| 质粒名称: | pAAVS1-PDi-CRISPRn |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 73500 |
| 存储实验室: | Bruce Conklin |
|
载体骨架基本信息 |
|
| 载体名称: | pAAVS1 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3356 bp |
| 完整质粒大小: | 12658 bp |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 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: | Cas9,SpCas9 |
| 物种来源: | Synthetic; S. pyogenes |
| 插入大小: | 4266 bp |
| 启动子 | TRE |
| 标签/融合蛋白: |
NLS（插入件上的N端子）
3xFLAG（插入件上的N端子）
NLS（插接件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | PacI（未销毁） |
| 3' 克隆位点: | AgeI （未销毁） |
| 5' 测序引物 | GCTCGTTTAGTGAACCGTCAG |
| 3' 测序引物 | TGTGGAATTGTGAGCGGATA |
|
文献引用方法 |
|
| 材料和方法部分: | pAAVS1-PDi-CRISPRn was a gift from Bruce Conklin (Addgene plasmid # 73500 ; http://n2t.net/addgene:73500 ; RRID:Addgene_73500) |
| 参考文献部分: | CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, Weissman JS, Qi LS, So PL, Conklin BR. Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 10.1016/j.stem.2016.01.022. Epub 2016 Mar 10. 10.1016/j.stem.2016.01.022 PubMed 26971820 |
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 pAAVS1-PDi-CRISPRn 12658 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Dox-inducible CRISPR nuclease (CRISPRn) knock in construct into the
AAVS1 locus.
ACCESSION .
VERSION .
KEYWORDS pAAVS1-PDi-CRISPRn
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12658)
AUTHORS Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen
TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP,
Weissman JS, Qi LS, So PL, Conklin BR
TITLE CRISPR Interference Efficiently Induces Specific and Reversible Gene
Silencing in Human iPSCs.
JOURNAL Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi:
10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
PUBMED 26971820
REFERENCE 2 (bases 1 to 12658)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..12658
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 54..914
/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(272..291)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 1085..1673
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1574..1593
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
misc_feature 1721..2557
/label=HA-R
/note="right homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
protein_bind 2590..2608
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2626..2644
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2662..2680
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2698..2716
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2734..2752
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2770..2788
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2806..2824
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2869..2893
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2977..2986
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2986..3051
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 3058..3078
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3103..7203
/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
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 7204..7251
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
polyA_signal 7286..7407
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7323..7342)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 7377..7396
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(7456..7472)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(7456..7472)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(7469..7491)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 7480..7496
/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(7504..7534)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7549..7570
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7687..7704)
/label=L4440
/note="L4440 vector, forward primer"
protein_bind complement(7760..7793)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 8124..8143
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
polyA_signal 8142..8197
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind 8243..8262
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
CDS complement(8352..9098)
/codon_start=1
/product="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/label=Tet-On(R) 3G
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
primer_bind complement(9130..9149)
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
intron 9157..10174
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind complement(9211..9233)
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
promoter complement(10176..10453)
/label=chicken beta-actin promoter
enhancer 10455..10834
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
polyA_signal 10840..11064
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 11053..11070
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
CDS complement(11102..11701)
/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(11185..11205)
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind 11682..11701
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
CDS complement(11711..11764)
/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 11788..11813
/label=SA
/note="splice acceptor site"
protein_bind complement(11820..11853)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 11855..12658
/label=HA-L
/note="left homology arm from the adeno-associated virus
integration site (AAVS1) within intron 1 of the human
PPP1R12C gene"
ORIGIN
1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
1021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
1201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
2941 ccacttccta ccctcgtaaa cttaaggtta attaacgcca ccatggacta taaggaccac
3001 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
3061 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
3121 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
3181 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
3241 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
3301 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
3361 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
3421 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
3481 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
3541 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
3601 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
3661 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
3721 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
3781 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
3841 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
3901 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
3961 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
4021 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
4081 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
4141 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
4201 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
4261 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
4321 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
4381 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
4441 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
4501 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
4561 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
4621 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
4681 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
4741 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
4801 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
4861 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
4921 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
4981 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
5041 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
5101 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
5161 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
5221 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
5281 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
5341 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
5401 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
5461 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
5521 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
5581 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
5641 aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
5701 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
5761 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
5821 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
5881 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
5941 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
6001 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
6061 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
6121 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
6181 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
6241 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
6301 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
6361 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
6421 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
6481 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
6541 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
6601 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
6661 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
6721 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
6781 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
6841 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
6901 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
6961 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
7021 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
7081 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
7141 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
7201 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaaaccggt
7261 gcggccgcaa tcgatcgccc accccaactt gtttattgca gcttataatg gttacaaata
7321 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
7381 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
7441 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
7501 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
7561 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
7621 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
7681 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
7741 tcactcaaag gacggccgca taacttcgta taatgtatgc tatacgaagt tataagcttg
7801 ggctgcaggt cgagggatct ccataagaga agagggacag ctatgactgg gagtagtcag
7861 gagaggagga aaaatctggc tagtaaaaca tgtaaggaaa attttaggga tgttaaagaa
7921 aaaaataaca caaaacaaaa tataaaaaaa atctaacctc aagtcaaggc ttttctatgg
7981 aataaggaat ggacagcagg gggctgtttc atatactgat gacctcttta tagccaacct
8041 ttgttcatgg cagccagcat atgggcatat gttgccaaac tctaaaccaa atactcattc
8101 tgatgtttta aatgatttgc cctcccatat gtccttccga gtgagagaca caaaaaattc
8161 caacacacta ttgcaatgaa aataaatttc ctttattagc cagaagtcag atgctcaagg
8221 ggcttcatga tgtccccata atttttggca gagggaaaaa gatctcagtg gtatttgtga
8281 gccagggcat tggccacacc agccaccacc ttctgatagg cagcctgcac ctgaggagtg
8341 aattcacgcg tttagcctgg cagcatatcg agatcgaagt catcgagagc gtcagcaggg
8401 agcatatcca gatcaaaatc atccagggca tcggcgggca gcatgtccag gtcgaagtcg
8461 tccagggcgt cggtgggtcc gccgctctcg cacttcagct gcttctcgag gccgcagatg
8521 atcagttcca ggccgaacag gaaggcgggc tcggcgccct gtctgtcgaa cagctcgatg
8581 gcctgcttca gcaggggggg catgctgtcg gtggtgggtg tctctctttc ctctttggcg
8641 acctggtgtt cctgttcttc cagcacgcag cccagggtga agtggcccac ggcgctcagg
8701 gcgtacaggg cgttttccag gctgaagccc tgctggcaca ggaaggccag ctggttttcc
8761 agtgtctcgt actgcttctc ggtgggtctg gtgcccaggt gcactttggc gccgtcccgg
8821 tggctcagca gggcgcatct gtagctcttg gcgttgttcc gcaggaagtc ctgccagctc
8881 tcgccttcca gagggcagct gtgggtgtgg tgccggtcca gcatctcgat gggcagggcg
8941 tccagcaggg cccgcttgtt cttcacgtgc cagtacaggg tgggctgttc cacgcccagt
9001 ttctgggcca gcttccgggt ggtcaggccc tcgatgccca cgccgttcag cagttccagg
9061 gcgctgttga tcactttgct cttgtccagc cggctcatgt cgacattctt tgccaaaatg
9121 atgagacagc acaataacca gcacgttgcc caggagctgt aggaaaaaga agaaggcatg
9181 aacatggtta gcagaggctc tagagccgcc ggtcacacgc cagaagccga accccgccct
9241 gccccgtccc ccccgaaggc agccgtcccc ccgcggacag ccccgaggct ggagagggag
9301 aaggggacgg cggcgcggcg acgcacgaag gccctccccg cccatttcct tcctgccggc
9361 gccgcaccgc ttcgccccgc gcccgctaga gggggtgcgg cggcgcctcc cagatttcgg
9421 ctccgcacag atttgggaca aaggaagtcc ctgcgccctc tcgcacgatt accataaaag
9481 gcaatggctg cggctcgccg cgcctcgaca gccgccggcg ctccgggggc cgccgcgccc
9541 ctcccccgag ccctccccgg cccgaggcgg ccccgccccg cccggcaccc ccacctgccg
9601 ccaccccccg cccggcacgg cgagccccgc gccacgcccc gtacggagcc ccgcacccga
9661 agccgggccg tgctcagcaa ctcggggagg ggggtgcagg gggggttgca gcccgaccga
9721 cgcgcccaca ccccctgctc acccccccac gcacacaccc cgcacgcagc ctttgttccc
9781 ctcgcagccc cccccgcacc gcggggcacc gcccccggcc gcgctcccct cgcgcacact
9841 gcggagcgca caaagccccg cgccgcgccc gcagcgctca cagccgccgg gcagcgcgga
9901 gccgcacgcg gcgctcccca cgcacacaca cacgcacgca ccccccgagc cgctcccccc
9961 gcacaaaggg ccctcccgga gcccctcaag gctttcacgc agccacagaa aagaaacaag
10021 ccgtcattaa accaagcgct aattacagcc cggaggagaa gggccgtccc gcccgctcac
10081 ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg
10141 gggcacgggg cgaaggcagc gtcgcagcga ctccccgccc gccgcgcgct tcgcttttta
10201 tagggccgcc gccgccgccg cctcgccata aaaggaaact ttcggagcgc gccgctctga
10261 ttggctgccg ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg ccccgccccg
10321 cctggcgcgc gccccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata
10381 aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg
10441 gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
10501 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
10561 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
10621 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
10681 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
10741 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
10801 gattactatt aataactagt caataatcaa tgtcgagacc catagagccc accgcatccc
10861 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
10921 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
10981 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
11041 atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag
11101 atcaggcacc gggcttgcgg gtcatgcacc aggtgcgcgg tccttcgggc acctcgacgt
11161 cggcggtgac ggtgaagccg agccgctcgt agaaggggag gttgcggggc gcggaggtct
11221 ccaggaaggc gggcaccccg gcgcgctcgg ccgcctccac tccggggagc acgacggcgc
11281 tgcccagacc cttgccctgg tggtcgggcg agacgccgac ggtggccagg aaccacgcgg
11341 gctccttggg ccggtgcggc gccaggaggc cttccatctg ttgctgcgcg gccagccggg
11401 aaccgctcaa ctcggccatg cgcgggccga tctcggcgaa caccgccccc gcttcgacgc
11461 tctccggcgt ggtccagacc gccaccgcgg cgccgtcgtc cgcgacccac accttgccga
11521 tgtcgagccc gacgcgcgtg aggaagagtt cttgcagctc ggtgacccgc tcgatgtggc
11581 ggtccggatc gacggtgtgg cgcgtggcgg ggtagtcggc gaacgcggcg gcgagggtgc
11641 gtacggccct ggggacgtcg tcgcgggtgg cgaggcgcac cgtgggcttg tactcggtca
11701 tctcgagcct agggccggga ttctcctcca cgtcaccgca tgttagaaga cttcctctgc
11761 cctctccgct gccagatctc tcgaggccct gtgggaggaa gagaagaggt cagaagctta
11821 taacttcgta taatgtatgc tatacgaagt tattgcccca ctgtggggtg gaggggacag
11881 ataaaagtac ccagaaccag agccacatta accggccctg ggaatataag gtggtcccag
11941 ctcggggaca caggatccct ggaggcagca aacatgctgt cctgaagtgg acataggggc
12001 ccgggttgga ggaagaagac tagctgagct ctcggacccc tggaagatgc catgacaggg
12061 ggctggaaga gctagcacag actagagagg taaggggggt aggggagctg cccaaatgaa
12121 aggagtgaga ggtgacccga atccacagga gaacggggtg tccaggcaaa gaaagcaaga
12181 ggatggagag gtggctaaag ccagggagac ggggtacttt ggggttgtcc agaaaaacgg
12241 tgatgatgca ggcctacaag aaggggaggc gggacgcaag ggagacatcc gtcggagaag
12301 gccatcctaa gaaacgagag atggcacagg ccccagaagg agaaggaaaa gggaacccag
12361 cgagtgaaga cggcatgggg ttgggtgagg gaggagagat gcccggagag gacccagaca
12421 cggggaggat ccgctcagag gacatcacgt ggtgcagcgc cgagaaggaa gtgctccgga
12481 aagagcatcc ttgggcagca acacagcaga gagcaagggg aagagggagt ggaggaagac
12541 ggaacctgaa ggaggcggca gggaaggatc tgggccagcc gtagaggtga cccaggccac
12601 aagctgcaga cagaaagcgg cacaggccca ggggagagaa tgctggtcag agaaagca
//
