pCDF1-MCS2-EF1-copGFP

pCDF1-MCS2-EF1-copGFP

载体基本信息

出品公司:	Ybscience
载体名称:	pCDF1-MCS2-EF1-copGFP
质粒类型:	慢病毒表达载体；cDNA表达载体
克隆方法:	多克隆位点，限制性内切酶
启动子:	CMV
载体大小:	6771 bp
5' 测序引物及序列:	LNCX-F AGCTCGTTTAGTGAACCGTCAGATC
3' 测序引物及序列:	EF1a-R
载体标签:	无
载体抗性:	氨苄青霉素（Ampicillin）
筛选标记:	GFP
克隆菌株:	stbl3 或者E. coli(RecA-)：OmniMAX 2
宿主细胞（系）:	常用细胞系(e.g.HeLa, HEK293, HT1080, H1299)
备注:	pCDF1-MCS2-EF1-copGFP慢病毒表达载体是基于FIV的慢病毒载体； 用于cDNA表达和克隆；高效转染细胞，建立稳定细胞系，表达水平高；
稳定性:	稳表达
组成型/诱导型:	组成型
病毒/非病毒:	慢病毒（FIV)

 

载体质粒图谱和多克隆位点信息

载体简介

 

背景简介：
A. Purpose of this Manual
This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDF lentivectors. Specifically, it provides critical instructions on amplification and cloning the cDNA into the pCDF Vectors, and verifying final expression constructs. This manual does not include information on packaging the pCDF expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. 

B. Advantages of the Lentivector Expression System Lentiviral expression vectors are the most effective vehicles for delivering and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-tomedium efficiency using conventional transfection protocols.
However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.
The lentiviral cDNA expression system consists of three main components:
(1) The lentiviral expression vector (e.g., pCDF1-MCS2-EF1-Puro)
(2) The lentiviral packaging plasmids (e.g., pPACKF1 Packaging Plasmid mix)
(3) A pseudoviral particle producer cell line (e.g., 293TN cells) 

The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently cotransfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system, please refer to the Lentivector Expression Systems user manual.
SBI’s novel pCDF Vectors are derived from feline immunodeficiency virus (FIV; Poeschla, 2003; for Safety Guidelines when working with these vectors, see section G). These pCDF Vectors, developed at SBI, are self-inactivating as a result of a deletion in the U3 region of 3’ ΔLTR (see Appendix for Vector Features). Upon integration into the genome, the 5’ LTR promoter is inactivated, which prevents formation of replication-competent viral particles.
When expressed, the hybrid CMV/FIV 5’ LTR drives high level transcription of the viral construct and produces a transcript that contains all the necessary functional elements (i.e., Psi, RRE, and cPPT) for efficient packaging. When this construct is expressed in HEK 293 cells that also express viral coat proteins (i.e., a packaging cell line), the pCDF transcripts are efficiently packaged into pseudoviral particles. After isolation, these pseudoviral particles containing the RNA version of the pCDF expression cassette can be efficiently transduced into any mammalian target cells. Following transduction into the target cells, this expression cassette is reverse transcribed and integrated into the genome of the target cell. The pCDF Vectors also contain a bacterial origin of replication and ampicillin resistance (AmpR) gene for propagation and selection in E.coli. 

The pCDF1-MCS2-EF1-Puro Vector (Cat. # CD110B-1) contains a puromycin resistance gene, under the control of a constitutive EF1 promoter and a WPRE regulatory element, to enable selection of target cells stably expressing the cDNA template. The pCDF1-MCS2-EF1-copGFP Vector (Cat. # CD111B-1) contains a copGFP gene under the control of a EF1 promoter and WPRE element. CopGFP is a novel fluorescent protein ,derived from copepod plankton (Panalina sp.), which is similar to EGFP but has a brighter color This gene serves as a reporter for the transfected or transduced cells. 

pCDF Cloning and Expression Lentivectors
The FIV derived pCDF vectors contain the following features:
 CMV promoter—promotes a high level of expression of your gene of interest in a wide variety of cell lines.
 Multiple Cloning Site (MCS)—for cloning the gene of interest in MCS located downstream of CMV promoter.
 WPRE element—enhances stability and translation of the CMVdriven transcripts.
 SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts. 
Optional second expression cassette—provides expression of puromycin resistance gene or copGFP reporter under control of constitutive elongation factor 1 (EF1) promoter for selection or FACS analysis of transduced cells.
 Hybrid CMV-5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.
 Genetic elements (cPPT, GAG, LTRs)—necessary for packaging, transducing, and stably integrating the viral expression construct into genomic DNA.
 SV40 origin—for stable propagation of the pCDF plasmid in mammalian cells.
 pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.
 Ampicillin resistance gene—for selection in E.coli cells. 

 

载体序列

LOCUS       pCDF1-MCS2-EF1-copGFP	6771 bp 	DNA	SYN
DEFINITION  pCDF1-MCS2-EF1-copGFP
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..6771
                     /organism="pCDF1-MCS2-EF1-copGFP"
                     /mol_type="other DNA"
     misc_feature    287..307
                     /label="CMV_fwd_primer"
     misc_feature    1165..1180
                     /label="cPPT"
     misc_feature    1633..1653
                     /label="CMV_fwd_primer"
     promoter        1634..1703
                     /label="CMV_promoter"
     misc_feature    1677..1696
                     /label="pCEP_fwd_primer"
     misc_feature    1679..1703
                     /label="LNCX_primer"
     CDS             2366..3124
                     /label="ORF frame 2"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK
                     MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG
                     VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS
                     FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY
                     QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*"
     misc_feature    3133..3708
                     /label="WPRE"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK
                     MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG
                     VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS
                     FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY
                     QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*"
     CDS             3221..3961
                     /label="ORF frame 2"
                     /translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVF
                     ADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGK
                     SSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRIS
                     LWAASPPVPMTELEDRFRKLFGTTSTTGDSTVDSEDEPPKKEKRVDWDEYWNPEIDSF
                     QCFVKLRGVSLLRTFEFSLEAPTDTINI*"
     CDS             3234..3731
                     /label="ORF frame 3"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     misc_feature    3818..3843
                     /label="U3PPT"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     misc_feature    3820..3843
                     /label="U3PPT"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     terminator      4058..4177
                     /label="SV40_PA_terminator"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     misc_feature    4146..4165
                     /label="EBV_rev_primer"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     rep_origin      4195..4272
                     /label="SV40_origin"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     misc_feature    4257..4276
                     /label="SV40pro_F_primer"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     promoter        complement(4364..4382)
                     /label="M13_reverse_primer"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     misc_feature    complement(4381..4403)
                     /label="M13_pUC_rev_primer"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     promoter        complement(4417..4446)
                     /label="lac_promoter"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     gene            complement(5529..6389)
                     /label="Ampicillin"
                     /gene="Ampicillin"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
                     PRRYR*"
     CDS             complement(5529..6389)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(6431..6459)
                     /label="AmpR_promoter"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     misc_feature    complement(6618..6640)
                     /label="pGEX_3_primer"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
ORIGIN
    1 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
   61 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
  121 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
  181 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
  241 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
  301 aggcgtgtac ggtgggaggt ctatataagc agagcttgtg aaacttcgag gagtctcttt
  361 gttgaggact tttgagttct cccttgaggc tcccacagat acaataaata tttgagattg
  421 aaccctgtcg agtatctgtg taatcttttt tacctgtgag gtctcggaat ccgggccgag
  481 aacttcgcag ttggcgcccg aacagggact tgattgagag tgattgagga agtgaagcta
  541 gagcaataga aagctgttaa gcagaactcc tgctgaccta aatagggaag cagtagcaga
  601 cgctgctaac agtgagtatc tctagtgaag cagactcgag ctcataatca agtcattgtt
  661 taaaggccca gataaattac atctggtgac tcttcgcgga ccttcaagcc aggagattcg
  721 ccgagggaca gtcaacaagg taggagagat tctacagcaa catggggaat ggacaggggc
  781 gagattggaa aatggccatt aagagatgta gtaatgttgc tgtaggagta ggggggaaga
  841 gtaaaaaatt tggagaaggg aatttcagat gggccattag aatggctaat gtatctacag
  901 gacgagaacc tggtgatata ccagagactt tagatcaact aaggttggtt atttgcgatt
  961 tacaagaaag aagagaaaaa tttggatcta gcaaagaaat tgatatggca attcctgcat
 1021 tgaggagaaa tggtaggcaa tgtggcatgt ctgaaaaaga ggaggaatga tgaagtatct
 1081 cagacttatt ttataaggga gatactgtgc tgagttcttc cctttgagga aggtatgtca
 1141 tatcctagac atagtctcaa ttttaaaaga agaggtagga taggagggat ggccccttat
 1201 gaattattag cacaacaaga atccttaaga atacaagatt atttttctgc aataccacaa
 1261 aaattgcaag cacagtggat ttattataaa gatcaaaaag ataagaaatg gaaaggacca
 1321 atgagagtag aatactgggg acagggatca gtattattaa aggatgaaga gaagggatat
 1381 tttcttataa tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt
 1441 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
 1501 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
 1561 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
 1621 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
 1681 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
 1741 gaagattcta gagcccgggc gcgccggatc cagatcttaa ttaatttaaa tgaattcgcg
 1801 gccgcgaagg atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
 1861 agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg
 1921 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
 1981 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
 2041 cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac
 2101 ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc
 2161 ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc
 2221 ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct
 2281 tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg
 2341 accggcgcct acgctagacg ccaccatgga gagcgacgag agcggcctgc ccgccatgga
 2401 gatcgagtgc cgcatcaccg gcaccctgaa cggcgtggag ttcgagctgg tgggcggcgg
 2461 agagggcacc cccaagcagg gccgcatgac caacaagatg aagagcacca aaggcgccct
 2521 gaccttcagc ccctacctgc tgagccacgt gatgggctac ggcttctacc acttcggcac
 2581 ctaccccagc ggctacgaga accccttcct gcacgccatc aacaacggcg gctacaccaa
 2641 cacccgcatc gagaagtacg aggacggcgg cgtgctgcac gtgagcttca gctaccgcta
 2701 cgaggccggc cgcgtgatcg gcgacttcaa ggtggtgggc accggcttcc ccgaggacag
 2761 cgtgatcttc accgacaaga tcatccgcag caacgccacc gtggagcacc tgcaccccat
 2821 gggcgataac gtgctggtgg gcagcttcgc ccgcaccttc agcctgcgcg acggcggcta
 2881 ctacagcttc gtggtggaca gccacatgca cttcaagagc gccatccacc ccagcatcct
 2941 gcagaacggg ggccccatgt tcgccttccg ccgcgtggag gagctgcaca gcaacaccga
 3001 gctgggcatc gtggagtacc agcacgcctt caagaccccc atcgccttcg ccagatcccg
 3061 cgctcagtcg tccaattctg ccgtggacgg caccgccgga cccggctcca ccggatctcg
 3121 ctaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
 3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
 3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
 3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
 3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
 3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
 3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
 3541 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
 3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
 3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
 3721 ggtaccgatg acagagttag aagatcgctt caggaagcta tttggcacga cttctacaac
 3781 gggagacagc acagtagatt ctgaagatga acctcctaaa aaagaaaaaa gggtggactg
 3841 ggatgagtat tggaaccctg aaatcgatag cttccagtgc tttgtgaaac ttcgaggagt
 3901 ctctttgttg aggacttttg agttctccct tgaggctccc acagatacaa taaatatttg
 3961 agattgaacc ctgtcgagta tctgtgtaat cttttttacc tgtgaggtct cggaatccgg
 4021 gccgagaact tcgcagcgag ctcattgtac cgcgaacttg tttattgcag cttataatgg
 4081 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
 4141 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
 4201 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
 4261 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
 4321 tttggaggcc tagacttttg cagagacggc ccaaattcgt aatcatggtc atagctgttt
 4381 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
 4441 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
 4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
 4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
 4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
 4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
 4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
 4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
 4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
 4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
 4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
 5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
 5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
 5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
 5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
 5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
 5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
 5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
 5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
 5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
 5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
 5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
 5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
 5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
 5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
 5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
 5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
 6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
 6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
 6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
 6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
 6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
 6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
 6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
 6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
 6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
 6541 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
 6601 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
 6661 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
 6721 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca c