| 质粒名称: | AAVS1-idCas9-vpr |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 89985 |
| 存储实验室: | Jie Na |
|
载体骨架基本信息 |
|
| 载体名称: | AAVS1 homologous recombineering donor plasmid |
| 完整质粒大小: | 13040 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Puromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | dCas9-VPR |
| 启动子: | TRE-tight |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | T7 |
|
文献引用方法 |
|
| 材料和方法部分: | AAVS1-idCas9-vpr was a gift from Jie Na (Addgene plasmid # 89985 ; http://n2t.net/addgene:89985 ; RRID:Addgene_89985) |
| 参考文献部分: | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells. Guo J, Ma D, Huang R, Ming J, Ye M, Kee K, Xie Z, Na J. Protein Cell. 2017 Jan 23. doi: 10.1007/s13238-016-0360-8. 10.1007/s13238-016-0360-8 [pii] PubMed 28116670 |
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 AAVS1-idCas9-vpr 13040 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION inducible CRISPR-ON system for controllable gene activation; this
plasmid is used to insert dCas9-VPR casette in one allele of AAVS1
locus.
ACCESSION .
VERSION .
KEYWORDS AAVS1-idCas9-vpr
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13040)
AUTHORS Guo J, Ma D, Huang R, Ming J, Ye M, Kee K, Xie Z, Na J
TITLE An inducible CRISPR-ON system for controllable gene activation in
human pluripotent stem cells.
JOURNAL Protein Cell. 2017 Jan 23. doi: 10.1007/s13238-016-0360-8.
PUBMED 28116670
REFERENCE 2 (bases 1 to 13040)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..13040
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 424..443
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
CDS complement(453..506)
/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"
misc_feature 530..555
/label=SA
/note="splice acceptor site"
misc_feature 562..1365
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter complement(1400..1418)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(1439..1455)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1439..1455)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1452..1474)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1463..1479
/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(1487..1517)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1532..1553
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1670..1687)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1841..2429)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1921..1940)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS 2553..3413
/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 complement(2771..2790)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
primer_bind complement(3774..3793)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 4388..4407
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
CDS complement(4810..5112)
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind complement(4912..4931)
/label=ccdB-fwd
/note="ccdB gene, forward primer"
primer_bind 5235..5257
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 5249..5266
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 5250..5266
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 5273..5292
/label=T7
/note="T7 promoter, forward primer"
promoter 5273..5291
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 5326..6162
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
promoter 6179..6493
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 6187..6205
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6223..6241
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6258..6276
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6294..6312
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6330..6348
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6365..6383
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 6401..6419
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 6466..6490
/label=LNCX
/note="Human CMV promoter, forward primer"
protein_bind 6510..6534
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 6557..6577
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6596..10699
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in the RuvC
catalytic domain and the H840A mutation in the HNH
catalytic domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 10715..10735
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 10760..10909
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 11348..11704
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
protein_bind complement(12326..12350)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
polyA_signal 12534..12615
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12571..12590)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
polyA_signal 12622..12846
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 12835..12852
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
CDS complement(12884..443)
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(12967..12987)
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
ORIGIN
1 actccgggga gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg
61 acggtggcca ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc
121 tgttgctgcg cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg
181 aacaccgccc ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg
241 tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc
301 tcggtgaccc gctcgatgtg gcggtccgga tcgacggtgt ggcgcgtggc ggggtagtcg
361 gcgaacgcgg cggcgagggt gcgtacggcc ctggggacgt cgtcgcgggt ggcgaggcgc
421 accgtgggct tgtactcggt catctcgagc ctagggccgg gattctcctc cacgtcaccg
481 catgttagaa gacttcctct gccctctccg ctgccagatc tctcgaggcc ctgtgggagg
541 aagagaagag gtcagaagct tgccccactg tggggtggag gggacagata aaagtaccca
601 gaaccagagc cacattaacc ggccctggga atataaggtg gtcccagctc ggggacacag
661 gatccctgga ggcagcaaac atgctgtcct gaagtggaca taggggcccg ggttggagga
721 agaagactag ctgagctctc ggacccctgg aagatgccat gacagggggc tggaagagct
781 agcacagact agagaggtaa ggggggtagg ggagctgccc aaatgaaagg agtgagaggt
841 gacccgaatc cacaggagaa cggggtgtcc aggcaaagaa agcaagagga tggagaggtg
901 gctaaagcca gggagacggg gtactttggg gttgtccaga aaaacggtga tgatgcaggc
961 ctacaagaag gggaggcggg acgcaaggga gacatccgtc ggagaaggcc atcctaagaa
1021 acgagagatg gcacaggccc cagaaggaga aggaaaaggg aacccagcga gtgaagacgg
1081 catggggttg ggtgagggag gagagatgcc cggagaggac ccagacacgg ggaggatccg
1141 ctcagaggac atcacgtggt gcagcgccga gaaggaagtg ctccggaaag agcatccttg
1201 ggcagcaaca cagcagagag caaggggaag agggagtgga ggaagacgga acctgaagga
1261 ggcggcaggg aaggatctgg gccagccgta gaggtgaccc aggccacaag ctgcagacag
1321 aaagcggcac aggcccaggg gagagaatgc tggtcagaga aagcaaaggg cgaattcgtt
1381 taaacctgca ggactagtcc ctttagtgag ggttaattct gagcttggcg taatcatggt
1441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
1501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
1561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
1621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
1681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
1741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
1801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
1861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
1921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
1981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
2041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
2101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
2161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
2221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
2281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
2341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
2401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
2461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagacaa taaccctgat
2521 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
2581 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
2641 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
2701 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
2761 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
2821 gtcgccgcat acactattct cagaatgact tggttgagta ttcaccagtc acagaaaagc
2881 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
2941 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
3001 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
3061 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
3121 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
3181 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
3241 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
3301 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
3361 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
3421 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
3481 tctaggtgaa gatccttttt gataatctca tgagcggata catatttgaa tgtatttaga
3541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gatgcggtgt
3601 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta agcgttaata
3661 attcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
3721 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
3781 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
3841 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgcatg
3901 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc
3961 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
4021 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
4081 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
4141 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
4201 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
4261 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
4321 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
4381 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
4441 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagagcttga
4501 tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt
4561 cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac
4621 cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc tctttgcgct
4681 tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt
4741 ctgcggactg gctttctacg tgaaaaggat ctaggtgaag atcctttttg ataatctcat
4801 gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt cattttcgcg
4861 gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg ccatatcggt
4921 ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt cacgggagac
4981 tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc ccggcgtgtc
5041 aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt tataggtgta
5101 aaccttaaac tgccgtacgt ataggctgcg caactgttgg gaagggcgat cggtgcgggc
5161 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
5221 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga
5281 ctcactatag ggcgaattga atttagcggc cgcgaattcg cccttagagc agagccagga
5341 acccctgtag ggaaggggca ggagagccag gggcatgaga tggtggacga ggaaggggga
5401 cagggaagcc tgagcgcctc tcctgggctt gccaaggact caaacccaga agcccagagc
5461 agggccttag ggaagcggga ccctgctctg ggcggaggaa tatgtcccag atagcactgg
5521 ggactcttta aggaaagaag gatggagaaa gagaaaggga gtagaggcgg ccacgacctg
5581 gtgaacacct aggacgcacc attctcacaa agggagtttt ccacacggac acccccctcc
5641 tcaccacagc cctgccagga cggggctggc tactggcctt atctcacagg taaaactgac
5701 gcacggagga acaatataaa ttggggacta gaaaggtgaa gagccaaagt tagaactcag
5761 gaccaactta ttctgatttt gtttttccaa actgcttctc ctcttgggaa gtgtaaggaa
5821 gctgcagcac caggatcagt gaaacgcacc agacagccgc gtcagagcag ctcaggttct
5881 gggagagggt agcgcagggt ggccactgag aaccgggcag gtcacgcatc ccccccttcc
5941 ctcccacccc ctgccaagct ctccctccca ggatcctctc tggctccatc gtaagcaaac
6001 cttagaggtt ctggcaagga gagagatggc tccaggaaat gggggtgtgt caccagataa
6061 ggaatctgcc taacaggagg tgggggttag acccaatatc aggagactag gaaggaggag
6121 gcctaaggat ggggcttttc tgtcaccaat cctgtcccta gtaaagctta gtactgtcga
6181 gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
6241 aacgatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat
6301 cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag
6361 tttatcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa
6421 cgtatgtcga ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa
6481 ccgtcagatc gcctggagaa ttccgatcaa caagtttgta caaaaaagca ggctccgcgg
6541 ttttcaggtt ggaatgccaa agaagaagcg gaaggtcggt ggcggctcac ccgggatgga
6601 caagaagtac tccattggcc tcgccatcgg aacaaatagc gtgggctggg ctgtcatcac
6661 agatgagtac aaggtgccta gcaagaaatt taaggtgctg ggaaatacag acagacatag
6721 catcaagaag aacctcattg gcgctctcct gtttgactcc ggcgaaacag ccgaagctac
6781 cagactcaag agaaccgcta ggagaaggta caccagaagg aaaaacagga tttgctacct
6841 gcaggaaatt ttttccaacg agatggccaa ggtggacgat tccttcttcc ataggctgga
6901 agagagcttc ctcgtggagg aagacaagaa acacgagagg catcctattt ttggcaatat
6961 tgtggatgag gtcgcctacc atgagaagta tcccacaatc tatcatctga gaaaaaaact
7021 ggtggatagc accgacaagg ccgatctcag gctcatttat ctcgctctgg ctcacatgat
7081 caagtttagg ggccacttcc tgatcgaagg cgacctgaat cccgacaact ccgacgtgga
7141 caaactgttc atccagctcg tccagaccta caatcaactc ttcgaggaga accccatcaa
7201 tgcttccggc gtggatgcca aggccatcct gagcgctagg ctctccaagt ccaggaggct
7261 ggaaaatctg atcgcccaac tccctggaga gaagaagaac ggcctgtttg gcaatctgat
7321 tgccctgagc ctcggactca cccccaactt caagagcaac ttcgatctcg ccgaagacgc
7381 caaactccaa ctgagcaagg atacctacga cgacgatctc gataatctcc tcgcccagat
7441 cggcgatcaa tatgccgacc tctttctggc cgccaaaaac ctgagcgacg ctattctgct
7501 cagcgacatt ctcagggtga atacagaaat cacaaaagcc cccctgtccg ccagcatgat
7561 caaaaggtac gatgaacacc atcaggacct caccctgctg aaggctctgg tcaggcagca
7621 actccccgaa aagtacaagg agattttctt tgatcagtcc aagaatggat atgctggcta
7681 tattgatgga ggcgcctccc aggaggaatt ttataaattc atcaagccca ttctcgaaaa
7741 gatggacgga accgaagagc tgctggtcaa actcaatagg gaggatctgc tgaggaagca
7801 aaggaccttc gacaatggca gcatccccca ccagatccac ctcggcgaac tccacgctat
7861 cctcaggagg caggaagact tctacccttt cctgaaggat aacagggaga aaatcgagaa
7921 aatcctgacc ttcagaatcc cctactacgt cggacctctc gccaggggca attccagatt
7981 cgcctggatg acaaggaaga gcgaggaaac aatcacacca tggaacttcg aagaagtggt
8041 cgataagggc gccagcgccc agagcttcat tgaaaggatg accaactttg ataagaacct
8101 gcccaatgag aaggtgctgc ctaagcactc cctgctgtat gagtatttca ccgtgtataa
8161 tgagctgacc aaggtcaagt acgtcaccga gggaatgaga aagcctgctt ttctctccgg
8221 cgagcagaaa aaagccatcg tggacctgct gttcaagacc aacaggaagg tgaccgtcaa
8281 gcaactcaag gaggactact ttaagaagat tgagtgcttt gatagcgtgg aaattagcgg
8341 agtcgaggac aggttcaatg cctccctcgg aacatatcac gacctgctga aaatcatcaa
8401 agacaaagat tttctggata acgaggagaa tgaagacatt ctggaggaca ttgtcctcac
8461 cctgaccctg tttgaggaca gagagatgat tgaagagagg ctgaaaacct atgcccacct
8521 gttcgacgac aaggtgatga agcagctcaa aagaaggaga tataccggct ggggcagact
8581 gtccaggaag ctgatcaacg gcattaggga caagcagagc ggcaagacca ttctcgactt
8641 tctcaagtcc gacggattcg ccaacagaaa ctttatgcaa ctgatccacg atgacagcct
8701 cacctttaag gaagatattc agaaggctca ggtcagcggc caaggcgatt ccctccatga
8761 gcacatcgct aatctggctg gctcccctgc tatcaaaaag ggcatcctcc agacagtcaa
8821 agtcgtcgat gagctggtca aggtgatggg caggcataaa cccgagaaca ttgtgattga
8881 gatggctagg gagaaccaga ccacccagaa aggccagaaa aacagcagag aaagaatgaa
8941 gaggatcgag gagggcatca aagaactggg cagccaaatc ctcaaggagc accccgtcga
9001 aaatacacaa ctccagaacg aaaaactcta cctctactat ctgcagaacg gcagagacat
9061 gtacgtggac caggaactgg acatcaacag gctctccgat tacgatgtgg acgccatcgt
9121 ccctcagtcc tttctgaaag atgatagcat cgacaacaag gtgctgacca ggtccgacaa
9181 gaataggggc aagagcgata atgtgccctc cgaggaggtc gtcaaaaaaa tgaaaaacta
9241 ctggagacaa ctcctcaacg ctaagctcat cacccaaaga aagttcgaca atctgaccaa
9301 agccgagagg ggcggcctct ccgaactgga caaggccggc ttcatcaaaa ggcaattggt
9361 ggaaaccagg cagattacaa agcatgtcgc tcaaattctc gatagcagga tgaataccaa
9421 atatgacgag aacgacaagc tgatcagaga ggtcaaggtc atcacactca agtccaagct
9481 cgtgagcgac ttcagaaaag atttccaatt ttataaagtc agggagatca acaattacca
9541 ccacgctcac gacgcttatc tcaacgctgt cgtgggaacc gccctgatca aaaaataccc
9601 caagctggaa agcgagttcg tgtatggcga ttataaagtg tacgacgtga ggaagatgat
9661 cgctaaaagc gagcaggaaa tcggcaaggc tacagccaag tactttttct actccaacat
9721 tatgaacttc ttcaagaccg agattaccct cgccaacggc gaaattagga agaggcccct
9781 gattgaaaca aatggagaaa caggcgaaat cgtctgggac aagggcaggg acttcgccac
9841 agtcagaaaa gtgctgtcca tgcctcaagt caacatcgtc aaaaagaccg aggtgcagac
9901 cggcggcttt agcaaagaaa gcatcctgcc caagagaaac tccgacaagc tcatcgctag
9961 gaagaaggac tgggacccta agaaatacgg aggatttgac tcccctaccg tcgcctattc
10021 cgtcctcgtc gtcgctaagg tggagaaggg caagagcaag aagctcaaga gcgtcaagga
10081 gctgctggga atcaccatca tggagaggag ctccttcgaa aaaaacccta ttgatttcct
10141 ggaggccaag ggctacaagg aggtcaagaa ggacctcatc atcaagctgc ccaaatacag
10201 cctcttcgaa ctggaaaatg gcaggaagag aatgctcgct agcgccggcg agctccagaa
10261 aggaaatgag ctggctctgc ccagcaagta cgtcaacttc ctctatctcg ccagccacta
10321 tgaaaagctc aagggcagcc ccgaagacaa tgagcagaag cagctcttcg tcgagcagca
10381 caagcactac ctcgatgaaa tcatcgagca aatcagcgag ttttccaaaa gggtgattct
10441 cgccgacgct aacctcgata aggtcctctc cgcttacaac aagcatagag acaagcccat
10501 cagagaacag gccgagaaca tcatccacct gtttacactc acaaacctcg gagcccctgc
10561 cgcttttaaa tacttcgata caaccattga taggaagagg tacacctcca ccaaggaggt
10621 gctggatgct accctgattc atcaatccat cacaggactc tacgaaacaa ggattgacct
10681 gtcccaactg ggaggcgaca ctagtagagc tgaccccaag aagaagagga aggtggaggc
10741 cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc tgggaagtga
10801 cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg actttgacct
10861 cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga ttaactctag
10921 aagccaatac ctcccagaca cagacgacag gcataggatt gaggaaaaaa gaaagaggac
10981 gtatgaaact ttcaagagca taatgaaaaa aagtccgttc tctggtccta cagaccccag
11041 gcccccacct agacgaatcg cggttccgag ccgatcatca gcgagtgttc ctaagccagc
11101 tccacagccc tatccattta cgtctagtct gtctacgatc aactacgacg agttccccac
11161 tatggtattc ccgtcaggcc agataagcca agctagtgcg ttggcacccg cacccccaca
11221 ggtgctccca caagctcccg ctcctgcgcc agcaccagct atggttagtg ccctcgcgca
11281 agcgcctgcc ccggttcctg ttctcgcacc tgggccaccg caggcagttg caccgccagc
11341 gccaaagccc acacaggcag gggaaggtac gttgagcgaa gcccttctgc aactccaatt
11401 tgacgatgag gacctcggag cacttcttgg caattcaacg gaccccgcag tatttactga
11461 tcttgccagc gtggacaatt ccgaatttca acagctcctg aaccagggaa tcccggtggc
11521 tccccatacg acagagccca tgctcatgga gtacccggag gccattacac gccttgtgac
11581 gggcgcacag cgaccccctg acccggcacc ggctcccttg ggggctcctg ggcttcccaa
11641 cgggttgctg tcaggcgacg aggactttag ctcaatcgcc gacatggatt ttagcgccct
11701 gttgggtagc ggaagtggta gccgcgacag ccgggaaggc atgttccttc cgaaaccgga
11761 agctggtagc gccataagcg acgtatttga gggtagggag gtgtgtcagc ctaagcggat
11821 tcgaccattc cacccccctg gctctccttg ggcaaatcga cccctcccgg cgagccttgc
11881 ccctacgccg accggacccg tgcatgagcc cgtcggtagc ctcacgccag cccccgtgcc
11941 gcaacctctc gaccctgctc cggcggttac gccagaagca agccaccttt tggaggaccc
12001 ggacgaagaa acaagccagg cggtaaaggc gctccgcgaa atggccgaca ccgtgatacc
12061 ccagaaggag gaagccgcca tttgcggcca aatggacctt tcccaccctc caccacgcgg
12121 ccacctggac gagctgacga ccaccctgga aagcatgacc gaggatttga atctggacag
12181 cccgttgacc cccgaattga acgaaatcct ggacacgttc ctgaatgatg agtgtttgtt
12241 gcacgctatg cacatcagca ccgggtttag tatatttgac acaagtttgt tttaactcga
12301 gactagataa ctgatcggcg cgccgaccca gctttcttgt acaaagtggt tgatccgggg
12361 atcctctagt cagctgacgc gtgctagcgc ggccgcatcg ataagcttgt cgacgatatc
12421 tctagaggat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
12481 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
12541 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
12601 catttttttc actgcctcga cccatagagc ccaccgcatc cccagcatgc ctgctattgt
12661 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
12721 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
12781 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
12841 gcacagtcga ggctgatcag cgggtttaaa cgggccctct agatcaggca ccgggcttgc
12901 gggtcatgca ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc
12961 cgagccgctc gtagaagggg aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc
13021 cggcgcgctc ggccgcctcc
//
