pIREShyg3

pIREShyg3

载体基本信息

出品公司:	Ybscience
载体名称:	pIREShyg3
质粒类型:	哺乳动物细胞表达载体
启动子:	CMV
表达水平:	高
克隆方法:	多克隆位点，限制性内切酶
载体大小:	5752 bp
5' 测序引物:	CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'
3' 测序引物:	pIRES-R:5'-GCCCTAGATGCATGCTCG-3'
载体标签:	无
载体抗性:	氨苄青霉素（Ampicillin）
筛选标记:	潮霉素（Hygromycin）
备注:	pIREShyg3载体含有IRES元件，目的基因与抗性基因以双顺反子的形式共转录。
稳定性:	稳表达 或 瞬表达
组成型:	组成型
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

Description
pIREShyg3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with hygromycin B, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the hygromycin B phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIREShyg3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the hygromycin B phosphotransferase gene, and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker.  

Use
When using the pIREShyg3 Vector, the antibiotic exerts selective pressure on the entire expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable over time in culture. Unless your expression experiments require a pure population of cells, you can use the pool of cells surviving selection instead of isolating and characterizing clonal cell lines. We recommend selecting mammalian cultures in 250–600 μg/ml Hygromycin B (Cat. No. 631309) depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of Hygromycin B to determine optimal selection concentration.

载体序列

LOCUS       pIREShyg3    5752 bp     DNA circular    SYN
DEFINITION  pIREShyg3
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|biofeng.com|
FEATURES             Location/Qualifiers
     source          1..5752
                     /organism="pIREShyg3"
                     /mol_type="other DNA"
     promoter        236..852
                     /label="CMV_immearly_promoter"
     misc_feature    315..602
                     /label="CAG_enhancer"
     misc_feature    769..789
                     /label="CMV_fwd_primer"
     promoter        863..881
                     /label="T7_promoter"
     misc_feature    1002..1297
                     /label="synth_int"
     misc_feature    1334..1898
                     /label="IRES"
     CDS             1819..2967
                     /label="ORF frame 1"
                     /translation="MLYMCLVEVKKTSRPPEPRGRGFPLKNTMISLPQPVPKMDRSGK
                     PELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDR
                     YVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETELPAVLQPVAEAM
                     DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQA
                     LDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFW
                     RPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRC
                     DAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE*"
     CDS             complement(1834..2700)
                     /label="ORF frame 2"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     gene            1933..2964
                     /label="hygroB"
                     /gene="hygroB"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     terminator      3266..3494
                     /label="SV40_PA_terminator"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     misc_feature    3464..3483
                     /label="EBV_rev_primer"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     misc_feature    3516..3535
                     /label="EBV_rev_primer"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     promoter        complement(3591..3609)
                     /label="M13_reverse_primer"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     misc_feature    complement(3608..3630)
                     /label="M13_pUC_rev_primer"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     promoter        complement(3644..3673)
                     /label="lac_promoter"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     rep_origin      complement(3982..4601)
                     /label="pBR322_origin"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     gene            complement(4756..5616)
                     /label="Ampicillin"
                     /gene="Ampicillin"
                     /translation="MPPLEVARLLLHTSQPRPPEEDVGDLVLGIPEHRLAPVNDRCYA
                     AIVRQDIVGAEIRVHEVPDFGAVLGPKHQLIESLRDGRTDGVVHHSLPVIHMGISNRA
                     YEITPCSVLTDSLRSEWAEPARLAKIGRSDRIHGLRDRLQNSGQFGFRQVLQRDTLCT
                     AGDAIGQALAEFPNVKHFRNRERGRCKVPINITIFVETIGAAIYPQDISTPSYIEAES
                     TRFFALRELHQVGDAVELFDQKLLDRRRGEFRLSGSIHLWYGLWQAYHRVFQRKTTSP
                     WFGGPRRFFNLD*"
     CDS             complement(4756..5616)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(5658..5686)
                     /label="AmpR_promoter"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
ORIGIN
    1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG
   61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG
  121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC
  181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT
  241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA
  301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC
  361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC
  421 ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT
  481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT
  541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA
  601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG
  661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC
  721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG
  781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA
  841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTTGGTACC
  901 GAGCTCGGAT CTGTACAGGC CTTAAGCCGG CGCGCTAGCG CTTCGAAGGC GCCCGGGATC
  961 CGATATCGTA CGTTAACATA GATAACTGAT CCAGTGTGCT GGAATTAATT CGCTGTCTGC
 1021 GAGGGCCAGC TGTTGGGGTG AGTACTCCCT CTCAAAAGCG GGCATGACTT CTGCGCTAAG
 1081 ATTGTCAGTT TCCAAAAACG AGGAGGATTT GATATTCACC TGGCCCGCGG TGATGCCTTT
 1141 GAGGGTGGCC GCGTCCATCT GGTCAGAAAA GACAATCTTT TTGTTGTCAA GCTTGAGGTG
 1201 TGGCAGGCTT GAGATCTGGC CATACACTTG AGTGACAATG ACATCCACTT TGCCTTTCTC
 1261 TCCACAGGTG TCCACTCCCA GGTCCAACTG CAGGTCGAGC ATGCATCTAG GGCGGCCGCA
 1321 CTAGAGGAAT TCCGCCCCTC TCCCTCCCCC CCCCCTAACG TTACTGGCCG AAGCCGCTTG
 1381 GAATAAGGCC GGTGTGTGTT TGTCTATATG TTATTTTCCA CCATATTGCC GTCTTTTGGC
 1441 AATGTGAGGG CCCGGAAACC TGGCCCTGTC TTCTTGACGA GCATTCCTAG GGGTCTTTCC
 1501 CCTCTCGCCA AAGGAATGCA AGGTCTGTTG AATGTCGTGA AGGAAGCAGT TCCTCTGGAA
 1561 GCTTCTTGAA GACAAACAAC GTCTGTAGCG ACCCTTTGCA GGCAGCGGAA CCCCCCACCT
 1621 GGCGACAGGT GCCTCTGCGG CCAAAAGCCA CGTGTATAAG ATACACCTGC AAAGGCGGCA
 1681 CAACCCCAGT GCCACGTTGT GAGTTGGATA GTTGTGGAAA GAGTCAAATG GCTCTCCTCA
 1741 AGCGTAGTCA ACAAGGGGCT GAAGGATGCC CAGAAGGTAC CCCATTGTAT GGGAATCTGA
 1801 TCTGGGGCCT CGGTGCACAT GCTTTACATG TGTTTAGTCG AGGTTAAAAA AACGTCTAGG
 1861 CCCCCCGAAC CACGGGGACG TGGTTTTCCT TTGAAAAACA CGATGATAAG CTTGCCACAA
 1921 CCCGTACCAA AGATGGATAG ATCCGGAAAG CCTGAACTCA CCGCGACGTC TGTCGAGAAG
 1981 TTTCTGATCG AAAAGTTCGA CAGCGTCTCC GACCTGATGC AGCTCTCGGA GGGCGAAGAA
 2041 TCTCGTGCTT TCAGCTTCGA TGTAGGAGGG CGTGGATATG TCCTGCGGGT AAATAGCTGC
 2101 GCCGATGGTT TCTACAAAGA TCGTTATGTT TATCGGCACT TTGCATCGGC CGCGCTCCCG
 2161 ATTCCGGAAG TGCTTGACAT TGGGGAATTC AGCGAGAGCC TGACCTATTG CATCTCCCGC
 2221 CGTGCACAGG GTGTCACGTT GCAAGACCTG CCTGAAACCG AACTGCCCGC TGTTCTGCAG
 2281 CCGGTCGCGG AGGCCATGGA TGCGATCGCT GCGGCCGATC TTAGCCAGAC GAGCGGGTTC
 2341 GGCCCATTCG GACCGCAAGG AATCGGTCAA TACACTACAT GGCGTGATTT CATATGCGCG
 2401 ATTGCTGATC CCCATGTGTA TCACTGGCAA ACTGTGATGG ACGACACCGT CAGTGCGTCC
 2461 GTCGCGCAGG CTCTCGATGA GCTGATGCTT TGGGCCGAGG ACTGCCCCGA AGTCCGGCAC
 2521 CTCGTGCACG CGGATTTCGG CTCCAACAAT GTCCTGACGG ACAATGGCCG CATAACAGCG
 2581 GTCATTGACT GGAGCGAGGC GATGTTCGGG GATTCCCAAT ACGAGGTCGC CAACATCTTC
 2641 TTCTGGAGGC CGTGGTTGGC TTGTATGGAG CAGCAGACGC GCTACTTCGA GCGGAGGCAT
 2701 CCGGAGCTTG CAGGATCGCC GCGGCTCCGG GCGTATATGC TCCGCATTGG TCTTGACCAA
 2761 CTCTATCAGA GCTTGGTTGA CGGCAATTTC GATGATGCAG CTTGGGCGCA GGGTCGATGC
 2821 GACGCAATCG TCCGATCCGG AGCCGGGACT GTCGGGCGTA CACAAATCGC CCGCAGAAGC
 2881 GCGGCCGTCT GGACCGATGG CTGTGTAGAA GTACTCGCCG ATAGTGGAAA CCGACGCCCC
 2941 AGCACTCGTC CGAGGGCAAA GGAATAGAGT AGATGCCGAC CGAACAAGAG CTGATTTCGA
 3001 GAACGCCTCA GCCAGCAACT CGCGCGAGCC TAGCAAGGCA AATGCGAGAG AACGGCCTTA
 3061 CGCTTGGTGG CACAGTTCTC GTCCACAGTT CGCTAAGCTC GCTCGGCTGG GTCGCGGGAG
 3121 GGCCGGTCGC AGTGATTCAG GCCCTTCTGG ATTGTGTTGG TCCCCAGGGC ACGATTGTCA
 3181 TGCCCACGCA CTCGGGTGAT CTGACTGATC CCGCAGATTG GAGATCGCCG CCCGTGCCTG
 3241 CCGATTGGGT GCAGATCTAG ATAACTGATC ATAATCAGCC ATACCACATT TGTAGAGGTT
 3301 TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA
 3361 ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC
 3421 ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC
 3481 ATCAATGTAT CTTAACGCTC GAGTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT
 3541 ATCTTATCAT GTCTGTATAC CGTCGACCTC TAGCTAGAGC TTGGCGTAAT CATGGTCATA
 3601 GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG
 3661 CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG
 3721 CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA
 3781 ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC
 3841 GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG
 3901 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA
 3961 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA
 4021 CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG
 4081 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT
 4141 TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG
 4201 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC
 4261 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT
 4321 AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA
 4381 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC
 4441 AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC
 4501 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT
 4561 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC
 4621 TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT
 4681 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA
 4741 AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT
 4801 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG
 4861 CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA
 4921 TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT
 4981 ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT
 5041 TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT
 5101 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT
 5161 GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC
 5221 CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC
 5281 CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT
 5341 GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG
 5401 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT
 5461 ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC
 5521 TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA
 5581 GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG
 5641 AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA
 5701 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TC