pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)

LOCUS       pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)                9258 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  This plasmid contains expression cassette for NmCas9 with N and C 
            NLS and an HA tag, a cassette for expression of tracrRNA, a cassette
            for cloning crRNA under the control of U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9258)
  AUTHORS   Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, 
            Thomson JA
  TITLE     Efficient genome engineering in human pluripotent stem cells using 
            Cas9 from Neisseria meningitidis.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 12.
  PUBMED    23940360
REFERENCE   2  (bases 1 to 9258)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9258
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(44..284)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     322..340
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     promoter        complement(359..377)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        complement(419..511)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(519..759)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(824..843)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        993..2166
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1224..2157
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2114..2134
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2172..2181
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2184..2204
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2205..5447
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF
                     ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKS
                     LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAG
                     NAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFG
                     NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
                     LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
                     AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
                     DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
                     EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
                     IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
                     LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
                     ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
                     FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
                     GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEK
                     LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
                     EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
                     GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
                     IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
                     IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             5448..5474
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5481..5507
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(5523..5540)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5529..5753
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5942..7055
                     /label=mini-oriP
                     /note="internally truncated variant of the Epstein-Barr 
                     virus oriP replication origin (Tanaka et al., 1999)"
     primer_bind     complement(7101..7117)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7101..7117)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7114..7136)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7125..7141
                     /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(7149..7179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7194..7215
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7332..7349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7503..8091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7583..7602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(8262..9122)
                     /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     8885..8904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9123..9227)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtttaaacaa aaaaatgaga cgtggaatcg tctccggtgt ttcgtccttt ccacaagata
       61 tataaagcca agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta
      121 aaacataatt ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt
      181 tgtactaata tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg
      241 tatcgtatat gcaaatatga aggaatcatg ggaaataggc cctcttcctg cccgacctta
      301 gatccgaatc catatcgata tgctagttat tgctcagcgg tggcagcagc caactcagcc
      361 tatagtgagt cgtattaatt tcgcgggatg tttaaacgcg gccgcaaaaa aaccgaaata
      421 aacgatgccc cttaaagcag aagctttaag gggcagagcg ttgcggcaca tcttttcaga
      481 cggccttatt gtagcaacgg ttctcatttc ggtgtttcgt cctttccaca agatatataa
      541 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
      601 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
      661 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg
      721 tatatgcaaa tatgaaggaa tcatgggaaa taggccctct tcctgcccga ccttgacgtc
      781 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
      841 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      901 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      961 ttagggttag gcgttttgcg ctgcttcgcg gaggctccgg tgcccgtcag tgggcagagc
     1021 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     1081 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     1141 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     1201 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
     1261 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctccag tacgtgattc
     1321 ttgatcccga gctggagcca ggggcgggcc ttgcgcttta ggagcccctt cgcctcgtgc
     1381 ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg
     1441 cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc
     1501 gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caggatctgc acactggtat
     1561 ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc
     1621 gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtcggacg ggggtagtct
     1681 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     1741 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     1801 cctgctccag ggggctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     1861 cccacacaaa ggaaaggggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     1921 taccgggcgc cgtccaggca cctcgattag ttctggagct tttggagtac gtcgtcttta
     1981 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     2041 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     2101 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     2161 tcgtgaacac cgccaccatg gtgcctaaga agaagagaaa ggtggctgcc ttcaaaccta
     2221 attcaatcaa ctacatcctc ggcctcgata tcggcatcgc atccgtcggc tgggcgatgg
     2281 tagaaattga cgaagaagaa aaccccatcc gcctgattga tttgggcgtg cgcgtatttg
     2341 agcgtgccga agtaccgaaa acaggcgact cccttgccat ggcaaggcgt ttggcgcgca
     2401 gtgttcgccg cctgacccgc cgtcgcgccc accgcctgct tcggacccgc cgcctattga
     2461 aacgcgaagg cgtattacaa gccgccaatt ttgacgaaaa cggcttgatt aaatccttac
     2521 cgaatacacc atggcaactt cgcgcagccg cattagaccg caaactgacg cctttagagt
     2581 ggtcggcagt cttgttgcat ttaatcaaac atcgcggcta tttatcgcaa cggaaaaacg
     2641 agggcgaaac tgccgataag gagcttggcg ctttgcttaa aggcgtagcc ggcaatgccc
     2701 atgccttaca gacaggcgat ttccgcacac cggccgaatt ggctttaaat aaatttgaga
     2761 aagaaagcgg ccatatccgc aatcagcgca gcgattattc gcatacgttc agccgcaaag
     2821 atttacaggc ggagctgatt ttgctgtttg aaaaacaaaa agaatttggc aatccgcatg
     2881 tttcaggcgg ccttaaagaa ggtattgaaa ccctactgat gacgcaacgc cctgccctgt
     2941 ccggcgatgc cgttcaaaaa atgttggggc attgcacctt cgaaccggca gagccgaaag
     3001 ccgctaaaaa cacctacaca gccgaacgtt tcatctggct gaccaagctg aacaacctgc
     3061 gtattttaga gcaaggcagc gagcggccat tgaccgatac cgaacgcgcc acgcttatgg
     3121 acgagccata cagaaaatcc aaactgactt acgcacaagc ccgtaagctg ctgggtttag
     3181 aagataccgc ctttttcaaa ggcttgcgct atggtaaaga caatgccgaa gcctcaacat
     3241 tgatggaaat gaaggcctac catgccatca gccgtgcact ggaaaaagaa ggattgaaag
     3301 acaaaaaatc cccattaaac ctttctcccg aattacaaga cgaaatcggc acggcattct
     3361 ccctgttcaa aaccgatgaa gacattacag gccgtctgaa agaccgtata cagcccgaaa
     3421 tcttagaagc gctgttgaaa cacatcagct tcgataagtt cgtccaaatt tccttgaaag
     3481 cattgcgccg aattgtgcct ctaatggaac aaggcaaacg ttacgatgaa gcctgcgccg
     3541 aaatctacgg agaccattac ggcaagaaga atacggaaga aaagatttat ctgccgccga
     3601 ttcccgccga cgaaatccgc aaccccgtcg tcttgcgcgc cttatctcaa gcacgtaagg
     3661 tcattaacgg cgtggtacgc cgttacggct ccccagctcg tatccatatt gaaactgcaa
     3721 gggaagtagg taaatcgttt aaagaccgca aagaaattga gaaacgccaa gaagaaaacc
     3781 gcaaagaccg ggaaaaagcc gccgccaaat tccgagagta tttccccaat tttgtcggag
     3841 aacccaaatc caaagatatt ctgaaactgc gcctgtacga gcaacaacac ggcaaatgcc
     3901 tgtattcggg caaagaaatc aacttaggcc gtctgaacga aaaaggctat gtcgaaatcg
     3961 accatgccct gccgttctcg cgcacatggg acgacagttt caacaataaa gtactggtat
     4021 tgggcagcga aaaccaaaac aaaggcaatc aaacccctta cgaatacttc aacggcaaag
     4081 acaacagccg cgaatggcag gaatttaaag cgcgtgtcga aaccagccgt ttcccgcgca
     4141 gtaaaaaaca acggattctg ctgcaaaaat tcgatgaaga cggctttaaa gaacgcaatc
     4201 tgaacgacac gcgctacgtc aaccgtttcc tgtgtcaatt tgttgccgac cgtatgcggc
     4261 tgacaggtaa aggcaagaaa cgtgtctttg catccaacgg acaaattacc aatctgttgc
     4321 gcggcttttg gggattgcgc aaagtgcgtg cggaaaacga ccgccatcac gccttggacg
     4381 ccgtcgtcgt tgcctgctcg accgttgcca tgcagcagaa aattacccgt tttgtacgct
     4441 ataaagagat gaacgcgttt gacggtaaaa ccatagacaa agaaacagga gaagtgctgc
     4501 atcaaaaaac acacttccca caaccttggg aatttttcgc acaagaagtc atgattcgcg
     4561 tcttcggcaa accggacggc aaacccgaat tcgaagaagc cgatacccta gaaaaactgc
     4621 gcacgttgct tgccgaaaaa ttatcatctc gccccgaagc cgtacacgaa tacgttacgc
     4681 cactgtttgt ttcacgcgcg cccaatcgga agatgagcgg gcaagggcat atggagaccg
     4741 tcaaatccgc caaacgactg gacgaaggcg tcagcgtgtt gcgcgtaccg ctgacacagt
     4801 taaaactgaa agacttggaa aaaatggtca atcgggagcg cgaacctaag ctatacgaag
     4861 cactgaaagc acggctggaa gcacataaag acgatcctgc caaagccttt gccgagccgt
     4921 tttacaaata cgataaagca ggcaaccgca cccaacaggt aaaagccgta cgcgtagagc
     4981 aagtacagaa aaccggcgta tgggtgcgca accataacgg tattgccgac aacgcaacca
     5041 tggtgcgcgt agatgtgttt gagaaaggcg acaagtatta tctggtaccg atttacagtt
     5101 ggcaggtagc gaaagggatt ttgccggata gggctgttgt acaaggaaaa gatgaagaag
     5161 attggcaact tattgatgat agtttcaact ttaaattctc attacaccct aatgatttag
     5221 tcgaggttat aacaaaaaaa gctagaatgt ttggttactt tgccagctgc catcgaggca
     5281 caggtaatat caatatacgc attcatgatc ttgatcataa aattggcaaa aatggaatac
     5341 tggaaggtat cggcgtcaaa accgcccttt cattccaaaa ataccaaatt gacgaactgg
     5401 gcaaagaaat cagaccatgc cgtctgaaaa aacgcccgcc tgtccgttac ccatacgatg
     5461 ttccagatta cgctgcagct ccagcagcga agaaaaagaa gctggattaa ctcgctgatc
     5521 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     5581 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     5641 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     5701 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     5761 ggcggaaaga accagctggg gctcgaccga tgcccttgag agccttcaac ccagtcagct
     5821 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     5881 tgcaactcgt aggacaggtg cctggccggg gtcccccgga aactcggccg tggtgaccat
     5941 gcaggaaaag gacaagcagc gaaaattcac gcccccttgg gaggtggcgg catatgcaaa
     6001 ggatagcact cccactctac tactgggtat catatgctga ctgtatatgc atgaggatag
     6061 catatgctac ccggatacag attaggatag catatactac ccagatatag attaggatag
     6121 catatgctac ccagatatag attaggatag cctatgctac ccagatataa attaggatag
     6181 catatactac ccagatatag attaggatag catatgctac ccagatatag attaggatag
     6241 cctatgctac ccagatatag attaggatag catatgctac ccagatatag attaggatag
     6301 catatgctat ccagatattt gggtagtata tgctacccag atataaatta ggatagcata
     6361 tactacccta atctctatta ggatagcata tgctacccgg atacagatta ggatagcata
     6421 tactacccag atatagatta ggatagcata tgctacccag atatagatta ggatagccta
     6481 tgctacccag atataaatta ggatagcata tactacccag atatagatta ggatagcata
     6541 tgctacccag atatagatta ggatagccta tgctacccag atatagatta ggatagcata
     6601 tgctatccag atatttgggt agtatatgct acccatggca acattagccc accgtgctct
     6661 cagcgacctc gtgaatatga ggaccaacaa ccctgtgctt ggcgctcagg cgcaagtgtg
     6721 tgtaatttgt cctccagatc gcagcaatcg cgcccctatc ttggcccgcc cacctactta
     6781 tgcaggtatt ccccggggtg ccattagtgg ttttgtgggc aagtggtttg accgcagtgg
     6841 ttagcggggt tacaatcagc caagttatta cacccttatt ttacagtcca aaaccgcagg
     6901 gcggcgtgtg ggggctgacg cgatcgctgt tccttaggac ccttttacta accctaattc
     6961 gatagcatat gcttcccgtt gggtaacata tgctattgaa ttagggttag tctggatagt
     7021 atatactact acccgggaag catatgctac ccgtttaggg ttataccgtc gacctctagc
     7081 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     7141 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     7201 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     7261 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     7321 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     7381 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     7441 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     7501 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     7561 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     7621 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     7681 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7741 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7801 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7861 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7921 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     7981 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     8041 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     8101 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     8161 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     8221 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     8281 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     8341 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     8401 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     8461 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     8521 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     8581 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     8641 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     8701 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     8761 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     8821 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     8881 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     8941 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     9001 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     9061 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     9121 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     9181 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     9241 aaagtgccac ctgacgtc
//