| 质粒名称: | pPB-US-ECasE |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 83961 |
| 存储实验室: | Eleanor Chen |
|
载体骨架基本信息 |
|
| 载体名称: | Piggybac Transposon |
| 载体出品公司: | Sytem Biosciences |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | ERT2-spCas9-ERT2 |
| 物种来源: | -- |
| 插入大小: | 7970 bp |
| 基因突变: | please see depositor comments below |
| GenBank编号 | -- |
| 启动子 | U6 (gRNA), EF1a (ERT2-Cas9-ERT2) |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pPB-US-ECasE was a gift from Eleanor Chen (Addgene plasmid # 83961 ; http://n2t.net/addgene:83961 ; RRID:Addgene_83961) |
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
载体序列
LOCUS pPB-US-ECasE 13381 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Piggyac transposon containing tamoxifen inducible Cas9 and a gRNA
cassette..
ACCESSION .
VERSION .
KEYWORDS pPB-US-ECasE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13381)
TITLE Chen lab CRISPR plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 13381)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13381
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter complement(1..105)
/gene="bla"
/label=AmpR promoter
rep_origin complement(131..586)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(268..289)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 480..499
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 713..735
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 727..744
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 728..744
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 874..936
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 1423..1663
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 1423..1443
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 1594..1613
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 1690..1765
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 1857..3035
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2088..3026
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 2983..3003
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
promoter 3060..3107
/label=EM7 promoter
/note="synthetic bacterial promoter "
regulatory 3132..3141
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3141..4076
/codon_start=1
/product="mutated ligand-binding domain of the human
estrogen receptor (Feil et al., 1997)"
/label=ERT2
/translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
CDS 4089..8189
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIWKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 8196..9131
/codon_start=1
/product="mutated ligand-binding domain of the human
estrogen receptor (Feil et al., 1997)"
/label=ERT2
/translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
CDS 9144..9197
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 9198..9794
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(9198..9217)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind complement(9885..9905)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 9951..10072
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9988..10007)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10042..10061
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 10835..10869
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(11360..11376)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11360..11376)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11373..11395)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11384..11400
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(11408..11438)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11453..11474
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11591..11608)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11762..12350)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11842..11861)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12521..13381)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 13144..13163
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
ORIGIN
1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
1321 cacctggggg gatacgggga aaaggcctcc acggccagac tagaggtacc aacgcggcct
1381 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtgagggcct atttcccatg
1441 attccttcat atttgcatat acgatacaag gctgttagag agataattgg aattaatttg
1501 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
1561 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
1621 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtcttcg
1681 agaagacctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
1741 tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa
1801 taaggctagt ccgtttttag cgctcgagct tctaggtctt gaaaggagtg ggaattggct
1861 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
1921 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
1981 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
2041 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
2101 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
2161 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
2221 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
2281 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
2341 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
2401 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
2461 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
2521 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
2581 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
2641 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
2701 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
2761 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
2821 tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
2881 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
2941 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
3001 ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tagcttggta ccggaccgtg
3061 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
3121 aaaccggatc cgccaccatg gctggagaca tgagagctgc caacctttgg ccaagcccgc
3181 tcatgatcaa acgctctaag aagaacagcc tggccttgtc cctgacggcc gaccagatgg
3241 tcagtgcctt gttggatgct gagcccccca tactctattc cgagtatgat cctaccagac
3301 ccttcagtga agcttcgatg atgggcttac tgaccaacct ggcagacagg gagctggttc
3361 acatgatcaa ctgggcgaag agggtgccag gctttgtgga tttgaccctc catgatcagg
3421 tccaccttct agaatgtgcc tggctagaga tcctgatgat tggtctcgtc tggcgctcca
3481 tggagcaccc agtgaagcta ctgtttgctc ctaacttgct cttggacagg aaccagggaa
3541 aatgtgtaga gggcatggtg gagatcttcg acatgctgct ggctacatca tctcggttcc
3601 gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaa atctattatt ttgcttaatt
3661 ctggagtgta cacatttctg tccagcaccc tgaagtctct ggaagagaag gaccatatcc
3721 accgagtcct ggacaagatc acagacactt tgatccacct gatggccaag gcaggcctga
3781 ccctgcagca gcagcaccag cggctggccc agctcctcct catcctctcc cacatcaggc
3841 acatgagtaa caaaggcatg gagcatctgt acagcatgaa gtgcaagaac gtggtgcccc
3901 tctatgacct gctgctggag gcggcggacg cccaccgcct acatgcgccc actagccgtg
3961 gaggggcatc cgtggaggag acggaccaaa gccacttggc cactgcgggc tctacttcat
4021 cgcattcctt gcaaaagtat tacatcacgg gggaggcaga gggtttccct gccacagctt
4081 accggtttga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
4141 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
4201 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
4261 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
4321 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
4381 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
4441 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
4501 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
4561 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
4621 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
4681 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
4741 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
4801 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
4861 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
4921 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
4981 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
5041 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
5101 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
5161 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
5221 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
5281 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
5341 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
5401 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
5461 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
5521 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
5581 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
5641 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
5701 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
5761 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
5821 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
5881 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
5941 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
6001 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
6061 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
6121 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
6181 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
6241 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
6301 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
6361 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
6421 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
6481 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
6541 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
6601 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
6661 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
6721 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
6781 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
6841 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
6901 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
6961 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
7021 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
7081 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
7141 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
7201 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatctgga
7261 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
7321 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
7381 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
7441 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
7501 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
7561 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
7621 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
7681 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
7741 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
7801 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
7861 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
7921 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
7981 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
8041 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
8101 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
8161 ggatcgacct gtctcagctg ggaggcgaca ccggtgctgg agacatgaga gctgccaacc
8221 tttggccaag cccgctcatg atcaaacgct ctaagaagaa cagcctggcc ttgtccctga
8281 cggccgacca gatggtcagt gccttgttgg atgctgagcc ccccatactc tattccgagt
8341 atgatcctac cagacccttc agtgaagctt cgatgatggg cttactgacc aacctggcag
8401 acagggagct ggttcacatg atcaactggg cgaagagggt gccaggcttt gtggatttga
8461 ccctccatga tcaggtccac cttctagaat gtgcctggct agagatcctg atgattggtc
8521 tcgtctggcg ctccatggag cacccagtga agctactgtt tgctcctaac ttgctcttgg
8581 acaggaacca gggaaaatgt gtagagggca tggtggagat cttcgacatg ctgctggcta
8641 catcatctcg gttccgcatg atgaatctgc agggagagga gtttgtgtgc ctcaaatcta
8701 ttattttgct taattctgga gtgtacacat ttctgtccag caccctgaag tctctggaag
8761 agaaggacca tatccaccga gtcctggaca agatcacaga cactttgatc cacctgatgg
8821 ccaaggcagg cctgaccctg cagcagcagc accagcggct ggcccagctc ctcctcatcc
8881 tctcccacat caggcacatg agtaacaaag gcatggagca tctgtacagc atgaagtgca
8941 agaacgtggt gcccctctat gacctgctgc tggaggcggc ggacgcccac cgcctacatg
9001 cgcccactag ccgtggaggg gcatccgtgg aggagacgga ccaaagccac ttggccactg
9061 cgggctctac ttcatcgcat tccttgcaaa agtattacat cacgggggag gcagagggtt
9121 tccctgccac agcttatgtc gacgagggca gaggaagtct gctaacatgc ggtgacgtcg
9181 aggagaatcc tggcccaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
9241 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
9301 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
9361 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
9421 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
9481 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
9541 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
9601 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
9661 gggtgcccgc cttcctggag acctccgcgc cccacaacct ccccttctac gagcggctcg
9721 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
9781 gcaagcccgg tgccacgcgt taactaagta agcggccgcc ccggtatcga caatcaacct
9841 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
9901 ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaacta aacttgttta
9961 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
10021 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
10081 ggaattgact caaatgatgt caattagtct atcagaagct catctggtct cccttccggg
10141 ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc
10201 ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa
10261 gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt
10321 ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc
10381 gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc
10441 cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg
10501 ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct
10561 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
10621 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
10681 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
10741 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
10801 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
10861 tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt
10921 catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc
10981 gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag
11041 cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt
11101 gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc
11161 cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc
11221 cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac
11281 gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg
11341 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
11401 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
11461 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
11521 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
11581 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
11641 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
11701 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
11761 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
11821 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
11881 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
11941 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
12001 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
12061 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
12121 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
12181 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
12241 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
12301 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
12361 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
12421 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
12481 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
12541 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
12601 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
12661 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
12721 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
12781 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
12841 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
12901 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
12961 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
13021 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
13081 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
13141 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
13201 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
13261 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
13321 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
13381 t
//
