pB-CAGGS-dCas9-KRAB-MeCP2

LOCUS       pB-CAGGS-dCas9-KRAB-MeCP2               12885 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac compatible plasmid expressing dCas9-KRAB-MeCP2.
ACCESSION   .
VERSION     .
KEYWORDS    pB-CAGGS-dCas9-KRAB-MeCP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12885)
  AUTHORS   Yeo NC, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D, Kuo CC,
            Guo X, Sharma S, Tung A, Cecchi RJ, Tuttle M, Pradhan S, Lim ET, 
            Davidsohn N, Ebrahimkhani MR, Collins JJ, Lewis NE, Kiani S, Church 
            GM
  TITLE     An enhanced CRISPR repressor for targeted mammalian gene regulation.
  JOURNAL   Nat Methods. 2018 Jul 16. pii: 10.1038/s41592-018-0048-5. doi: 
            10.1038/s41592-018-0048-5.
  PUBMED    30013045
REFERENCE   2  (bases 1 to 12885)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..12885
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1357..1736
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2016..3032
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2956..2978
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3040..3059
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3129..3138
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3135..7238
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7251..7271
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7332..7526
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             7560..7580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7590..8447
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(8502..8521)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8546..8594
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        8826..9037
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      9047..9056
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             9053..9451
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    9455..9576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9492..9511)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9546..9565
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10339..10373
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(10864..10880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10864..10880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10877..10899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10888..10904
                     /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(10912..10942)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10957..10978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11095..11112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11266..11854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11346..11365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12025..12885)
                     /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     12648..12667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccgaca ttgattattg actagttatt
     1381 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1441 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1501 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1561 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1621 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1681 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1741 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1801 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggs
     1861 gcgcgccggg gggggggggg gggggggggg gggggggggg ggsgrggsgg rgaggtgcgg
     1921 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     1981 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     2041 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     2101 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt
     2161 aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc
     2221 ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg
     2281 cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttttg
     2341 cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     2401 gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg
     2461 tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg
     2521 gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg
     2581 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggrgggct
     2641 cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca
     2701 gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct
     2761 gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc
     2821 ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc
     2881 gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg
     2941 gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta
     3001 accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc
     3061 tgtctcatca ttttggcaaa gaatctagat accctcgtaa aggtctagag ctagcggatc
     3121 gaacccttgc caccatggac aagaagtact ccattgggct cgctatcggc acaaacagcg
     3181 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
     3241 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
     3301 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
     3361 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
     3421 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
     3481 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
     3541 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
     3601 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
     3661 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
     3721 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     3781 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     3841 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     3901 tcgacctggc cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     3961 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     4021 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     4081 cgctgagcgc tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga
     4141 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     4201 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     4261 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     4321 aagatctgtt gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc
     4381 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     4441 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     4501 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct
     4561 ggaacttcga ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     4621 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     4681 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     4741 agccagcatt cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga
     4801 accggaaagt taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg
     4861 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     4921 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     4981 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     5041 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
     5101 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     5161 gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt
     5221 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     5281 agggggacag tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     5341 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     5401 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
     5461 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     5521 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     5581 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     5641 acgacgtggc tgctatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     5701 tgttgacaag atccgataaa gctagaggga agagtgataa cgtcccctca gaagaagttg
     5761 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     5821 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
     5881 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     5941 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     6001 ttactctgaa gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga
     6061 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     6121 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     6181 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     6241 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     6301 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     6361 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     6421 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
     6481 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     6541 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     6601 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     6661 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     6721 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     6781 gtgcgggcga gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     6841 tgtatctggc cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc
     6901 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     6961 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     7021 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     7081 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
     7141 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     7201 atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga
     7261 agaggaaggt gagtggtgga ggaagtggcg ggtcagggtc gatggacgcg aaatcactta
     7321 cggcatggtc gagaacactg gttacgttca aggacgtgtt tgtggacttt acacgtgagg
     7381 agtggaaatt gctggatact gcgcaacaaa ttgtgtatcg aaatgtcatg cttgagaatt
     7441 acaagaacct cgtcagtctc ggataccagt tgacgaaacc ggatgtgatc cttaggctcg
     7501 aaaaggggga agaaccttgg ctggtatcgg gaggtggttc gggtggctct ggatcaagcc
     7561 caaagaagaa acggaaggtg gaagcctcag tgcaggtgaa aagggtgctg gaaaaatccc
     7621 ccggcaaact cctcgtgaag atgcccttcc aggcttcccc tggcggaaaa ggtgaagggg
     7681 gtggcgcaac cacatctgcc caggtcatgg tcatcaagcg acctggaagg aaaagaaagg
     7741 ccgaggctga ccctcaggcc attccaaaga aacggggacg caagccaggg tccgtggtcg
     7801 cagctgcagc agctgaggct aagaaaaagg cagtgaagga aagctccatc cgcagtgtgc
     7861 aggagactgt cctgcccatc aagaagagga agactaggga gaccgtgtcc atcgaggtca
     7921 aagaagtggt caagcccctg ctcgtgtcca ccctgggcga aaaatctgga aaggggctca
     7981 aaacatgcaa gtcacctgga cggaaaagca aggagtctag tccaaagggg cgctcaagct
     8041 ccgcttctag tccccctaaa aaggaacacc atcaccatca ccatcacgcc gagtctccta
     8101 aggctcctat gccactgctc ccaccacctc caccacctga gccacagtca agcgaagacc
     8161 ccatcagccc acccgagcct caggatctgt cctctagtat ttgcaaagag gaaaagatgc
     8221 ccagagcagg cagcctggag agtgatggct gtccaaaaga acccgccaag acccagccta
     8281 tggtggcagc cgctgcaact accaccacaa ccacaactac cacagtggcc gaaaaataca
     8341 agcatcgcgg cgagggcgaa cgaaaggaca ttgtgtcaag ctccatgccc agacctaacc
     8401 gggaggaacc agtcgatagt aggacacccg tgactgagag agtctcatag aagaattcga
     8461 tccctaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca
     8521 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
     8581 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     8641 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     8701 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     8761 agatctgcga aatcggatcc gcggccgcac tgattctagc tgcagaggct ccggtgcccg
     8821 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     8881 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     8941 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     9001 cgttcttttt cgcaacgggt ttgccgccag aacacaggat cctcgagcca ccatggccaa
     9061 gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat
     9121 ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt
     9181 cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg
     9241 cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa
     9301 caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg
     9361 gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt
     9421 gctgccctct ggttatgtgt gggagggcta agttaacttg tttattgcag cttataatgg
     9481 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     9541 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg
     9601 atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg
     9661 tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac
     9721 caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca
     9781 ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc
     9841 cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga
     9901 tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg
     9961 ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag
    10021 ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga
    10081 aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt
    10141 atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt
    10201 cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc
    10261 aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag
    10321 agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct
    10381 agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc
    10441 gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat
    10501 ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta
    10561 ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg
    10621 ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga
    10681 agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga
    10741 ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga
    10801 tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc
    10861 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
    10921 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
    10981 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
    11041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
    11101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
    11161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
    11221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
    11281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
    11341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
    11401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
    11461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
    11521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
    11581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
    11641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
    11701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
    11761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
    11821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
    11881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
    11941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
    12001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
    12061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
    12121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
    12181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
    12241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
    12301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
    12361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
    12421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
    12481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
    12541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
    12601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
    12661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
    12721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
    12781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
    12841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat
//