pCDH-CMV-MCS-EF1-copGFP-T2A-Pu

pCDH-CMV-MCS-EF1-copGFP-T2A-Puro

载体基本信息

出品公司:	Ybscience
载体名称:	pCDH-CMV-MCS-EF1-copGFP-T2A-Puro
质粒类型:	慢病毒表达载体；cDNA表达载体；双启动子载体
克隆方法:	多克隆位点，限制性内切酶
启动子:	CMV
载体大小:	--
5' 测序引物及序列:	CMV-F :CGCAAATGGGCGGTAGGCGTG
3' 测序引物及序列:	--
载体标签:	无
载体抗性:	氨苄青霉素（Ampicillin）
筛选标记:	GFP、Puromycin
克隆菌株:	E.coli cells(RecA-)推荐: Stbl2 ,OmniMAX 2 T1R
宿主细胞（系）:	常用细胞系，如HeLa, HEK293, HT1080, H1299
备注:	pCDH-CMV-MCS-EF1-copGFP-T2A-Puro慢病毒表达载体是基于HIV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； CMV启动子驱动目的基因的高水平表达，EF1a启动子驱动报告基因的中 等水平的表达。
稳定性:	稳表达
组成型/诱导型:	组成型
病毒/非病毒:	慢病毒（HIV)

 

载体质粒图谱和多克隆位点信息

 

载体简介

 

背景简介：

This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.

基于HIV-1的pCDH 慢病毒载体特征：

 Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.
 WPRE element—enhances stability and translation of the CMV-driven transcripts.
 SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.
 Hybrid RSV/5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.
 Genetic elements (cPPT, gag, env, LTRs)—necessary for packaging, transducing, and stably integrating the vira expression construct into genomic DNA.
 SV40 origin—for stable propagation of the pCDH 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.

pCDH 慢病毒表达载体的优势：

Lentiviral expression vectors are the most effective vehicles for the delivery 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-to-medium 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.

pCDH 慢病毒载体的包装载体及细胞系

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 co-transfected 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 System user manual.

启动子的选择：

SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.

The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.
The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).

The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.

MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.

SBI第三代慢病毒载体

SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three maincomponents:
(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)
(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)
(3) A pseudoviral particle producer cell line (e.g., 293TN cells)

2A Peptide-enabled dual expression system
Coexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgenes with an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines— however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).
The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006). The 2A-like sequences exist in several  viruses  and are used to mediate protein cleavage from a single open reading frame. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reporter gene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.

 

载体序列

此序列来自于测序结果，仅供参考。

LOCUS       Exported                8201 bp ds-DNA     circular SYN 25-JUL-2016
DEFINITION  pCDH-CMV-MCS-EF1-CoGFP-T2A-puro.
ACCESSION   .
VERSION     .
KEYWORDS    pCDH-CMV-MCS-EF1-CoGFP-T2A-puro
SOURCE      http://www.biofeng.com
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8201)
  AUTHORS   Biofeng Lab.
  TITLE     Direct Submission
  JOURNAL   Exported Monday, July 25, 2016 from SnapGene Viewer 3.1.4
            http://www.snapgene.com
COMMENT     ORIGDB|GenBank
FEATURES             Location/Qualifiers
     source          1..8201
                     /organism="pCDH-CMV-MCS-EF1-Puro"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     misc_feature    7..234
                     /note="RSV"
                     /note="color: #993366"
     misc_feature    234..414
                     /note="3'LTR"
                     /note="truncHIV-1  3'LTR"
                     /note="color: #66cc99; direction: RIGHT"
     misc_feature    235..414
                     /note="5'LTR"
                     /note="HIV-1 5'LTR"
                     /note="color: #ffcc99; direction: RIGHT"
     misc_feature    522..566
                     /note="psi pack"
                     /note="HIV-1 psi pack"
                     /note="color: #3366cc"
     gene            567..919
                     /product="gag"
                     /note="gag"
                     /note="color: #666699; direction: RIGHT"
     CDS             954..1454
                     /codon_start=1
                     /note="ORF frame 3"
                     /note="color: #ff9966"
                     /translation="MRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALF
                     LGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQA
                     RILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIGITRPGWSGTEKLTI
                     TQA"
     misc_feature    1076..1308
                     /note="RRE"
                     /note="color: #66cc99; direction: RIGHT"
     CDS             1309..1797
                     /codon_start=1
                     /note="Env"
                     /note="color: #333399"
                     /translation="WGFGVALENSFAPLLCLGMLVGVINLWNRLESHDLDGVGQRN*QL
                     HKLNTLLN*RIAKPARKE*TRIIGIR*MGKFVELV*HNKLAVVYKIIHNDSRRLGRFKN
                     SFCCTFYSE*S*AGIFTIIVSDPPPNPEGTRQARRNRRRRWRERQRQIHSISERISTVS
                     "
     CDS             1415..2056
                     /codon_start=1
                     /note="ORF frame 2"
                     /note="color: #ff9966"
                     /translation="MEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
                     NITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEG
                     IEEEGGERDRDRSIRLVNGSRRYRLTFKRKGGIGGYSAGERIVDIIATDIQTKELQKQI
                     TKFKILSILVLCPVHDLMGLSYLAVHLRISHRYYHGDAVLAVHQWAWIAV"
     misc_feature    1798..1916
                     /note="cPPT"
                     /note="color: #ffcc00"
     promoter        1922..2271
                     /note="CMV promoter"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2164..2184
                     /note="CMV_fwd_primer"
                     /note="color: #c0c0c0"
     misc_feature    2208..2227
                     /note="pCEP_fwd_primer"
                     /note="color: #c0c0c0"
     misc_feature    2210..2234
                     /note="LNCX_primer"
                     /note="color: #c0c0c0"
     misc_feature    2278..2320
                     /note="MCS"
                     /note="color: #00ffff"
     promoter        2315..2860
                     /note="EF1-alpha promoter"
                     /note="EF1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2873..3631
                     /note="CoGFP"
                     /note="color: #00ff00; direction: RIGHT"
     misc_feature    3630..3683
                     /note="T2A"
                     /note="color: #ff0000"
     misc_feature    3690..4286
                     /note="Puro R"
                     /note="color: #99ccff; direction: RIGHT"
     misc_feature    4298..4873
                     /note="WPRE"
                     /note="color: #66cc99; direction: RIGHT"
     CDS             4386..4931
                     /codon_start=1
                     /note="ORF frame 2"
                     /note="color: #ff9966"
                     /translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVFA
                     DATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGKSS
                     SFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRISLWA
                     ASPPGTFKTNDLQGSCRS"
     CDS             4399..4989
                     /codon_start=1
                     /note="ORF frame 3"
                     /note="color: #ff9966"
                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQPP
                     LVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPFLG
                     CSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPPPRL
                     VPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRK"
     misc_feature    4941..4962
                     /note="U3PPT"
                     /note="color: #ffcc00"
     misc_feature    4941..4956
                     /note="cPPT"
                     /note="color: #ffcc00"
     misc_feature    4958..5010
                     /note="Delta-U3"
                     /note="delta U3 "
                     /note="color: #3366cc"
     misc_feature    5011..5191
                     /note="HIV-1_5_LTR"
                     /note="color: #66cc99; direction: RIGHT"
     misc_feature    5011..5191
                     /note="Delta-3'LTR"
                     /note="truncHIV-1  Delta-3'LTR"
                     /note="color: #ffcc99; direction: RIGHT"
     terminator      5266..5385
                     /note="SV40 PA terminator"
                     /note="SV40_PA_terminator"
                     /note="color: #c0c0c0"
     misc_feature    5354..5373
                     /note="EBV_rev_primer"
                     /note="color: #3366cc"
     rep_origin      5403..5480
                     /direction=RIGHT
                     /note="SV40 ori"
                     /note="color: #ffff00"
     misc_feature    5465..5484
                     /note="SV40pro_F_primer"
                     /note="color: #3366cc"
     promoter        complement(5572..5590)
                     /note="M13_reverse_primer"
                     /note="color: #800000; direction: LEFT"
     misc_feature    5589..5611
                     /note="M13_pUC_rev_primer"
                     /note="color: #3366cc"
     promoter        complement(5625..5654)
                     /note="lac promoter"
                     /note="color: #000000; direction: LEFT"
     repeat_region   complement(5912..6584)
                     /note="pUC Ori"
                     /note="pUC origin"
                     /note="color: #ffff00; direction: LEFT"
     gene            complement(6737..7597)
                     /gene="Ampicillin"
                     /note="Ampicillin"
                     /note="color: #ccffcc; direction: LEFT"
     CDS             complement(6737..7597)
                     /codon_start=1
                     /note="ORF frame 2"
                     /note="color: #ff9966"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7639..7667)
                     /note="AmpR promoter"
                     /note="color: #000000; direction: LEFT"
     misc_feature    7826..7848
                     /note="pGEX_3_primer"
                     /note="color: #3366cc"
     misc_feature    complement(8047..8190)
                     /note="lacZ a"
                     /note="color: #808080; direction: LEFT"
     misc_feature    8161..8183
                     /note="M13_pUC_fwd_primer"
                     /note="color: #3366cc"
     promoter        8176..8192
                     /note="M13_forward20_primer"
                     /note="color: #800000; direction: RIGHT"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
      481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
      781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
      841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
      901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
      961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
     1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
     1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
     1921 atcgatacta gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     1981 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     2041 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     2101 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     2161 tgacgcaaat gggcggtagg cgtgtacggt gggaggttta tataagcaga gctcgtttag
     2221 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagattct
     2281 agagctagcg aattcgaatt taaatcggat ccgcggccgc gaaggatctg cgatcgctcc
     2341 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     2401 gtcggcaatt gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     2461 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     2521 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg
     2581 gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg
     2641 agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta
     2701 agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga
     2761 ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg
     2821 ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctacgct agatggagag
     2881 cgacgagagc ggcctgcccg ccatggagat cgagtgccgc atcaccggca ccctgaacgg
     2941 cgtggagttc gagctggtgg gcggcggaga gggcaccccc aagcagggcc gcatgaccaa
     3001 caagatgaag agcaccaaag gcgccctgac cttcagcccc tacctgctga gccacgtgat
     3061 gggctacggc ttctaccact tcggcaccta ccccagcggc tacgagaacc ccttcctgca
     3121 cgccatcaac aacggcggct acaccaacac ccgcatcgag aagtacgagg acggcggcgt
     3181 gctgcacgtg agcttcagct accgctacga ggccggccgc gtgatcggcg acttcaaggt
     3241 ggtgggcacc ggcttccccg aggacagcgt gatcttcacc gacaagatca tccgcagcaa
     3301 cgccaccgtg gagcacctgc accccatggg cgataacgtg ctggtgggca gcttcgcccg
     3361 caccttcagc ctgcgcgacg gcggctacta cagcttcgtg gtggacagcc acatgcactt
     3421 caagagcgcc atccacccca gcatcctgca gaacgggggc cccatgttcg ccttccgccg
     3481 cgtggaggag ctgcacagca acaccgagct gggcatcgtg gagtaccagc acgccttcaa
     3541 gacccccatc gccttcgcca gatcccgcgc tcagtcgtcc aattctgccg tggacggcac
     3601 cgccggaccc ggctccaccg gatctcgctg agggcagagg aagtcttcta acatgcggtg
     3661 acgtggagga gaatcccggc ccttccggga tgaccgagta caagcccacg gtgcgcctcg
     3721 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     3781 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     3841 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     3901 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     3961 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     4021 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     4081 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     4141 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     4201 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     4261 ggtgcatgac ccgcaagccc ggtgcctgag tcgacaatca acctctggat tacaaaattt
     4321 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4381 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4441 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4501 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4561 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4621 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4681 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     4741 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4801 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4861 tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta caaggcagct
     4921 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     4981 cgaaaataag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     5041 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     5101 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     5161 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     5221 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     5281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     5401 ctatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5521 gaggcttttt tggaggccta gacttttgca gagacggccc aaattcgtaa tcatggtcat
     5581 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5641 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5701 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5761 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5821 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5881 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5941 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6001 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6061 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6121 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6181 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6241 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6301 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6361 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6421 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6481 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6541 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6601 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6661 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6721 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6781 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6841 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6901 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6961 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7021 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7081 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7141 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7201 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7261 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7321 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7381 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7441 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7501 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7561 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7621 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7681 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     7741 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     7801 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     7861 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     7921 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     7981 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     8041 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     8101 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     8161 cccagtcacg acgttgtaaa acgacggcca gtgccaagct g