pRetroX-Tet-On Advanced载体描述
pRetroX-Tet-On Advanced is a Retro-X Q retroviral vector that expresses rtTA-Advanced, an improved version of the reverse tetracycline(Tet)-controlled transactivator protein rtTA (1–4). rtTA-Advanced is more sensitive to doxycycline (Dox) and yields lower background expression than the original rtTA. The rtTA-Advanced protein is a fusion of the Tet repressor (TetR) DNA-binding domain, and three minimal “F”-type transcriptional activation domains from the herpes simplex virus VP16 protein. The gene encoding rtTA-Advanced is completely synthetic, lacks cryptic splice sites, and utilizes human codon preferences for stable expression in mammalian cells. Expression of rtTA-Advanced is driven by the powerful, constitutively active CMV promoter.
As with all of our Retro-X Q vectors, pRetroX-Tet-On Advanced uses LTR self-inactivation to eliminate promoter interference. In LTR self-inactivation, the mechanism of viral integration is used to introduce a deletion into the 5’ LTR. As a result of this mutation, the 5’ LTR CMV/MSV promoter, which drives high expression of the complete viral genome in packaging cells, is inactive in stably transduced target cells. This allows the expression of rtTA-Advanced (from PCMV IE) to proceed unimpeded (5, 6). The vector also contains a neomycin resistance gene (Neor) that allows G418 selection of stably transduced cells. rtTA-Advanced and the Neor marker are coexpressed from a bicistronic transcript containing an internal ribosome entry site (IRES).This ensures that a high frequency of G418 resistant clones express the Tet-On Advanced transactivator.
pRetroX-Tet-On Advanced contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, a packaging signal (Ψ+), and a tRNA primer binding site. The vector also contains an SV40 origin of replication for plasmid propagation in mammalian cells that express SV40 T antigen. In addition, a pUC origin of replication and an E. coli Ampr gene allow for propagation and selection in bacteria.
The pRetroX-Tet-On Advanced vector is used in conjunction with Tet response vectors to create double stable Retro-X Tet-On Advanced cell lines. Such cell lines are essentially Doxycycline (Dox)-controlled gene expression systems in which the Tet response vector expresses a gene of interest under the control of a Tet-responsive element (TRE; e.g. PTight; 7, 8). In the presence of Dox, rtTA-Advanced binds to the TRE and activates expression of the gene of interest. In the absence of Dox, rtTA-Advanced is unable to bind to the TRE, and the system is inactive. Additional information on TRE-containing vectors and protocols describing how to construct a Retro-X Tet-On Advanced cell line can be found in the Retro-X Tet-On Advanced Inducible Gene Expression System User manual (PT3958-1).
Selection of Stable Transfectants
Selectable marker: plasmid confers resistance to G418.
Propagation in E. coli
Suitable host strains: DH5α, DH10B and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
LOCUS pRetroX-Tet-On Advanced 8152 bp DNA SYN
DEFINITION pRetroX-Tet-On Advanced
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..8152
/organism="pRetroX-Tet-On Advanced"
/mol_type="other DNA"
promoter 2..506
/label="CMV_immearly_promoter"
misc_feature 6..688
/label="5_LTR"
misc_feature 9..296
/label="CAG_enhancer"
misc_feature 463..483
/label="CMV_fwd_primer"
misc_feature 758..1567
/label="psi_plus_pack"
misc_feature 1154..1561
/label="gag"
misc_feature 1497..1519
/label="MSCV_primer"
misc_feature 1497..1519
/label="pLXSN_5_primer"
misc_feature 1535..1551
/label="pBABE_5_primer"
misc_feature 1608..1895
/label="CAG_enhancer"
promoter 1613..2105
/label="CMV_immearly_promoter"
misc_feature 2062..2082
/label="CMV_fwd_primer"
promoter 2063..2132
/label="CMV_promoter"
promoter 2075..2194
/label="CMV2_promoter"
misc_feature 2106..2125
/label="pCEP_fwd_primer"
misc_feature 2108..2132
/label="LNCX_primer"
CDS 2287..3033
/label="ORF frame 1"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLG
TRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERET
PTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLD
MLPADALDDFDLDMLPADALDDFDLDMLPG*"
misc_feature 3071..3622
/label="IRES"
/translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLG
TRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERET
PTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLD
MLPADALDDFDLDMLPADALDDFDLDMLPG*"
CDS 3647..4441
/label="ORF frame 2"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
gene 3662..4438
/label="NeoR/KanR"
/gene="NeoR/KanR"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
misc_feature complement(5386..5408)
/label="pGEX_3_primer"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
misc_feature complement(5544..5564)
/label="pBABE_3_primer"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
misc_feature complement(5550..5765)
/label="SV40_enhancer"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
promoter 5562..5830
/label="SV40_promoter"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
rep_origin 5729..5806
/label="SV40_origin"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
misc_feature 5791..5810
/label="SV40pro_F_primer"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
gene complement(6930..7790)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
CDS complement(6930..7790)
/label="ORF frame 3"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(7832..7860)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter 8082..506
/label="CMV_immearly_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
misc_feature 8146..688
/label="5_LTR"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC
61 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC
121 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA
181 TGCCAAGTAC GCCCCCTATT GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC
241 AGTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA
301 TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC
361 GGGGATTTCC AAGTCTCCAC CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC
421 AACGGGACTT TCCAAAATGT CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC
481 GTGTACGGTG GGAGGTCTAT ATAAGCAGAG CTCAATAAAA GAGCCCACAA CCCCTCACTC
541 GGCGCGCCAG TCTTCCGATA GACTGCGTCG CCCGGGTACC CGTATTCCCA ATAAAGCCTC
601 TTGCTGTTTG CATCCGAATC GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC TCTGAGTGAT
661 TGACTACCCA CGACGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATTTGG AGACCCCTGC
721 CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCCA GCAACTTATC TGTGTCTGTC
781 CGATTGTCTA GTGTCTATGT TTGATGTTAT GCGCCTGCGT CTGTACTAGT TAGCTAACTA
841 GCTCTGTATC TGGCGGACCC GTGGTGGAAC TGACGAGTTC TGAACACCCG GCCGCAACCC
901 TGGGAGACGT CCCAGGGACT TTGGGGGCCG TTTTTGTGGC CCGACCTGAG GAAGGGAGTC
961 GATGTGGAAT CCGACCCCGT CAGGATATGT GGTTCTGGTA GGAGACGAGA ACCTAAAACA
1021 GTTCCCGCCT CCGTCTGAAT TTTTGCTTTC GGTTTGGAAC CGAAGCCGCG CGTCTTGTCT
1081 GCTGCAGCGC TGCAGCATCG TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC
1141 TGAAAATTAG GGCCAGACTG TTACCACTCC CTTAAGTTTG ACCTTAGGTC ACTGGAAAGA
1201 TGTCGAGCGG ATCGCTCACA ACCAGTCGGT AGATGTCAAG AAGAGACGTT GGGTTACCTT
1261 CTGCTCTGCA GAATGGCCAA CCTTTAACGT CGGATGGCCG CGAGACGGCA CCTTTAACCG
1321 AGACCTCATC ACCCAGGTTA AGATCAAGGT CTTTTCACCT GGCCCGCATG GACACCCAGA
1381 CCAGGTCCCC TACATCGTGA CCTGGGAAGC CTTGGCTTTT GACCCCCCTC CCTGGGTCAA
1441 GCCCTTTGTA CACCCTAAGC CTCCGCCTCC TCTTCCTCCA TCCGCCCCGT CTCTCCCCCT
1501 TGAACCTCCT CGTTCGACCC CGCCTCGATC CTCCCTTTAT CCAGCCCTCA CTCCTTCTCT
1561 AGGCGCCGGA ATTGAAGATC TGGGGGATCG ATCCTCTAGA GTCCGTTACA TAACTTACGG
1621 TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT
1681 ATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC
1741 GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG
1801 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT
1861 TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT
1921 GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC
1981 CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC
2041 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA
2101 TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG
2161 ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCGG CCGGGAACGG TGCATTGGAA
2221 CGGACCTGCA GGAATTGATC CGCGGCCGCA CCGGTAGGCC TCGTACGCTT AATTAACGGA
2281 TCCACCATGT CTAGACTGGA CAAGAGCAAA GTCATAAACG GCGCTCTGGA ATTACTCAAT
2341 GGAGTCGGTA TCGAAGGCCT GACGACAAGG AAACTCGCTC AAAAGCTGGG AGTTGAGCAG
2401 CCTACCCTGT ACTGGCACGT GAAGAACAAG CGGGCCCTGC TCGATGCCCT GCCAATCGAG
2461 ATGCTGGACA GGCATCATAC CCACTTCTGC CCCCTGGAAG GCGAGTCATG GCAAGACTTT
2521 CTGCGGAACA ACGCCAAGTC ATTCCGCTGT GCTCTCCTCT CACATCGCGA CGGGGCTAAA
2581 GTGCATCTCG GCACCCGCCC AACAGAGAAA CAGTACGAAA CCCTGGAAAA TCAGCTCGCG
2641 TTCCTGTGTC AGCAAGGCTT CTCCCTGGAG AACGCACTGT ACGCTCTGTC CGCCGTGGGC
2701 CACTTTACAC TGGGCTGCGT ATTGGAGGAA CAGGAGCATC AAGTAGCAAA AGAGGAAAGA
2761 GAGACACCTA CCACCGATTC TATGCCCCCA CTTCTGAGAC AAGCAATTGA GCTGTTCGAC
2821 CGGCAGGGAG CCGAACCTGC CTTCCTTTTC GGCCTGGAAC TAATCATATG TGGCCTGGAG
2881 AAACAGCTAA AGTGCGAAAG CGGCGGGCCG GCCGACGCCC TTGACGATTT TGACTTAGAC
2941 ATGCTCCCAG CCGATGCCCT TGACGACTTT GACCTTGATA TGCTGCCTGC TGACGCTCTT
3001 GACGATTTTG ACCTTGACAT GCTCCCCGGG TAACTAGAAT TCCGCCCCCC CCCCCCCCCC
3061 CCCTCTCCCT CCCCCCCCCC TAACGTTACT GGCCGAAGCC GCTTGGAATA AGGCCGGTGT
3121 GCGTTTGTCT ATATGTTATT TTCCACCATA TTGCCGTCTT TTGGCAATGT GAGGGCCCGG
3181 AAACCTGGCC CTGTCTTCTT GACGAGCATT CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA
3241 ATGCAAGGTC TGTTGAATGT CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA
3301 ACAACGTCTG TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC
3361 TGCGGCCAAA AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC CCAGTGCCAC
3421 GTTGTGAGTT GGATAGTTGT GGAAAGAGTC AAATGGCTCT CCTCAAGCGT ATTCAACAAG
3481 GGGCTGAAGG ATGCCCAGAA GGTACCCCAT TGTATGGGAT CTGATCTGGG GCCTCGGTGC
3541 ACATGCTTTA CATGTGTTTA GTCGAGGTTA AAAAAACGTC TAGGCCCCCC GAACCACGGG
3601 GACGTGGTTT TCCTTTGAAA AACACGATGA TAAGCTTGCC ACAACCATGG CTGAACAAGA
3661 TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC
3721 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC
3781 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC
3841 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC
3901 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC
3961 TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC
4021 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG
4081 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT
4141 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT
4201 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG
4261 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC
4321 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG
4381 TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG
4441 AGCGGGATCG GCTAGAGTCG ACCCGGACGG GACGCTCCTG CGCCTGATAC AGAACGAATT
4501 GCTTGCAGGC ATCTCATGAG TGTGTCTTCC CGTTTTCCGC CTGAGGTCAC TGCGTGGATG
4561 GAGCGCTGGC GCCTGCTGCG CGACGGCGAG CTGCTCACCA CCCACTCGAG GGCTGCAGCG
4621 CTGCAGAGGC CGAGTGCAGA ACTGCTCCAA AGGGACCTCA AGGCTTTCCG AGGGACACTA
4681 GGCTGACTCC ATCGAGCCAG TGTAGAGATA AGCTTATCGA TTAGTCCAAT TTGTTAAAGA
4741 CAGGATATCA GTGGTCCAGG CTCTAGTTTT GACTCAACAA TATCACCAGC TGAAGCCTAT
4801 AGAGTACGAG CCATAGATAA AATAAAAGAT TTTATTTAGT CTCCAGAAAA AGGGGGGAAT
4861 GAAAGACCCC ACCTGTAGGT TTGGCAAGCT AGCTTAAGTA ACGCCATTTT GCAAGGCATG
4921 GAAAAATACA TAACTGAGAA TAGAGAAGTT CAGATCAAGG TCAGGAACAG ATGGAACAGG
4981 GTCGACCCTA GAGAACCATC AGATGTTTCC AGGGTGCCCC AAGGACCTGA AATGACCCTG
5041 TGCCTTATTT GAACTAACCA ATCAGTTCGC TTCTCGCTTC TGTTCGCGCG CTTCTGCTCC
5101 CCGAGCTCAA TAAAAGAGCC CACAACCCCT CACTCGGGGC GCCAGTCCTC CGATTGACTG
5161 AGTCGCCCGG GTACCCGTGT ATCCAATAAA CCCTCTTGCA GTTGCATCCG ACTTGTGGTC
5221 TCGCTGTTCC TTGGGAGGGT CTCCTCTGAG TGATTGACTA CCCGTCAGCG GGGGTCTTTC
5281 ATTTGGGGGC TCGTCCGGGA TCGGGAGACC CCTGCCCAGG GACCACCGAC CCACCACCGG
5341 GAGGTAAGCT GGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA
5401 GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA
5461 GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC ATGACCCAGT CACGTAGCGA
5521 TAGCGGAGTG TAGATCCGGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG
5581 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG
5641 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC
5701 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA
5761 TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC
5821 CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA
5881 GCTTACTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG
5941 TGTGAAATAC CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC
6001 TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA
6061 AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA
6121 AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG
6181 CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG
6241 ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT
6301 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT
6361 TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC
6421 TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT
6481 GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT
6541 AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC
6601 TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA
6661 AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT
6721 TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT
6781 ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA
6841 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA
6901 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC
6961 TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT
7021 ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC
7081 TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT
7141 GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA
7201 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTGCAGG CATCGTGGTG
7261 TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT
7321 ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC
7381 AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT
7441 ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC
7501 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAACACG GGATAATACC
7561 GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA
7621 CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC
7681 TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA
7741 AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT
7801 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA
7861 TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT
7921 GACGTCTAAG AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG
7981 CCCTTTCGTC TTCAAGAATT AGCTTGGCCA TTGCATACGT TGTATCCATA TCATAATATG
8041 TACATTTATA TTGGCTCATG TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT
8101 TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GT
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