| 出品公司: | Addgene |
|---|---|
| 试剂名称: | pCMV-VSV-G |
| 质粒类型: | 哺乳动物载体；逆病毒包装；双质粒包装系统；信封载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 克隆方法: | 限制性内切酶，多克隆位点 |
| 启动子: | CMV |
| 载体大小: | 6507 bp |
| 5' 测序引物及序列: | CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’ |
| 3' 测序引物及序列: | -- |
| 载体标签: | 无 |
| 载体抗性: | Ampicillin |
| 筛选标记: | 无 |
| 克隆菌株: | DH5α等 |
| 宿主细胞（系）: | 包装细胞系如293T |
| 备注: | 逆病毒包装载体pCMV-VSV-G需要与pUMVC共同使用，使用方法见下文。 |
| 产品目录号: | #8454 |
| 稳定性: | 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
双质粒系统逆病毒包装质粒pCMV-VSV-G使用方法
Day 1
1. Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if desired)
Day 2
1. Set up (use polypropylene tubes for this; polystyrene tubes DO NOT work!):
1 µg retroviral DNA encoding gene X
1 µg packaging plasmid Mix
the packaging plasmid (pUMVC3) at a 8:1 ratio with the envelope plasmid (pCVM-VSV-G)-a total of 1µg
DME without serum to 94μL total
6 μL Fugene
Mix and wait 15 to 30 minutes at room temperature
Add to 293T cells without touching the sides of the dish (DO NOT CHANGE MEDIA)
If you are using amphotropic virus then move immediately to BL2+ in a secondary container, which has an absorbent material.
(This does not mean a couple of hours; it means Immediately!). The rest of this protocol is the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic viruses
Day 3
1. Change the media to whatever media you wish to use when infecting target cells. 293T cells are easily detached so remember not to put the media directly onto to cells, but rather “run” it down the side of the dish. Remember that you will get the highest titer virus when your cells are “happy.”
2. Plate out your target cells
Day 4
1. Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virus
making it noninfectious
Note: After filtering, the filter should be removed and placed in the biohazard bag in the hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to place a plastic beaker with bleach in the hood in advance
2. Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target cells
3. Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media
Note: If you wish to do a second infection the following day, it is important to put fresh media on the cells and not let the virus remain on the cells overnight. The media contains huge amounts of envelope, both associated and unassociated with viral particles, which will bind all the cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if you don’t change the media after the initial infection, very few receptors will be available for the next round of infection. In addition, very few cells tolerate the presence of high levels of the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)
Day 5 +
1. Allow the cells to recover and begin to express the virus-encoded genes. The cells usually require 48 hours for this to occur
2. Add drug if you are scoring for the presence of a vector that carries a drug resistance marker. Prior to this step it is advisable that you titrate the drug to be used for selection in order to know precisely how much to add. In addition, it is necessary to bring an extra plate of uninfected cells (often referred to as “canaries”) which will function as a positive control in the kill assay. Add drug to both plates. When your canaries are dead, you can remove the drug.
载体序列
Please note that depositor's sequence is not 100% complete. The flanking sequence of VSV-G actually contains XhoI (at both the 5' and 3'end).
LOCUS pCMV-VSV-G Sequencing Result 6507 bp DNA SYN
DEFINITION pCMV-VSV-G Sequencing Result
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..6507
/organism="pCMV-VSV-G Sequencing Result"
/mol_type="other DNA"
promoter 84..660
/label="CMV_immearly_promoter"
misc_feature 148..974
/label="5_LTR"
misc_feature 163..450
/label="CAG_enhancer"
misc_feature 617..637
/label="CMV_fwd_primer"
promoter 618..687
/label="CMV_promoter"
promoter 630..749
/label="CMV2_promoter"
misc_feature 661..680
/label="pCEP_fwd_primer"
misc_feature 663..687
/label="LNCX_primer"
misc_feature 778..1350
/label="bGlob_int"
CDS 1322..2971
/label="ORF frame 2"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
gene 1436..2959
/label="vsv-G"
/gene="vsv-G"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
misc_feature 2936..2968
/label="VSVG Tag"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
terminator 3118..3644
/label="rb_glob_PA_terminator"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
misc_feature complement(3689..3711)
/label="M13_pUC_rev_primer"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
promoter complement(3725..3754)
/label="lac_promoter"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
gene complement(4837..5697)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
CDS complement(4837..5697)
/label="ORF frame 1"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(5739..5767)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
misc_feature complement(6284..6439)
/label="lacZ_a"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
misc_feature 6410..6432
/label="M13_pUC_fwd_primer"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter 6425..6441
/label="M13_forward20_primer"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter 6451..6469
/label="T7_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta tattggctca
61 tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt
121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
421 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
481 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
541 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
601 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
661 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
721 catagaagac accgggaccg atccagcctc cggtcgaccg atcctgagaa cttcagggtg
781 agtttgggga cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga
841 gggggcaaag ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga
901 ccctcatgat aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt
961 attttctttt cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt
1021 tttaaattca cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt
1081 caaggcaatc agggtatatt atattgtact tcagcacagt tttagagaac aattgttata
1141 attaaatgat aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt
1201 attggtagaa acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga
1261 tatacactgt ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac
1321 catgttcatg ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg
1381 tctcatcatt ttggcaaaga attcctcgac ggatccctcg aggaattctg acactatgaa
1441 gtgccttttg tacttagcct ttttattcat tggggtgaat tgcaagttca ccatagtttt
1501 tccacacaac caaaaaggaa actggaaaaa tgttccttct aattaccatt attgcccgtc
1561 aagctcagat ttaaattggc ataatgactt aataggcaca gccttacaag tcaaaatgcc
1621 caagagtcac aaggctattc aagcagacgg ttggatgtgt catgcttcca aatgggtcac
1681 tacttgtgat ttccgctggt atggaccgaa gtatataaca cattccatcc gatccttcac
1741 tccatctgta gaacaatgca aggaaagcat tgaacaaacg aaacaaggaa cttggctgaa
1801 tccaggcttc cctcctcaaa gttgtggata tgcaactgtg acggatgccg aagcagtgat
1861 tgtccaggtg actcctcacc atgtgctggt tgatgaatac acaggagaat gggttgattc
1921 acagttcatc aacggaaaat gcagcaatta catatgcccc actgtccata actctacaac
1981 ctggcattct gactataagg tcaaagggct atgtgattct aacctcattt ccatggacat
2041 caccttcttc tcagaggacg gagagctatc atccctggga aaggagggca cagggttcag
2101 aagtaactac tttgcttatg aaactggagg caaggcctgc aaaatgcaat actgcaagca
2161 ttggggagtc agactcccat caggtgtctg gttcgagatg gctgataagg atctctttgc
2221 tgcagccaga ttccctgaat gcccagaagg gtcaagtatc tctgctccat ctcagacctc
2281 agtggatgta agtctaattc aggacgttga gaggatcttg gattattccc tctgccaaga
2341 aacctggagc aaaatcagag cgggtcttcc aatctctcca gtggatctca gctatcttgc
2401 tcctaaaaac ccaggaaccg gtcctgcttt caccataatc aatggtaccc taaaatactt
2461 tgagaccaga tacatcagag tcgatattgc tgctccaatc ctctcaagaa tggtcggaat
2521 gatcagtgga actaccacag aaagggaact gtgggatgac tgggcaccat atgaagacgt
2581 ggaaattgga cccaatggag ttctgaggac cagttcagga tataagtttc ctttatacat
2641 gattggacat ggtatgttgg actccgatct tcatcttagc tcaaaggctc aggtgttcga
2701 acatcctcac attcaagacg ctgcttcgca acttcctgat gatgagagtt tattttttgg
2761 tgatactggg ctatccaaaa atccaatcga gcttgtagaa ggttggttca gtagttggaa
2821 aagctctatt gcctcttttt tctttatcat agggttaatc attggactat tcttggttct
2881 ccgagttggt atccatcttt gcattaaatt aaagcacacc aagaaaagac agatttatac
2941 agacatagag atgaaccgac ttggaaagta actcaaatcc tgcacaacag attcttcatg
3001 tttggaccaa atcaacttgt gataccatgc tcaaagaggc ctcaattata tttgagtttt
3061 taatttttat gaaaaaaaaa aaaaaaaacg gaattcctcg agggatccgt cgaggaattc
3121 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
3181 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc
3241 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
3301 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
3361 aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa
3421 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
3481 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
3541 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
3601 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cggatcggcc
3661 gcaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
3721 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
3781 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
3841 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
3901 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
3961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
4021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
4081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
4141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
4201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
4261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
4321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
4381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
4441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
4501 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
4561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
4621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
4681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
4741 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
4801 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
4861 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
4921 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
4981 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
5041 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
5101 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
5161 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
5221 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
5281 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
5341 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
5401 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
5461 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
5521 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
5581 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
5641 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
5701 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
5761 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
5821 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
5881 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
5941 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
6001 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
6061 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
6121 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
6181 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
6241 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
6301 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
6361 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
6421 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag
6481 ctccaccgcg gtggcggccg ctctaga
//
