LOCUS       40924_215        9626 bp DNA     circular SYN 13-MAY-2021
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            Cas9-D10A nickase linked via 2A peptide with puromycin resistant 
            marker to enhance efficient and accurate genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9626)
  AUTHORS   Chiang TW, le Sage C, Larrieu D, Demir M, Jackson SP
  TITLE     CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening 
            to enhance genome editing.
  JOURNAL   Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356.
  PUBMED    27079678
REFERENCE   2  (bases 1 to 9626)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9626)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2016-04-15"; pages: "
            10.1038/srep24356"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..247
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        283..358
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        448..465
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        483..723
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        758..833
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(842..860)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     enhancer        897..1182
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1184..1461
                     /label=chicken beta-actin promoter
     intron          1462..1690
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1702..1711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1711..1776
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1783..1803
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1828..5928
                     /codon_start=1
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5929..5976
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5992..6048
                     /codon_start=1
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6049..6642
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    6679..6886
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6895..7035
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      7110..7565
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7582..7601)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     7701..7723
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7761..7779)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        7847..7951
                     /label=AmpR promoter
     CDS             7952..8809
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      8983..9571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccggggt cttcggatcc atgcgaagac ctgttttaga gctagaaata
      301 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      361 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc
      421 gccaattctg cagacaaatg gctctagatt aaccctcact aaagggaaag gtcgggcagg
      481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      721 gacgaaacac cgggagacca tcgatgagag ggtctcagtt ttagagctag aaatagcaag
      781 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
      841 ttctatagtg tcacctaaat tgcagctctg gcccgtgtct caaaatctct ggtacccgtt
      901 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      961 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1021 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1081 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
     1141 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     1201 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     1261 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg
     1321 gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag
     1381 agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa
     1441 aagcgaagcg cgcggcgggc gggagtcgct gcgacgctgc cttcgccccg tgccccgctc
     1501 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
     1561 cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa
     1621 gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac
     1681 tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg agactacaag
     1741 gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
     1801 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggccatcggc
     1861 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     1921 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     1981 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     2041 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     2101 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     2161 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     2221 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     2281 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     2341 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     2401 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     2461 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     2521 aagaagaatg gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc
     2581 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     2641 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     2701 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     2761 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     2821 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     2881 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     2941 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     3001 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     3061 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     3121 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     3181 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     3241 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     3301 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     3361 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     3421 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     3481 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     3541 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     3601 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     3661 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     3721 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     3781 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     3841 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     3901 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     3961 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     4021 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     4081 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     4141 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     4201 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     4261 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     4321 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     4381 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     4441 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     4501 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     4561 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     4621 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     4681 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     4741 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     4801 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     4861 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     4921 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     4981 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     5041 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     5101 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     5161 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     5221 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     5281 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     5341 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     5401 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     5461 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     5521 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     5581 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     5641 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     5701 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     5761 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     5821 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     5881 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
     5941 gccacgaaaa aggccggcca ggcaaaaaag aaaaaggaat tcggctccgg agcaacaaac
     6001 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac cgagtacaag
     6061 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     6121 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     6181 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     6241 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     6301 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     6361 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     6421 gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     6481 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc
     6541 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     6601 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgagaatt ctaactagag
     6661 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     6721 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     6781 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     6841 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     6901 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     6961 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     7021 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     7081 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     7141 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     7201 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7261 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7321 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     7381 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     7441 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     7501 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     7561 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     7621 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     7681 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     7741 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     7801 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     7861 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     7921 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     7981 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     8041 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     8101 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     8161 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     8221 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     8281 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     8341 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     8401 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     8461 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     8521 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     8581 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     8641 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     8701 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     8761 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     8821 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     8881 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8941 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     9001 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     9061 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     9121 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     9181 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     9241 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     9301 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     9361 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     9421 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     9481 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     9541 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     9601 cctggccttt tgctggcctt ttgctc
//
