggybac-Tango systems in 4 monoclonal cell system verification result figures；
Fig. 8 is that β-arrestin and GFP express copy number results figure in 4 monoclonal cell systems.
Specific implementation mode
Explanation is further explained to the present invention by embodiment as follows.Described embodiment is merely to illustrate the present invention
Feature, be not so limited the present invention.Other people some non-intrinsically safes are replaced or improve within the scope of the present invention.In embodiment
The reagent of manufacturer or instrument, which is not specified, can be bought by market and be obtained.Not experimental method dated in detail, according to normal condition
Or the method implementation that reagent is recommended by the manufacturer.
Plasmid AC133-CAR piggyBac transposon vector (SEQ ID NO used in following embodiment：
1) prepared by the preparation method that, can be recorded according to following documents：Zhu, X., Prasad, S., Gaedicke, S., Hettich, M.,
Firat, E., and Niedermann, G. (2015) .Patient-derived glioblastoma stem cells are
killed by CD133-specific CAR T cells but induce the T cell aging marker
CD57.Oncotarget 6,171-184.
The cDNA of 293T cells used in following embodiment can be prepared according to following preparation methods：
1) RNA is extracted：293T cells (being purchased from ATCC) are inoculated in 12 orifice plates, after adherent, 1ml Trizol are added
(Life, 15596018) is blown and beaten, and is blown and beaten mixing after standing 5min and is transferred in 1.5ml centrifuge tubes, 200 μ l are added into centrifuge tube
Chloroform acutely shakes 15s, is incubated at room temperature 2-3min, 12000 × g, 4 DEG C of centrifugation 15min.Liquid is divided into three layers after centrifugation, carefully
300 μ l supernatant liquids are drawn, are transferred in new EP pipes.Equivalent isopropanol mixing is added, is stored at room temperature 10min.12000 × g,
4 DEG C of centrifugation 10min, remove supernatant, and the cleaning of 75% ethyl alcohol of 1ml, upper and lower mixing 6-8 times is added.Ethyl alcohol is absorbed completely, and 20 μ l are added
Nuclease-free water dissolutions detect RNA concentration.
2) RNA reverse transcriptions：Reverse transcription reagent box (Toyobo, FSQ-301) is used, is as follows：First will
4 × DN Master Mix of 440 μ l are mixed with the gDNA Remover of 8.8 μ l, RNA under the conditions of 65 DEG C thermal denaturation 5min
It is denaturalized, is immediately placed on cooled on ice.Genomic DNA is removed by following reaction：4 × DN Master Mix, 0.5 μ g of 2 μ l
RNA template, Nuclease-free Water polishings to 8 μ l.It will react and prepare on ice above, gently mixing, it
It is incubated 5 minutes under the conditions of 37 DEG C afterwards.Reverse transcription reaction is carried out later, and 5 × RT of 2 μ l is added in reaction solution before
Master Mix II, gently mixing, is reacted by following temperature：
37 DEG C, 15min
50 DEG C, 5min
98 DEG C, 5min
4 DEG C, continue
After reaction, prepared by the cDNA of 293T cells completes.
Embodiment
As shown in Figure 1, a kind of GPCR targeted drugs screening system Piggybac-Tango, including with Piggybac it is to carry
Body, by β-arrestin and tobacco etch virus protease (TEV protease) connection composition PB- β-arrestin-TEV
Nia；Using Piggybac as carrier, Tet response elements are connected to composition PB-TRE-GFP with GFP.
The construction method of above-mentioned GPCR targeted drug screening systems Piggybac-Tango is：
The structure of 1.Piggybac-Tango system plasmids：
(1) the PB- β-arrestin-TEV Nia plasmids of puromycin (Puromycin) selection markers are built, strategy is
By AC133-CAR piggyBac transposon vector (SEQ ID NO：1) be used as skeleton, by XbaI (NEB,
R0145S) and EcoRI (NEB, R3101L) double digestion obtains linearisation PB-T2A-Puro carriers.Using the method for bridging PCR,
Bridging primer is designed, with F1, R1 is primer (table 1), using the cDNA of 293T cells as template, is usedMax Super-
Fidelity DNA Polymerase (Vazyme, P505-d3) carry out PCR amplification, and system is as follows：
In the thermal cycler, 98 DEG C of thermal startings, 58 DEG C are annealed, 72 DEG C of extensions, 35 cycles of coreaction, 4 DEG C of holdings.
The PCR product come is amplified to purify by following step：PCR-A (the Axygen of three times volume are added：AP-PCR-250G), and
Column is crossed, 12000 revs/min centrifuge 1 minute；Waste liquid is abandoned, 500 μ L W2 (AP-PCR-250G) are added, is centrifuged 1 minute；1 point of idle running
Clock is added 20 μ L sterilizing water elutions, is identified by agarose gel electrophoresis and obtain segment β-arrestin.
With F2, R2 is PCR primer (table 1), with TEV Nia (SEQ ID NO：2, Jin Sirui bio tech ltd
(http：//www.genscript.com.cn/) synthesis) it is template, it usesMax Super-Fidelity DNA
Polymerase (Vazyme, P505-d3) carries out PCR amplification, and system is as follows：
In the thermal cycler, 98 DEG C of thermal startings, 58 DEG C are annealed, 72 DEG C of extensions, 35 cycles of coreaction, 4 DEG C of holdings.
The PCR product come is amplified to purify by following step：PCR-A (the Axygen of three times volume are added：AP-PCR-250G), and
Column is crossed, 12000 revs/min centrifuge 1 minute；Waste liquid is abandoned, 500 μ L W2 (AP-PCR-250G) are added, is centrifuged 1 minute；1 point of idle running
Clock is added 20 μ L sterilizing water elutions, is identified by agarose gel electrophoresis and obtain segment TEV Nia.
Using segment β-arrestin and segment TEV Nia as template, forward primer F1 (table 1) carries XbaI enzyme cutting site,
Reverse primer R2 (table 1) carries EcoRI restriction enzyme sites, carries out bridging PCR, usesMax Super-Fidelity
DNA Polymerase (Vazyme, P505-d3) carry out PCR amplification, and system is as follows：
In the thermal cycler, 98 DEG C of thermal startings, 58 DEG C are annealed, 72 DEG C of extensions, 35 cycles of coreaction, 4 DEG C of holdings.
The PCR product come is amplified to purify by following step：PCR-A (the Axygen of three times volume are added：AP-PCR-250G), and
Column is crossed, 12000 revs/min centrifuge 1 minute；Waste liquid is abandoned, 500 μ L W2 (AP-PCR-250G) are added, is centrifuged 1 minute；1 point of idle running
Clock is added 20 μ L sterilizing water elutions, is identified by agarose gel electrophoresis and obtain segment β-arrestin-TEV Nia.
Linearisation PB-T2A-Puro carriers are done into digestion with XbaI and EcoRI together with β-arrestin-TEV Nia, are led to
It crosses T4 ligases (NEB, M0202S) connection and obtains plasmid PB- β-arrestin-TEV Nia-T2A-Puro (Fig. 2, SEQ ID
NO：3).The connection product that above-mentioned steps are obtained converts DH5 α competent cells, and applies Amp+ tablets (50 μ g/mL), and picking
Clone.With restriction enzyme site upstream and downstream 100-300bp or so design primer T1-F, T1-R, T2-F and T2-R (table 1) and synthesize,
The method identification by PCR and being routinely sequenced obtains positive colony, and gel electrophoresis and sequencing result are as shown in Figure 3A.
(2) build hygromycin (hygromycin) selection markers PB-TRE-EGFP plasmids, strategy for
PB-TRE-NLS-3XFlag-Cas9-P2A-Hygro is carrier (SEQ ID NO：4, Jin Sirui biotechnologies are limited
Company (http：//www.genscript.com.cn/) synthesis), by XmaI (NEB, R0180S) and BamHI (NEB,
R0136S) double digestion obtains linearisation PB-TRE-T2A-Hygro carriers.It designs forward primer F3 and carries XmaI restriction enzyme sites, instead
BamHI restriction enzyme sites (table 1) are carried to primer R3, with Oct4-IRES-eGFP-PGK-Neo (SEQ ID NO：5, addgene,
48681) it is template, usesMax Super-Fidelity DNA Polymerase (Vazyme, P505-d3) into
Row PCR amplification, system are as follows：
In the thermal cycler, 98 DEG C of thermal startings, 58 DEG C are annealed, 72 DEG C of extensions, 35 cycles of coreaction, 4 DEG C of holdings.
The PCR product come is amplified to purify by following step：PCR-A (the Axygen of three times volume are added：AP-PCR-250G), and
Column is crossed, 12000 revs/min centrifuge 1 minute；Waste liquid is abandoned, 500 μ L W2 (AP-PCR-250G) are added：, centrifuge 1 minute；Idle running 1
Minute, 20 μ L sterilizing water elutions are added, is identified by agarose gel electrophoresis and obtains segment EGFP.By the PB-TRE- of linearisation
P2A-Hygro carriers do digestion together with EGFP with XmaI and BamHI, are connected by T4 ligases (NEB, M0202S) and obtain matter
Grain PB-TRE-EGFP-P2A-Hygro (Fig. 2, SEQ ID NO：6).The connection product conversion DH5 α impressions that above-mentioned steps are obtained
State cell, and Amp+ tablets (50 μ g/mL) are applied, and picked clones.It designs and draws with restriction enzyme site upstream and downstream 150-300bp or so
Object T3-F, T3-R, T4-F and T4-R (table 1) are simultaneously synthesized, and the method identification by PCR and being routinely sequenced obtains positive colony,
Gel electrophoresis and sequencing result are as shown in Figure 3B.
2. stablizing the 293T cell lines structure of expression Piggybac-Tango systems
We turn element 2 in Piggybac-Tango systems and 3 pairs of 293T cell progress electricity, and concrete operations are as follows：
1) HEK293T cells (coming from ATCC) recovery, culture, culture medium in 10cm culture dishes (Corning, 430167)
To be mixed with 10% fetal calf serum (Gemini, 100525), the DMEM of 1% dual anti-(Life, 15140122) (HyClone,
SH30243.01).Cultivation temperature is 37 DEG C, gas concentration lwevel 5%.Repeatedly after passage when cell density is 70-80%,
Digestion centrifugation is carried out, is resuspended with 5ml PBS, and count.
2) according to the operating procedure of SF Cell Line 4D X Kit L (Lonza-Amaxa, V4XC-2024), 1 is taken out
×106For a cell in sterile 1.5ml EP pipes, 200 × g room temperature centrifuges 10min.
3) electricity swivel system：100μl
Pipette tips are blown and beaten, and mild mixing cell avoids bubble.
4) electric revolving cup bottom slowly is added in cell suspension, pats and drive bubble out of, be put into electroporation, uses DS 150
Program carries out electricity and turns.
5) after, cell suspension is transferred to immediately in the 6cm wares for being placed in advance in and being preheated in incubator and is cultivated.
Culture medium is the fetal calf serum for being mixed with 10%, 1% dual anti-DMEM.
6) after electricity turns for 24 hours, add 2 μ g/ml (InvivoGen, ht-pr-1) of puromycin, handle 2 weeks, during which normally pass on
Change liquid.293T control groups are set, and cellular control unit is not survived, and electricity turns group survivaling cell and expands culture, and during which culture medium is mixed
There are 10% fetal calf serum, 1% dual anti-, 2 μ g/ml of puromycin DMEM.
7) by (2-3 weeks) after the above cell expansion culture, it is stronger that expression green fluorescence background is removed by airflow classification
Cell colony.
8) cell inoculation is changed when cell concentration is 80% with the DMEM culture mediums of 10% serum in 10cm culture dishes
Liquid, culture make cell state restore best for 2 hours.The plasmid of transfection is pTet-on Advanced (Clontech, 631069),
24 μ g plasmids are mixed in Opti-MEM (Gibco, 11058021) culture medium of 1.5ml, vortex mixing.By 60 μ l's
2000 transfection reagents of Lipofectamine (Thermo, 11668019) are mixed into the Opti-MEM culture mediums of 1.5ml, are vortexed mixed
It is even, stand 5 minutes respectively.
9) Opti-MEM for being mixed with plasmid is added to the Opti-MEM for being mixed with Lipofectamine 2000, at a slow speed speed piping and druming
Mixing stands 20 minutes.
10) Opti-MEM mixed above is slowly dropped into 10cm culture dishes.
11) liquid is changed with the DMEM of 10%FBS after transfecting 6 hours, and the fortimicin of 2 μ g/ml is added simultaneously.
12) after for 24 hours, fluorescence is observed, and collects the cell of expression green fluorescence by airflow classification, and expands culture, is trained
Foster base is the fetal calf serum for being mixed with 10%, 1% dual anti-, 2 μ g/ml of puromycin DMEM.
The verification of 3.Piggybac-Tango systems
1) by the above cell expansion culture until green fluorescence disappears.
2) part cell is taken, is collected in 1.5ml centrifuge tubes after trypsin digestion, 500ul Tissue lysates are added and (split
The ingredient for solving liquid is 50mM KCl, 1.5mM MgCl2, 10mM Tris pH 8.0,0.5%Nonidet P-40,0.5%
20,100 μ g/ml protease K of Tween) and 5ul Proteinase Ks (Yeasen, 10401ES60,10ug/ul) be placed on 55 DEG C
Overnight after cracking, genomic DNA, and measured concentration are extracted with phenol chloroform method for water-bath.
3) the positive anti-primer of T1, T2, T3, the T4 designed before carries out PCR, is sequenced by gel electrophoresis and routine
Determine that element 2 and 3 has been successively inserted into 293T cellular genomes in Piggybac-Tango systems, the results are shown in Figure 4.
4) after genotype determination, cell is spread into 24 orifice plates, cell density is transfected in 70%-80% using lipo2000
(Addgene, 1000000068, pass through tobacco etch virus protease (TEV protease) to ADORA1 receptor plasmids for GPCR
Cleavage site forms GPCR-TEV-tTA with tetracycline trans-acting factor tTA), after 6-8 hours, change liquid.
5) after transfecting 24 hours, 5 μM of NECA (agonist of ADORA1 receptors, Sigma, E2387) are added.
6) after 24 hours, fluorescing matter is observed, result is after NECA activates ADORA1 receptors, successfully to cause β-arrestin
Recruitment and TEV cutting, to start the expression of EGFP.Result above proves that we are successfully prepared high quality
Piggy-Tango-293T systems (Fig. 5)
7) flow sorting techniques are used, expression green cells are subjected to unicellular sorting, and be inoculated in 96 orifice plates.
And expand culture, genome is extracted respectively, carries out genotype identification.Determine that monoclonal is thin by gel electrophoresis and conventional sequencing
Born of the same parents system Cell A, Cell B, Cell C, Cell D stablize element 2 and 3 (Fig. 6) in expression Piggybac-Tango systems.
8) according to above-mentioned qualification result, above 4 monoclonal cell systems are subjected to ADORA1 transfections, for 24 hours after, 5 μM of excitements
Agent NECA is handled overnight.As a result such as Fig. 7, after ADORA1 transfections, low background is substantially consistent with blank group.Agonist NECA is added
Afterwards, activated receptor ADORA1, so that EGFP is expressed.Result above proves that we are successfully prepared high quality, high stability
Piggy-Tango systems, and be successfully prepared the monoclonal cell system of Piggy-Tango systems.
9) according to verification result, the RNA of four monoclonal cell systems is extracted respectively, and is carried out reverse transcription and obtained cDNA, it
After carry out Q-PCR, investigate the copy number of different monoclonal cell system Piggy-Tango reporter genes, as a result such as Fig. 8, monoclonal
The copy number of cell line Cell Line A, Cell Line B, Cell Line C, Cell Line D reporter genes is relative to just
Normal 293T cell lines are compared to respectively 38,17,15,34.Show the expressing gene of Piggy-Tango reporting system reporter genes
For multicopy signal-to-noise ratio is improved to enhance detection signal.
To sum up, for successfully building GPCR targeted drug screening system-Piggy-Tango, this is strategy of the invention
System it is embodied the characteristics of be：(1) high stability (using PiggyBac by constituent element be inserted into genome in, can table steady in a long-term
Up to)；(2) highly sensitive (exogenous origin gene integrator form is multicopy, and detection signal is amplified)；(3) signal-to-noise ratio is high；(4) it is low at
This (not needing detection reagent)；(5) In vivo detection (cytotoxicity that ligand to be measured can be reacted).PiggyBac-Tango systems
It is the effective tool of GPCR targeted drugs research and development.
1 primer sequence of table
Sequence table
<110>Shanghai Science and Technology Univ.
<120>A kind of GPCR targeted drugs screening system and its structure and application
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7857
<212> DNA
<213> Artificial Sequence
<400> 1
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatgctg 1920
ctgctggtca cttctctgct gctgtgcgaa ctgccccacc ccgcctttct gctgattccc 1980
caggtccagc tgcagcagtc tggagctgag ctggtcagac ccggcgcatc agtgaaactg 2040
agctgcaagg cttccggcta tactttctcc gactttgaga tgcactgggt caagcagacc 2100
ccagtgcatg gcctggaatg gatcggggac attgatcccg gcactgggga caccgcctat 2160
aacctgaagt tcaaaggcaa ggctaccctg accacagata agagctcctc tacagcctac 2220
atggagctga ggtctctgac tagtgaagat tcagcagtct actattgcac actgggggcc 2280
ttcgtgtact ggggacaggg cacactggtc accgtgagcg ccgctaaaac tacccccaag 2340
ctggaggaag gagagttcag cgaagcaaga gtggacgtgg tcgtgaccca gacacccctg 2400
tctctgcctg tcagttttgg cgatcaggtg agcatctcct gtaggagttc acagtcactg 2460
gccaacagct acgggaatac atatctgtct tggtacctgc acaagccagg acagagtccc 2520
cagctgctga tctatgggat ttccaatcgc ttctctggag tgcctgaccg attttctggg 2580
agtggatcag gcaccgattt cacactgaaa atcagcacca ttaagcccga ggacctgggc 2640
atgtactatt gtctgcaggg gacccatcag ccttacactt ttggcggggg aaccaaactg 2700
gagatcaagc gagcagacgc agcggccgca ggcagcgaac agaaactgat ttccgaggaa 2760
gatctgttcg tccccgtgtt cctgcctgcc aagccaacaa ctacccctgc tccacgacca 2820
cctactccag cacctaccat cgcaagtcag cccctgtcac tgcgacctga ggcttgccgg 2880
ccagcagctg gaggagcagt gcacacccga ggcctggact tcgcatgcga tatctacatt 2940
tgggcaccac tggctggaac ctgtggggtc ctgctgctga gcctggtcat caccctgtat 3000
tgtaaccaca gaaataggag caaacgctcc cgactgctgc attccgacta catgaacatg 3060
acacctcgga gaccaggccc cactagaaag cattaccagc catatgcccc acccagggat 3120
ttcgcagcct atcggagccg gttcagcgtc gtgaaaaggg ggcgcaagaa actgctgtac 3180
atcttcaagc agccttttat gcgcccagtg cagacaactc aggaggaaga cggatgctct 3240
tgtcggttcc cagaggagga ggaaggaggc tgcgagctga gagtgaagtt cagccggagc 3300
gccgatgcac cagcatatca gcagggacag aatcagctgt acaacgagct gaatctgggc 3360
aggcgcgagg aatatgacgt gctggataag cgacgaggac gggaccccga aatgggagga 3420
aaacccagaa ggaagaaccc tcaggagggg ctgtataatg aactgcagaa agacaagatg 3480
gctgaggcat acagcgaaat tggaatgaaa ggagagcgcc gacgggggaa gggacacgat 3540
gggctgtacc agggactgtc aaccgccact aaagatacct acgacgcact gcacatgcag 3600
gctctgcccc caagagaatt cgaaggatcc gcggccgctg agggcagagg aagtcttcta 3660
acatgcggtg acgtggagga gaatcccggc ccttccggga tgaccgagta caagcccacg 3720
gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc 3780
gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc 3840
gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg 3900
gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg 3960
ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa 4020
cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc 4080
gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga 4140
gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac 4200
ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga 4260
ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa tctaggtcga caatcaacct 4320
ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 4380
ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaaact tgtttattgc 4440
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 4500
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggaa 4560
ttgactcaaa tgatgtcaat tagtctatca gaagctcatc tggtctccct tccgggggac 4620
aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct tatatccctt 4680
gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc cctaaagaaa 4740
cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg cccattggta 4800
tggctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc 4860
agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc 4920
tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc 4980
ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct gtccctgata 5040
tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat gaaataacaa 5100
tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt 5160
gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac aagcacgcct 5220
cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga ttcgcgctat 5280
ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag actatctttc 5340
tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg ctcagtcatc 5400
gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt tcccgcgagg 5460
ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac gttgagcgaa 5520
aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt aacggtgaac 5580
tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca cagttccgga 5640
tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg aactgccgtg 5700
ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc gaggacgggt 5760
atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc ggcgggcgcg 5820
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 5880
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5940
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6000
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6060
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6120
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6180
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6240
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6300
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6360
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6420
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6480
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6540
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6600
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6660
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6720
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6780
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6840
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6900
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6960
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7020
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7080
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7140
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7200
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7260
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7320
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7380
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7440
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7500
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7560
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 7620
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7680
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7740
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7800
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcat 7857
<210> 2
<211> 720
<212> DNA
<213> Artificial Sequence
<400> 2
agcttgttta agggaccacg tgattacaac ccgatatcga gcaccatttg tcatttgacg 60
aatgaatctg atgggcacac aacatcgttg tatggtattg gatttggtcc cttcatcatt 120
acaaacaagc acttgtttag aagaaataat ggaacactgt tggtccaatc actacatggt 180
gtattcaagg tcaagaacac cacgactttg caacaacacc tcattgatgg gagggacatg 240
ataattattc gcatgcctaa ggatttccca ccatttcctc aaaagctgaa atttagagag 300
ccacaaaggg aagagcgcat atgtcttgtg acaaccaact tccaaactaa gagcatgtct 360
agcatggtgt cagacactag ttgcacattc ccttcatctg atggcatatt ctggaagcat 420
tggattcaaa ccaaggatgg gcagtgtggc agtccattag tatcaactag agatgggttc 480
attgttggta tacactcagc atcgaatttc accaacacaa acaattattt cacaagcgtg 540
ccgaaaaact tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg 600
cgattaaatg ctgactcagt attgtggggg ggccataaag ttttcatgag caaacctgaa 660
gagccttttc agccagttaa ggaagcgact caactcatga atgaattggt gtactcgcaa 720
<210> 3
<211> 8166
<212> DNA
<213> Artificial Sequence
<400> 3
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatgggg 1920
gagaaacccg ggaccagggt cttcaagaag tcgagcccta actgcaagct caccgtgtac 1980
ttgggcaagc gggacttcgt agatcacctg gacaaagtgg accctgtaga tggcgtggtg 2040
cttgtggacc ctgactacct gaaggaccgc aaagtgtttg tgaccctcac ctgcgccttc 2100
cgctatggcc gtgaagacct ggatgtgctg ggcttgtcct tccgcaaaga cctgttcatc 2160
gccacctacc aggccttccc cccggtgccc aacccacccc ggccccccac ccgcctgcag 2220
gaccggctgc tgaggaagct gggccagcat gcccacccct tcttcttcac cataccccag 2280
aatcttccat gctccgtcac actgcagcca ggcccagagg atacaggaaa ggcctgcggc 2340
gtagactttg agattcgagc cttctgtgct aaatcactag aagagaaaag ccacaaaagg 2400
aactctgtgc ggctggtgat ccgaaaggtg cagttcgccc cggagaaacc cggcccccag 2460
ccttcagccg aaaccacacg ccacttcctc atgtctgacc ggtccctgca cctcgaggct 2520
tccctggaca aggagctgta ctaccatggg gagcccctca atgtaaatgt ccacgtcacc 2580
aacaactcca ccaagaccgt caagaagatc aaagtctctg tgagacagta cgccgacatc 2640
tgcctcttca gcaccgccca gtacaagtgt cctgtggctc aactcgaaca agatgaccag 2700
gtatctccca gctccacatt ctgtaaggtg tacaccataa ccccactgct cagcgacaac 2760
cgggagaagc ggggtctcgc cctggatggg aaactcaagc acgaggacac caacctggct 2820
tccagcacca tcgtgaagga gggtgccaac aaggaggtgc tgggaatcct ggtgtcctac 2880
agggtcaagg tgaagctggt ggtgtctcga ggcggggatg tctctgtgga gctgcctttt 2940
gttcttatgc accccaagcc ccacgaccac atccccctcc ccagacccca gtcagccgct 3000
ccggagacag atgtccctgt ggacaccaac ctcattgaat ttgataccaa ctatgccaca 3060
gatgatgaca ttgtgtttga ggactttgcc cggcttcggc tgaaggggat gaaggatgac 3120
gactatgatg atcaactctg cggatccggc ggcgggggtt cgagcttgtt taagggacca 3180
cgtgattaca acccgatatc gagcaccatt tgtcatttga cgaatgaatc tgatgggcac 3240
acaacatcgt tgtatggtat tggatttggt cccttcatca ttacaaacaa gcacttgttt 3300
agaagaaata atggaacact gttggtccaa tcactacatg gtgtattcaa ggtcaagaac 3360
accacgactt tgcaacaaca cctcattgat gggagggaca tgataattat tcgcatgcct 3420
aaggatttcc caccatttcc tcaaaagctg aaatttagag agccacaaag ggaagagcgc 3480
atatgtcttg tgacaaccaa cttccaaact aagagcatgt ctagcatggt gtcagacact 3540
agttgcacat tcccttcatc tgatggcata ttctggaagc attggattca aaccaaggat 3600
gggcagtgtg gcagtccatt agtatcaact agagatgggt tcattgttgg tatacactca 3660
gcatcgaatt tcaccaacac aaacaattat ttcacaagcg tgccgaaaaa cttcatggaa 3720
ttgttgacaa atcaggaggc gcagcagtgg gttagtggtt ggcgattaaa tgctgactca 3780
gtattgtggg ggggccataa agttttcatg agcaaacctg aagagccttt tcagccagtt 3840
aaggaagcga ctcaactcat gaatgaattg gtgtactcgc aaggcggcgg gggttcgtac 3900
ccatacgacg tcccagacta cgctgaattc gaaggatccg cggccgctga gggcagagga 3960
agtcttctaa catgcggtga cgtggaggag aatcccggcc cttccgggat gaccgagtac 4020
aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 4080
gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 4140
cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 4200
tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg 4260
ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 4320
gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 4380
ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 4440
ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 4500
ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 4560
cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaat ctaggtcgac 4620
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 4680
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgcgtt aactaaactt 4740
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 4800
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 4860
tgtctggaat tgactcaaat gatgtcaatt agtctatcag aagctcatct ggtctccctt 4920
ccgggggaca agacatccct gtttaatatt taaacagcag tgttcccaaa ctgggttctt 4980
atatcccttg ctctggtcaa ccaggttgca gggtttcctg tcctcacagg aacgaagtcc 5040
ctaaagaaac agtggcagcc aggtttagcc ccggaattga ctggattcct tttttagggc 5100
ccattggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga 5160
gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac 5220
gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc 5280
tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg 5340
tccctgatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg 5400
aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg 5460
cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca 5520
agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat 5580
tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga 5640
ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct tttttccggc 5700
tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc cgtgcctttt 5760
cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc tgcattgacg 5820
ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg cacgccttta 5880
acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt ttccggacac 5940
agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc gacagccgga 6000
actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat tacgtcagcg 6060
aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt gagttacccg 6120
gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 6180
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 6240
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 6300
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6360
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6420
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6480
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6540
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6600
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6660
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6720
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6780
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6840
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6900
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 6960
gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 7020
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 7080
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 7140
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 7200
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 7260
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 7320
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 7380
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 7440
accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 7500
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 7560
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 7620
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 7680
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 7740
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 7800
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 7860
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 7920
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 7980
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 8040
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 8100
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 8160
actcat 8166
<210> 4
<211> 9890
<212> DNA
<213> Artificial Sequence
<400> 4
ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct 480
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600
ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgg 660
tattcacgac agcaggctga ataataaaaa aattagaaac tattatttaa ccctagaaag 720
ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta 780
tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt 840
acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa 900
aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa cattctctct 960
tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta taaaataagt 1020
aattagctta acctatacat aatagaaaca aattatactt attagtcagt cagaaacaac 1080
tttggcacat atcaatatta tgctctcgtt aatcgcataa cttcgtataa tgtatgctat 1140
acgaagttat aattcagatc tttcggcgcg ccgggtcgac gcttctgagg cggagtcgag 1200
tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga 1260
acgatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc 1320
agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt 1380
ttatccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac 1440
gtatgtcgag gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac 1500
cgtcagatcg cctggagctc cccgggtctg gctaactaga gaacccactg cttactggct 1560
tatcgaaatt aatacgactc actataggga gacccaagct ggctagcacc atgggaccta 1620
agaaaaagag gaaggtggcg gccgctgatt acaaagacca cgacggtgac tacaaggatc 1680
atgacattga ctataaggat gacgacgata agtctagaga caagaaatac tctattggac 1740
tggatatcgg gacaaactcc gttggctggg ccgtcataac cgacgagtat aaggtgccaa 1800
gcaagaaatt caaggtgctg ggtaatactg accgccattc aatcaagaag aacctgatcg 1860
gagcactcct cttcgactcc ggtgaaaccg ctgaagctac tcggctgaag cggaccgcaa 1920
ggcggagata cacccgccgc aagaatcgga tatgttatct gcaagagatc tttagcaacg 1980
aaatggctaa ggtggacgac tccttctttc accgcctgga agagagcttt ctggtggagg 2040
aggataagaa acacgagagg caccctatat tcggaaatat cgtggatgag gtggcttacc 2100
atgaaaagta tcctacaatc taccatctga ggaagaagct ggtggacagc accgataaag 2160
cagacctgag gctcatctat ctggccctgg ctcatatgat aaagtttaga ggacactttc 2220
tgatcgaggg cgacctgaat cccgataatt ccgatgtgga taaactcttc attcaactgg 2280
tgcagacata taaccaactg ttcgaggaga atcccataaa cgcttctggt gtggatgcca 2340
aggctattct gtccgctcgg ctgtccaagt cacgcagact ggagaatctg attgcccaac 2400
tgccaggaga aaagaagaac ggcctgtttg ggaacctcat cgccctgagc ctgggcctga 2460
cacctaactt caagtccaat tttgatctgg ccgaagatgc taaactccag ctctccaagg 2520
acacctatga cgatgatctg gacaacctgc tcgcacagat aggcgaccag tacgccgatc 2580
tctttctggc tgctaagaat ctctccgacg ccattctgct gagcgacata ctccgggtca 2640
acactgagat caccaaagca cctctgagcg cctccatgat aaaacgctat gatgaacacc 2700
atcaagacct gactctgctc aaagccctcg tgaggcaaca gctgccagag aagtacaaag 2760
agatattctt cgaccagagc aagaatggat atgccggata catcgatggc ggagcatcac 2820
aggaagaatt ttacaagttc atcaaaccaa tcctcgagaa gatggacggt actgaagagc 2880
tgctggtgaa gctgaacagg gaggacctgc tgaggaagca gaggaccttt gataatggct 2940
ccattccaca tcagatacac ctgggagagc tgcatgcaat cctccgcagg caggaggatt 3000
tctatccttt cctgaaggat aaccgggaga agatagagaa gatcctgacc ttcaggatcc 3060
cttattacgt cggccctctg gctagaggca actcccgctt cgcttggatg accaggaaat 3120
ctgaggagac aattactcct tggaacttcg aagaggtcgt ggataagggc gcaagcgccc 3180
agtcattcat cgaacggatg accaatttcg ataagaacct gcccaacgag aaggtcctgc 3240
ccaaacattc actcctgtac gagtatttca ccgtctataa cgagctgact aaagtgaagt 3300
acgtgaccga gggcatgagg aagcctgcct tcctgtccgg agagcagaag aaggctatcg 3360
ttgatctgct cttcaagact aatagaaagg tgacagtgaa gcagctcaag gaggattact 3420
ttaagaagat cgaatgcttt gactcagtgg aaatctctgg cgtggaggac cgctttaatg 3480
ccagcctggg cacttaccat gatctgctga agataatcaa agacaaagat ttcctcgata 3540
atgaggagaa cgaggacatc ctggaagata tcgtgctgac cctgactctg ttcgaggata 3600
gagagatgat cgaagagcgc ctgaagacct atgcccatct gtttgacgat aaagtcatga 3660
aacagctcaa gcggcggcgc tacactgggt ggggtagact ctccaggaaa ctcataaacg 3720
gcatccgcga caaacagagc ggaaagacca tcctggattt cctgaaatcc gacggattcg 3780
ctaacaggaa cttcatgcaa ctgattcacg atgactctct gacatttaaa gaggacatcc 3840
agaaggcaca ggtgagcggt caaggcgaca gcctgcacga gcacatcgcc aacctcgctg 3900
gatcacccgc cataaagaag ggaatactgc agacagtcaa ggtcgtggac gaactcgtca 3960
aagtgatggg tcggcacaag ccagagaata tcgttatcga aatggcaagg gagaaccaaa 4020
ccacccagaa gggccagaag aactctcggg aacggatgaa aagaatcgaa gagggaatta 4080
aggagctggg atctcagata ctgaaggagc accctgtgga gaatacacag ctccagaacg 4140
agaaactcta cctgtactac ctccagaacg ggcgggacat gtacgttgac caggaactcg 4200
acatcaaccg gctgtccgat tatgacgtgg accatattgt tccacagtcc ttcctcaaag 4260
atgactccat tgacaacaag gtgctgacca gatccgataa gaatcgcggt aagtctgaca 4320
atgttccatc agaagaggtg gtcaagaaga tgaagaatta ctggcggcag ctcctcaacg 4380
ccaaactgat cacccagcgg aagtttgaca atctgactaa ggcagaaaga ggaggtctga 4440
gcgaactcga caaggccggc tttattaaga ggcaactggt cgaaacacgc cagattacca 4500
aacacgtggc acaaatcctc gactctagga tgaacactaa gtacgatgag aacgataagc 4560
tgatcaggga agtgaaagtg ataactctga agagcaagct ggtgtctgac ttccggaagg 4620
actttcaatt ctacaaagtt cgcgaaataa acaattacca tcatgctcac gatgcctatc 4680
tcaatgctgt cgttggcacc gccctgatca agaaataccc taaactggag tctgagttcg 4740
tgtacggtga ctataaagtc tacgatgtga ggaagatgat agcaaagtct gagcaagaga 4800
ttggcaaagc caccgccaag tacttcttct actctaatat catgaatttc tttaagactg 4860
agataaccct ggctaacggc gaaatccgga agcgcccact gatcgaaaca aacggagaaa 4920
caggagaaat cgtgtgggat aaaggcaggg acttcgcaac tgtgcggaag gtgctgtcca 4980
tgccacaagt caatatcgtg aagaagaccg aagtgcagac cggcggattc tcaaaggaga 5040
gcatcctgcc aaagcggaac tctgacaagc tgatcgccag gaagaaagat tgggacccaa 5100
agaagtatgg cggtttcgat tcccctacag tggcttattc cgttctggtc gtggcaaaag 5160
tggagaaagg caagtccaag aaactcaagt ctgttaagga gctgctcgga attactatta 5220
tggagagatc cagcttcgag aagaatccaa tcgatttcct ggaagctaag ggctataaag 5280
aagtgaagaa agatctcatc atcaaactgc ccaagtactc tctctttgag ctggagaatg 5340
gtaggaagcg gatgctggcc tccgccggag agctgcagaa aggaaacgag ctggctctgc 5400
cctccaaata cgtgaacttc ctgtatctgg cctcccacta cgagaaactc aaaggtagcc 5460
ctgaagacaa tgagcagaag caactctttg ttgagcaaca taaacactac ctggacgaaa 5520
tcattgaaca gattagcgag ttcagcaagc gggttattct ggccgatgca aacctcgata 5580
aagtgctgag cgcatataat aagcacaggg acaagccaat tcgcgaacaa gcagagaata 5640
ttatccacct ctttactctg actaatctgg gcgctcctgc tgccttcaag tatttcgata 5700
caactattga caggaagcgg tacacctcta ccaaagaagt tctcgatgcc accctgatac 5760
accagtcaat taccggactg tacgagactc gcatcgacct gtctcagctc ggcggcgacg 5820
gttctcccaa gaagaagagg aaagtctcga ccggtggatc cggcgcaaca aacttctctc 5880
tgctgaaaca agccggagat gtcgaagaga atcctggacc gatgaaaaag cctgaactca 5940
ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc 6000
agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg 6060
tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact 6120
ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc 6180
tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg 6240
aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc 6300
ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat 6360
ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg 6420
acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg 6480
actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg 6540
acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat 6600
acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc 6660
gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc 6720
tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag 6780
cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta 6840
cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg 6900
atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaataggtt taaacaactt 6960
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 7020
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 7080
tgtctggatc aagcttgcgg aacccttact cgagataact tcgtataatg tatgctatac 7140
gaagttatgc tagccaacaa gctcgtcatc gctttgcaga agagcagaga ggatatgctc 7200
atcgtctaaa gaactaccca ttttattata tattagtcac gatatctata acaagaaaat 7260
atatatataa taagttatca cgtaagtaga acatgaaata acaatataat tatcgtatga 7320
gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca cgcggtcgtt 7380
atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg agctccaagc 7440
ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa agagagagca 7500
atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt taaaaaagat 7560
ttgcgcttta ctcgacctaa actttaaaca cgtcatagaa tcttcgtttg acaaaaacca 7620
cattgtggcc aagctgtgtg acgcgacgcg cgctaaagaa tggcaaacca agtcgcgcga 7680
ggtacccagc ttttgttccc tttagtgagg gttaattccg agcttggcgt aatcatggtc 7740
atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 7800
aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 7860
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 7920
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 7980
ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 8040
acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 8100
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 8160
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 8220
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 8280
gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 8340
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 8400
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 8460
ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 8520
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 8580
gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 8640
ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 8700
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 8760
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 8820
cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 8880
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 8940
tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 9000
gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 9060
agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 9120
tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 9180
agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 9240
gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 9300
catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 9360
ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 9420
atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 9480
tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 9540
cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 9600
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 9660
atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 9720
aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 9780
ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 9840
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 9890
<210> 5
<211> 7379
<212> DNA
<213> Artificial Sequence
<400> 5
tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 60
tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 120
cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 180
gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 240
ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300
tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360
cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420
ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480
gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540
ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600
catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660
tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720
ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780
cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc 840
cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta 900
gcgtttaaac ttaagcttgg taccgagctc ggatccacca tgggggagaa acccgggacc 960
agggtcttca agaagtcgag ccctaactgc aagctcaccg tgtacttggg caagcgggac 1020
ttcgtagatc acctggacaa agtggaccct gtagatggcg tggtgcttgt ggaccctgac 1080
tacctgaagg accgcaaagt gtttgtgacc ctcacctgcg ccttccgcta tggccgtgaa 1140
gacctggatg tgctgggctt gtccttccgc aaagacctgt tcatcgccac ctaccaggcc 1200
ttccccccgg tgcccaaccc mccccggccc cccacccgcc tgcaggaccg gctgctgagg 1260
aagctgggcc agcatgccca ccccttcttc ttcaccatac cccagaatct tccatgctcc 1320
gtcacactgc agccaggccc agaggataca ggaaaggcct gcggcgtaga ctttgagatt 1380
cgagccttct gtgctaaatc actagaagag aaaagccaca aaaggaactc tgtgcggctg 1440
gtgatccgaa aggtgcagtt cgccccggag aaacccggcc cccagccttc agccgaaacc 1500
acacgccact tcctcatgtc tgaccggtcc ctgcacctcg aggcttccct ggacaaggag 1560
ctgtactacc atggggagcc cctcaatgta aatgtccacg tcaccaacaa ctccaccaag 1620
accgtcaaga agatcaaagt ctctgtgaga cagtacgccg acatctgcct cttcagcacc 1680
gcccagtaca agtgtcctgt ggctcaactc gaacaagatg accaggtatc tcccagctcc 1740
acattctgta aggtgtacac cataacccca ctgctcagcg acaaccggga gaagcggggt 1800
ctcgccctgg atgggaaact caagcacgag gacaccaacc tggcttccag caccatcgtg 1860
aaggagggtg ccaacaagga ggtgctggga atcctggtgt cctacagggt caaggtgaag 1920
ctggtggtgt ctcgaggcgg ggatgtctct gtggagctgc cttttgttct tatgcacccc 1980
aagccccacg accacatccc cctccccaga ccccagtcag ccgctccgga gacagatgtc 2040
cctgtggaca ccaacctcat tgaatttgat accaactatg ccacagatga tgacattgtg 2100
tttgaggact ttgcccggct tcggctgaag gggatgaagg atgacgacta tgatgatcaa 2160
ctctgcggat ccggtgagag tctcttcaaa gggccgcgcg attataaccc catctcatcc 2220
acaatttgtc acctcacaaa tgagtccgat ggacatacaa caagccttta cggcattggc 2280
ttcggtccat tcatcatcac caacaagcat cttttcagac gcaataacgg caccttgctg 2340
gtgcagtccc tccacggcgt ctttaaagtc aagaacacca cgacccttca gcaacacctg 2400
atcgacggaa gggatatgat tatcattaga atgcccaaag atttcccgcc atttcctcag 2460
aaactcaagt ttagagagcc acagagggaa gaaaggatct gcttggtgac aacaaatttt 2520
caaactaagt ctatgtcatc catggtatca gataccagct gtacgtttcc tagttcagac 2580
ggaatattct ggaagcattg gatacagaca aaggacggcc aatgcggtag tccactggtg 2640
tctactcggg atggtttcat cgtcggcatt catagcgcca gcaacttcac caatactaat 2700
aactacttca ccagcgtgcc caagaatttc atggagcttc tgacaaacca ggaagcccag 2760
cagtgggttt ctggatggcg cttgaacgct gactccgttc tgtggggagg ccataaagtg 2820
tttatgagta aacctgaaga accctttcag ccggtgaaag aggccactca gcttatgaat 2880
gagctggttt atagccagta ggaattctgc agatatccag cacagtggcg gccgctcgag 2940
tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca 3000
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3060
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3120
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3180
ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 3240
tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3300
gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3360
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3420
cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 3480
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3540
aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 3600
gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 3660
aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 3720
gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 3780
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3840
caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 3900
attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 3960
cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 4020
agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt 4080
ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 4140
tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 4200
tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 4260
aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 4320
ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 4380
ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 4440
caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 4500
atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 4560
acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 4620
ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 4680
aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 4740
aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 4800
gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 4860
ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 4920
caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 4980
aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 5040
cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 5100
cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5160
catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 5220
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5280
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5340
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 5400
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 5460
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5520
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 5580
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 5640
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 5700
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 5760
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 5820
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 5880
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 5940
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6000
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6060
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6120
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6180
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6240
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6300
aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6360
atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 6420
gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 6480
acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 6540
ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 6600
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 6660
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 6720
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 6780
atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 6840
taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 6900
catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 6960
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7020
acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7080
aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7140
ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7200
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 7260
atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 7320
ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacg 7379
<210> 6
<211> 6284
<212> DNA
<213> Artificial Sequence
<400> 6
ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct 480
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600
ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tggagctcgg 660
tattcacgac agcaggctga ataataaaaa aattagaaac tattatttaa ccctagaaag 720
ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc atgtgtttta 780
tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt 840
acacttacat actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa 900
aaaaaaacaa aaactcaaaa tttcttctat aaagtaacaa aacttttaaa cattctctct 960
tttacaaaaa taaacttatt ttgtacttta aaaacagtca tgttgtatta taaaataagt 1020
aattagctta acctatacat aatagaaaca aattatactt attagtcagt cagaaacaac 1080
tttggcacat atcaatatta tgctctcgtt aatcgcataa cttcgtataa tgtatgctat 1140
acgaagttat aattcagatc tttcggcgcg ccgggtcgac gcttctgagg cggagtcgag 1200
tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga 1260
acgatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc 1320
agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt 1380
ttatccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac 1440
gtatgtcgag gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac 1500
cgtcagatcg cctggagctc cccggggcca ccatggtgag caagggcgag gagctgttca 1560
ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 1620
tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 1680
ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 1740
agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 1800
ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 1860
gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 1920
acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 1980
acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 2040
acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 2100
gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 2160
aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 2220
tcactctcgg catggacgag ctgtacaagg gatccggcgc aacaaacttc tctctgctga 2280
aacaagccgg agatgtcgaa gagaatcctg gaccgatgaa aaagcctgaa ctcaccgcga 2340
cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct 2400
cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc 2460
gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat 2520
cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct 2580
attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc 2640
ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc 2700
agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg 2760
atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca 2820
ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc 2880
ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg 2940
gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg 3000
tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact 3060
tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca 3120
ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg 3180
cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa 3240
tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg 3300
gaaaccgacg ccccagcact cgtccgaggg caaaggaata ggtttaaaca acttgtttat 3360
tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 3420
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 3480
gatcaagctt gcggaaccct tactcgagat aacttcgtat aatgtatgct atacgaagtt 3540
atgctagcca acaagctcgt catcgctttg cagaagagca gagaggatat gctcatcgtc 3600
taaagaacta cccattttat tatatattag tcacgatatc tataacaaga aaatatatat 3660
ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa 3720
tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt 3780
caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac 3840
tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt 3900
caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaaaaa agatttgcgc 3960
tttactcgac ctaaacttta aacacgtcat agaatcttcg tttgacaaaa accacattgt 4020
ggccaagctg tgtgacgcga cgcgcgctaa agaatggcaa accaagtcgc gcgaggtacc 4080
cagcttttgt tccctttagt gagggttaat tccgagcttg gcgtaatcat ggtcatagct 4140
gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 4200
aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 4260
actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 4320
cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 4380
gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 4440
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 4500
caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 4560
gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 4620
ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 4680
cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 4740
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4800
cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4860
acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 4920
aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 4980
atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5040
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 5100
gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 5160
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 5220
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 5280
ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 5340
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 5400
accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 5460
atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 5520
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 5580
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 5640
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 5700
gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 5760
agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 5820
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 5880
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 5940
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 6000
gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 6060
tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 6120
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 6180
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 6240
acaaataggg gttccgcgca catttccccg aaaagtgcca cctg 6284
<210> 7
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 7
gtgaccggcg cctactctag agccaccatg ggggagaaac ccgggac 47
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 8
cccgccgccg gatccgcaga gttgatcatc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 9
gatgatcaac tctgcggatc cggcggcggg 30
<210> 10
<211> 75
<212> DNA
<213> Artificial Sequence
<400> 10
ccgcggatcc ttcgaattca gcgtagtctg ggacgtcgta tgggtacgaa cccccgccgc 60
cttgcgagta cacca 75
<210> 11
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 11
tggagctccc cggggccacc atggtgagca agggc 35
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 12
gcgccggatc ccttgtacag ctcgtcc 27
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 13
ctccacgctt tgcctgaccc tgctt 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 14
ccagcacatc caggtcttca cggcc 25
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 15
ggcatattct ggaagcattg gattc 25
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 16
gagttcttgc agctcggtga 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 17
cttcgggcat ggcggacttg 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 18
cttcgggcat ggcggacttg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 19
gtgctgctgc ccgacaacca 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 20
cgccctccga gagctgcatc 20
Claims (7)
1. a kind of GPCR targeted drugs screening system, which is characterized in that including：By β-arrestin and etch virus of tobacco toxalbumin
PB- β-arrestin-TEV the Nia of enzyme connection composition；And Tet response elements are connected to the PB-TRE-GFP of composition with GFP.
2. GPCR targeted drugs screening system as described in claim 1, which is characterized in that the PB- β-arrestin-
TEV Nia and PB-TRE-GFP are using Piggybac as carrier.
3. GPCR targeted drugs screening system as described in claim 1, which is characterized in that the PB- β-arrestin-
TEV Nia and PB-TRE-GFP sequences are respectively SEQ ID NO.2 and SEQ ID NO.5.
4. GPCR targeted drugs screening system as described in claim 1, which is characterized in that the reporter gene of the system is GFP,
Result visualization.
5. GPCR targeted drugs screening system as described in claim 1, which is characterized in that the GPCR targeted drugs screening
System further includes ADORA1 receptor plasmids.
6. a kind of structure of the cell line for the GPCR targeted drug screening systems that can be expressed described in any one of claim 1-5
Method, which is characterized in that including：Using PiggyBac transposons as carrier, by PB- β-arrestin-TEV Nia and PB-TRE-
GFP is imported in cell strain genome and is integrated, and obtains the cell line that can express Piggybac-Tango systems.
7. GPCR targeted drugs screening system described in any one of claim 1-5 or can express in claim 1-5 is appointed
Application of the cell line of GPCR targeted drug screening systems described in one in the screening of GPCR targeted drugs.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810600546.9A CN108753727A (en) | 2018-06-11 | 2018-06-11 | A kind of GPCR targeted drugs screening system and its structure and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810600546.9A CN108753727A (en) | 2018-06-11 | 2018-06-11 | A kind of GPCR targeted drugs screening system and its structure and application |
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|---|---|
| CN108753727A true CN108753727A (en) | 2018-11-06 |
Family
ID=64021149
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810600546.9A Pending CN108753727A (en) | 2018-06-11 | 2018-06-11 | A kind of GPCR targeted drugs screening system and its structure and application |
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| CN (1) | CN108753727A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996214A (en) * | 2020-07-29 | 2020-11-27 | 温州医科大学 | Establishment and application of inducible high-efficiency expression gene system in human pluripotent stem cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086499A (en) * | 2006-06-07 | 2007-12-12 | 努特诺瓦营养产品及食品成分有限公司 | Screening methods for compounds that modulate the activity of g-protein coupled receptors |
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN102943092A (en) * | 2012-11-20 | 2013-02-27 | 西北农林科技大学 | General type PiggyBac transposon transgenosis carrier and preparation method thereof |
| WO2017207992A1 (en) * | 2016-06-01 | 2017-12-07 | Ucl Business Plc | Cell expressing car and gpcr |
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2018
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|---|---|---|---|---|
| CN101086499A (en) * | 2006-06-07 | 2007-12-12 | 努特诺瓦营养产品及食品成分有限公司 | Screening methods for compounds that modulate the activity of g-protein coupled receptors |
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN102943092A (en) * | 2012-11-20 | 2013-02-27 | 西北农林科技大学 | General type PiggyBac transposon transgenosis carrier and preparation method thereof |
| WO2017207992A1 (en) * | 2016-06-01 | 2017-12-07 | Ucl Business Plc | Cell expressing car and gpcr |
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| CN111996214A (en) * | 2020-07-29 | 2020-11-27 | 温州医科大学 | Establishment and application of inducible high-efficiency expression gene system in human pluripotent stem cells |
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Application publication date: 20181106 |