| 质粒名称: | iSpyMac |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 155014 |
| 存储实验室: | Joseph Jacobson |
|
载体骨架基本信息 |
|
| 载体名称: | Adapted from pCMV_AncBE4max (#112094) |
| 空载体大小: | 6031bp |
| 完整质粒大小: | 10111 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | iSpyMac |
| 物种来源: | Streptococcus macacae NCTC 11558 |
| 插入大小: | 4080 bp |
| 基因突变: | PID grafted onto SpyCas9 N-terminal domain, R221K, N394K |
| 启动子: | EF1ɑ |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | EF-1a Forward |
| 3’测序引物 | EGFP-N |
|
文献引用方法 |
|
| 材料和方法部分: | iSpyMac was a gift from Joseph Jacobson (Addgene plasmid # 155014 ; http://n2t.net/addgene:155014 ; RRID:Addgene_155014) |
| 参考文献部分: | A Cas9 with PAM recognition for adenine dinucleotides. Chatterjee P, Lee J, Nip L, Koseki SRT, Tysinger E, Sontheimer EJ, Jacobson JM, Jakimo N. Nat Commun. 2020 May 18;11(1):2474. doi: 10.1038/s41467-020-16117-8. 10.1038/s41467-020-16117-8 PubMed 32424114 |
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 iSpyMac 10111 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses the codon-optimized, engineered iSpyMac Cas9 nuclease in
mammalian cells.
ACCESSION .
VERSION .
KEYWORDS iSpyMac
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10111)
AUTHORS Chatterjee P, Lee J, Nip L, Koseki SRT, Tysinger E, Sontheimer EJ,
Jacobson JM, Jakimo N
TITLE A Cas9 with PAM recognition for adenine dinucleotides.
JOURNAL Nat Commun. 2020 May 18;11(1):2474. doi: 10.1038/s41467-020-16117-8.
PUBMED 32424114
REFERENCE 2 (bases 1 to 10111)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..10111
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..1179
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 232..1170
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1127..1147
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
CDS 1292..3010
/codon_start=1
/product="N-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(N)
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
CDS 5426..5446
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5456..5512
/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 5513..6229
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(5558..5579)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(5819..5838)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 6166..6187
/label=EGFP-C
/note="EGFP, forward primer"
CDS 6242..6262
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6271..6288
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
misc_feature 6321..6909
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(6374..6394)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6792..6803)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6981..7214
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7292..7413
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7329..7348)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 7383..7402
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 7453..7588
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 7515..7534
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(7608..7627)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(7609..7627)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(7637..7654)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(7637..7653)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(7760..7777)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(7931..8519)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8011..8030)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(8690..9550)
/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 9313..9332
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(9551..9655)
/gene="bla"
/label=AmpR promoter
primer_bind complement(9734..9753)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
misc_feature 9887..9958
/label=5' beta-globin insulator
/note="insulator upstream of the human beta-globin locus
(Farrell et al., 2002)"
terminator 9963..10009
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
polyA_signal 10027..10075
/note="synthetic polyadenylation signal"
ORIGIN
1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
181 ctagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
361 gagagttcgt ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgtggcctgg
421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
541 caagatagtc ttgtaaatgc gggccaagat cagcacactg gtatttcggt ttttggggcc
601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg cccggcctgc tctggtgcct
721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagcac aaaatggagg
841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
961 tagttctcca gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa aactacccct aaaagccaaa
1201 agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgct gccaagcttc
1261 cgagctctcg aattcaaagg aggtacccac catggacaag aagtacagca tcggcctgga
1321 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
1381 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
1441 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
1501 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
1561 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
1621 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
1681 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
1741 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
1801 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
1861 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
1921 catcctgtct gccagactga gcaagagcag aaagctggaa aatctgatcg cccagctgcc
1981 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
2041 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
2101 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
2161 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
2221 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
2281 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
2341 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
2401 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
2461 cgtgaagctg aagagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
2521 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
2581 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
2641 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
2701 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
2761 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
2821 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
2881 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
2941 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
3001 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
3061 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
3121 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
3181 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
3241 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
3301 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
3361 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
3421 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
3481 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
3541 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
3601 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
3661 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
3721 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
3781 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
3841 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
3901 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
3961 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
4021 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
4081 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
4141 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
4201 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctaaa
4261 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
4321 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
4381 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
4441 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
4501 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
4561 ccaagtgaat atcgtgaaaa agaccgagat ccagaccgtg ggacagaatg gaggcctgtt
4621 cgacgacaac cccaagagcc ccctggaggt gacccccagc aaactggtgc ccctgaagaa
4681 agagctgaac cccaagaaat acggcggcta ccagaagccc accaccgcct accccgtgct
4741 gctgatcacc gacaccaagc agctgatccc catcagcgtg atgaacaaga aacagttcga
4801 gcagaacccc gtgaagttcc tgcgggaccg gggctaccag caggtgggca agaacgactt
4861 catcaagctg cccaagtaca ccctggtgga catcggcgac ggcatcaagc ggctgtgggc
4921 cagcagcaag gagatccaca agggcaacca gctggtggtg agcaagaaga gccagatcct
4981 gctgtaccac gcccaccacc tggacagcga cctgagcaac gactacctgc agaaccacaa
5041 ccagcagttc gacgtgctgt tcaacgagat catcagcttc agcaaaaagt gcaagctggg
5101 caaggagcac atccagaaga tcgagaacgt gtacagcaac aagaagaaca gcgccagcat
5161 cgaggagctg gccgagagct ttatcaaact gctcggcttc acccagctgg gcgccaccag
5221 ccccttcaac ttcctgggcg tgaagctgaa ccagaagcag tacaagggca agaaggacta
5281 catcctgccc tgcaccgagg gcaccctgat cagacagagc atcaccggac tgtacgagac
5341 cagagtggat ctgagcaaga tcggcgagga tagcggcgga agcggaggca gcaagagaac
5401 cgccgatgga agcgagttcg agtctcccaa gaagaagagg aaagtcggaa gcggagctac
5461 taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatggtgag
5521 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
5581 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
5641 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
5701 caccctgacc tatggagtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
5761 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
5821 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
5881 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
5941 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
6001 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
6061 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
6121 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
6181 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ctggtggttc
6241 tcccaagaag aaaaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc
6301 cgctgatcag caaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
6361 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
6421 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
6481 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
6541 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
6601 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
6661 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
6721 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
6781 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
6841 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
6901 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
6961 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
7021 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
7081 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
7141 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
7201 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
7261 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
7321 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
7381 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
7441 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
7501 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
7561 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
7621 cgtattacgc gcgctcactg gccgtcgttt tacactcact gcccgctttc cagtcgggaa
7681 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc gggaagaggc ggtttgcgta
7741 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
7801 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
7861 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
7921 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
7981 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
8041 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
8101 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
8161 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
8221 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
8281 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
8341 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
8401 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
8461 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
8521 aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag
8581 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
8641 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
8701 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
8761 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
8821 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
8881 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
8941 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
9001 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
9061 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
9121 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
9181 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
9241 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
9301 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
9361 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
9421 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
9481 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
9541 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
9601 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
9661 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag
9721 ggtcgactct cagtacaatc tgctctgatg ccgctcgact ctggctctat cgaatctccg
9781 tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg
9841 tcagctatcg tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt
9901 agctccacag gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa
9961 ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc
10021 gttacacaca caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta
10081 actgctgatc gagtgtagcc agatctcgtg a
//
