| 质粒名称: | PB_tre_Cas9 |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 126029 |
| 存储实验室: | Mauro Calabrese |
|
载体骨架基本信息 |
|
| 载体名称: | piggyBac cargo vector |
| 载体出品公司: | System Biosciences |
| 空载体大小: | 11497 bp |
| 完整质粒大小: | 11497bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | Hygromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称1: | humanized S. pyrogenes Cas9 |
| 插入大小: | 4272 bp |
| 启动子: | TRE |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | GGGAAAAGGCCTCCACGG |
| 插入基因名称2: | Hygromycin resistance |
| 插入大小: | 1008bp |
| 启动子: | EF1 alpha |
| 克隆方法: | Restriction Enzyme |
| 5’测序站 | HpaI (destroyed during cloning) |
| 3’测序站 | SpeI (destroyed during cloning) |
| 5’测序引物 | NA |
|
文献引用方法 |
|
| 材料和方法部分: | PB_tre_Cas9 was a gift from Mauro Calabrese (Addgene plasmid # 126029 ; http://n2t.net/addgene:126029 ; RRID:Addgene_126029) |
| 参考文献部分: | A piggyBac-based toolkit for inducible genome editing in mammalian cells. Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM, Kim SO, Vitucci EM, Calabrese JM. RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118. 10.1261/rna.068932.118 PubMed 311016 |
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 PB_tre_Cas9 11497 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION PiggyBac cargo vector expressing doxycycline inducible Cas9.
ACCESSION .
VERSION .
KEYWORDS PB_tre_Cas9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11497)
AUTHORS Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM,
Kim SO, Vitucci EM, Calabrese JM
TITLE A piggyBac-based toolkit for inducible genome editing in mammalian
cells.
JOURNAL RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118.
PUBMED 31101683
REFERENCE 2 (bases 1 to 11497)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..11497
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..315
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 9..27
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 45..63
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 80..98
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 116..134
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 152..170
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 187..205
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 223..241
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 288..312
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 320..329
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 329..394
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 401..421
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 446..4546
/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 4547..4594
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
polyA_signal 4628..4852
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 4980..5070
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
promoter 5118..5329
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 5342..5610
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 5647..6672
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 6701..6822
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6738..6757)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6792..6811
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
repeat_region 7585..7619
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(8110..8126)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8110..8126)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8123..8145)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8134..8150
/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(8158..8188)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 8203..8224
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8341..8358)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8512..9100)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8592..8611)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(9271..10131)
/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 9894..9913
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(10132..10236)
/gene="bla"
/label=AmpR promoter
rep_origin complement(10262..10717)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10399..10420)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 10611..10630
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 10844..10866
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 10858..10875
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 10859..10875
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 11005..11067
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
ORIGIN
1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag
241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
301 aaccgtcaga tcgcctggag ccaccatgga ctataaggac cacgacggag actacaagga
361 tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt
421 cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac
481 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
541 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
601 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
661 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
721 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
781 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
841 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
901 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
961 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
1021 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
1081 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
1141 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
1201 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga
1261 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
1321 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
1381 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac
1441 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
1501 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
1561 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
1621 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca
1681 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
1741 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
1801 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
1861 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga
1921 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
1981 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
2041 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt
2101 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
2161 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
2221 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
2281 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
2341 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
2401 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
2461 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
2521 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
2581 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
2641 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
2701 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
2761 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
2821 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
2881 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
2941 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga
3001 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
3061 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
3121 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
3181 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
3241 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
3301 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
3361 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
3421 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
3481 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
3541 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
3601 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
3661 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
3721 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
3781 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
3841 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
3901 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
3961 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
4021 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
4081 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
4141 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
4201 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
4261 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
4321 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
4381 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
4441 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
4501 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc
4561 cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcctagagc tcgccaatgc
4621 gtttcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggaaacacgc
4861 ggcgacgcgt tcgaccgaat aaaacctgtg acggaagatc acttcgcaga ataaataaat
4921 cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt
4981 gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta
5041 ttttttgagt tgtcgagatt ttcaggagct aaagatctgg aagctaagga tctgcgatcg
5101 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
5161 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
5221 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
5281 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg
5341 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg
5401 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta
5461 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc
5521 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt
5581 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta caagctgcta
5641 gccaccatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
5701 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
5761 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
5821 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
5881 gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
5941 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
6001 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
6061 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
6121 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
6181 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
6241 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
6301 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
6361 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
6421 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
6481 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
6541 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
6601 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
6661 gcaaaggaat agttttacgt attgagtatt taactaacta aacttgttta ttgcagctta
6721 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
6781 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggaattgact
6841 caaatgatgt caattagtct atcagaagct catctggtct cccttccggg ggacaagaca
6901 tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc ccttgctctg
6961 gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa gaaacagtgg
7021 cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt ggtatggctt
7081 tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga
7141 aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg
7201 atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg
7261 ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct gatatctata
7321 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
7381 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
7441 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
7501 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
7561 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
7621 taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt catcgcccaa
7681 gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc gaggttgaag
7741 cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag cgaaaacgca
7801 cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt gaactgttcg
7861 ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc cggatggtca
7921 gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc cgtgccggtg
7981 tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac gggtatcctg
8041 gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg cgcgcttggc
8101 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
8161 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
8221 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
8281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
8341 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
8401 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
8461 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
8521 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
8581 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
8641 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
8701 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
8761 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
8821 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
8881 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
8941 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
9001 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
9061 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
9121 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
9181 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
9241 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
9301 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
9361 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
9421 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
9481 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
9541 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
9601 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
9661 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
9721 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
9781 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
9841 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
9901 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
9961 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
10021 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
10081 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
10141 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
10201 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
10261 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
10321 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
10381 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
10441 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
10501 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
10561 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
10621 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
10681 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
10741 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
10801 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
10861 aaaacgacgg ccagtgagcg cgcctcgttc attcacgttt ttgaacccgt ggaggacggg
10921 cagactcgcg gtgcaaatgt gttttacagc gtgatggagc agatgaagat gctcgacacg
10981 ctgcagaaca cgcagctaga ttaaccctag aaagataatc atattgtgac gtacgttaaa
11041 gataatcatg tgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta
11101 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca
11161 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa caaaaactca aaatttcttc
11221 tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat
11281 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
11341 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
11401 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
11461 ggatacgggg aaaaggcctc cacggccaga ctagttc
//
