| 质粒名称: | pLenti-Puro-sgAMPKa1 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 138684 |
| 存储实验室: | Reuben Shaw |
|
载体骨架基本信息 |
|
| 载体名称: | pLentiCRISPR v2 |
| 载体出品公司: | -- |
| 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: | 慢病毒载体 |
| 筛选标记: | -- |
| 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: | sgAMPKa1 human |
| 物种来源: | H. sapiens (human) |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | hU6 |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 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: | pLenti-Puro-sgAMPKa1 was a gift from Reuben Shaw (Addgene plasmid # 138684 ; http://n2t.net/addgene:138684 ; RRID:Addgene_138684) |
| 参考文献部分: | The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC. Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ. Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26. 10.1158/2159-8290.CD-18-1261 PubMed 31350328 |
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 pLenti-Puro-sgAMPKa1 13013 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses a human AMPKa1-targeting sgRNA and Cas9.
ACCESSION .
VERSION .
KEYWORDS pLenti-Puro-sgAMPKa1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13013)
AUTHORS Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera
LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE,
Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ
TITLE The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive
Effects of LKB1 in NSCLC.
JOURNAL Cancer Discov. 2019 Nov;9(11):1606-1627. doi:
10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26.
PUBMED 31350328
REFERENCE 2 (bases 1 to 13013)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13013
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..199
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 154..174
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 217..397
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 444..569
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1062..1295
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1480..1524
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 1822..1939
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 1990..2230
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 1990..2010
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2161..2180
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 2260..2335
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 2397..2608
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 2627..2636
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2633..6736
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 6737..6784
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 6785..6808
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 6818..6874
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 6875..7471
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 7368..7388
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 7487..8075
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(7540..7560)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(7958..7969)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 8147..8380
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(8406..8423)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 8412..8636
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 8682..9110
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(8769..8788)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 8979..9000
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(9119..9139)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 9124..9453
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 9304..9439
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 9366..9385
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 9501..9548
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 9567..9941
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 10071..10192
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10108..10127)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10162..10181
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(10241..10257)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10241..10257)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(10254..10276)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 10265..10281
/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(10289..10319)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 10334..10355
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(10472..10489)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(10643..11231)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(10723..10742)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(11402..12262)
/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 12025..12044
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(12263..12367)
/gene="bla"
/label=AmpR promoter
primer_bind complement(12442..12461)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 12633..13012
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
2221 tggaaaggac gaaacaccgc tggtgtggat tattgtcacg ttttagagct agaaatagca
2281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
2341 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
2401 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
2461 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
2521 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
2581 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
2641 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
2701 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
2761 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
2821 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
2881 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
2941 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
3001 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
3061 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
3121 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
3181 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
3241 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
3301 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
3361 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
3421 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
3481 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
3541 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
3601 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
3661 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
3721 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
3781 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
3841 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
3901 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
3961 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
4021 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
4081 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
4141 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
4201 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
4261 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
4321 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
4381 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
4441 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
4501 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
4561 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
4621 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
4681 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
4741 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
4801 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
4861 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
4921 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
4981 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
5041 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
5101 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
5161 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
5221 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
5281 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
5341 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
5401 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
5461 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
5521 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
5581 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
5641 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
5701 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
5761 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
5821 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
5881 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
5941 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
6001 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
6061 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
6121 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
6181 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
6241 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
6301 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
6361 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
6421 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
6481 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
6541 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
6601 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
6661 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
6721 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
6781 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
6841 acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct
6901 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
6961 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
7021 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
7081 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
7141 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
7201 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
7261 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
7321 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
7381 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
7441 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
7501 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
7561 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
7621 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
7681 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
7741 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
7801 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
7861 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
7921 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
7981 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
8041 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
8101 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
8161 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
8221 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
8281 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
8341 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
8401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
8461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
8521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
8581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
8641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
8701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
8761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
8821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
8881 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
8941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
9001 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
9061 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
9121 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
9181 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
9241 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
9301 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
9361 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
9421 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
9481 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
9541 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
9601 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
9661 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
9721 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
9781 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
9841 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
9901 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
9961 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
10021 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
10081 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
10141 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
10201 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
10261 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
10321 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
10381 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
10441 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
10501 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
10561 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
10621 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
10681 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
10741 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
10801 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
10861 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
10921 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
10981 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
11041 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
11101 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
11161 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
11221 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
11281 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
11341 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
11401 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
11461 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
11521 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
11581 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
11641 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
11701 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
11761 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
11821 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
11881 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
11941 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
12001 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
12061 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
12121 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
12181 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
12241 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
12301 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
12361 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
12421 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
12481 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
12541 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
12601 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
12661 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
12721 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
12781 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
12841 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
12901 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
12961 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg
//
