| 出品公司: | SBI |
|---|---|
| 载体名称: | pCDF1-MCS2-EF1-Puro |
| 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: | 6606 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: | 嘌呤霉素 |
| 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-Puro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； |
| 产品目录号: | CD110B-1 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 慢病毒（FIV) |
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
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-Puro 6606 bp DNA SYN
DEFINITION pCDF1-MCS2-EF1-Puro
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..6606
/organism="pCDF1-MCS2-EF1-Puro"
/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"
gene 2358..2957
/label="puro (variant)"
/gene="puro (variant)"
CDS 2358..2957
/label="ORF frame 3"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
misc_feature 2966..3541
/label="WPRE"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
CDS 3054..3566
/label="ORF frame 3"
/translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVF
ADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGK
SSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRIS
LWAASPPGYR*"
CDS 3067..3639
/label="ORF frame 1"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
misc_feature 3653..3678
/label="U3PPT"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
misc_feature 3655..3678
/label="U3PPT"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
terminator 3893..4012
/label="SV40_PA_terminator"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
misc_feature 3981..4000
/label="EBV_rev_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
rep_origin 4030..4107
/label="SV40_origin"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
misc_feature 4092..4111
/label="SV40pro_F_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
promoter complement(4199..4217)
/label="M13_reverse_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
misc_feature complement(4216..4238)
/label="M13_pUC_rev_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
promoter complement(4252..4281)
/label="lac_promoter"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
gene complement(5364..6224)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"
CDS complement(5364..6224)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(6266..6294)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
misc_feature complement(6453..6475)
/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 acgctagatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
2401 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
2461 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
2521 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
2581 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
2641 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
2701 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
2761 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
2821 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
2881 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
2941 gcaagcccgg tgcctgagtc gacaatcaac ctctggatta caaaatttgt gaaagattga
3001 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
3061 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
3121 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
3181 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
3241 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
3301 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
3361 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
3421 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
3481 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
3541 ccgcctcccc gcctgggtac cgatgacaga gttagaagat cgcttcagga agctatttgg
3601 cacgacttct acaacgggag acagcacagt agattctgaa gatgaacctc ctaaaaaaga
3661 aaaaagggtg gactgggatg agtattggaa ccctgaaatc gatagcttcc agtgctttgt
3721 gaaacttcga ggagtctctt tgttgaggac ttttgagttc tcccttgagg ctcccacaga
3781 tacaataaat atttgagatt gaaccctgtc gagtatctgt gtaatctttt ttacctgtga
3841 ggtctcggaa tccgggccga gaacttcgca gcgagctcat tgtaccgcga acttgtttat
3901 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
3961 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
4021 gctctagcta tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
4081 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
4141 tagtgaggag gcttttttgg aggcctagac ttttgcagag acggcccaaa ttcgtaatca
4201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
4261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
4321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
4381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
4441 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
4501 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
4561 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
4621 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
4681 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
4741 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
4801 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
4861 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
4921 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
4981 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
5041 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
5101 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
5161 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
5221 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
5281 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
5341 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
5401 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
5461 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
5521 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
5581 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
5641 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
5701 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
5761 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
5821 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
5881 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
5941 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
6001 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
6061 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
6121 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
6181 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
6241 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
6301 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
6361 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
6421 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
6481 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
6541 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
6601 gtgcac
//
