LentiRCas9-CUG

LOCUS       LentiRCas9-CUG               13752 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Lentiviral transfer vector that carries U6-driven sgRNA targeting 
            CUG repeats using a modified scaffold (Chen et al. Cell 2013) and 
            CMV-driven PIN-dCas9. Derived from LentiCRISPR v2 (Zhang lab).
ACCESSION   .
VERSION     .
KEYWORDS    LentiRCas9-CUG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13752)
  AUTHORS   Batra R, Nelles DA, Pirie E, Blue SM, Marina RJ, Wang H, Chaim IA, 
            Thomas JD, Zhang N, Nguyen V, Aigner S, Markmiller S, Xia G, Corbett
            KD, Swanson MS, Yeo GW
  TITLE     Elimination of Toxic Microsatellite Repeat Expansion RNA by 
            RNA-Targeting Cas9.
  JOURNAL   Cell. 2017 Aug 24;170(5):899-912.e10. doi: 
            10.1016/j.cell.2017.07.010. Epub 2017 Aug 10.
  PUBMED    28803727
REFERENCE   2  (bases 1 to 13752)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..13752
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2371..2611
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2371..2391
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2542..2561
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2748..2764)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2748..2764)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2801..2821)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2801..2819)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2881..3092
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3720..7820
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7824..7850
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7869..7889
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7896..7916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7938..7994
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7995..8591
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     8488..8508
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8607..9195
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8660..8680)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9078..9089)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9267..9500
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9526..9543)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9532..9756
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9802..10230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9889..9908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10099..10120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10239..10259)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10244..10573
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10424..10559
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10486..10505
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10621..10668
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10687..11061
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    11191..11312
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11228..11247)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11282..11301
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11361..11377)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11361..11377)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11374..11396)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11385..11401
                     /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(11409..11439)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11454..11475
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11592..11609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11763..12351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11843..11862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12522..13382)
                     /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     13145..13164
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13383..13487)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13562..13581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat
     2401 ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca
     2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg agcagcagca gcagcagcag
     2641 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     2701 ttgaaaaagt ggcaccgagt cggtgctttt tttcctgcag cccgggggat ccactagttc
     2761 tagagcggcc gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg
     2821 cgcgcgaatt cgctagctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
     2881 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
     2941 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     3001 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     3061 tttttcgcaa cgggtttgcc gccagaacac aggaccggtt ctagagcgct ctcgagagaa
     3121 ccatgcagat ggagctcgaa atcaggccgc tgttcctcgt gccggacact aatggtttta
     3181 tagatcactt ggcgtccttg gctagacttc tggaaagccg aaagtatata ttggtagtgc
     3241 cgttgattgt aattaacgaa ttggatgggt tggcgaaagg acaagagact gatcacagag
     3301 caggaggcta cgcgagggtc gtccaagaga aggcgcgaaa aagcatcgag ttcctggagc
     3361 agcgatttga gagcagggac tcatgcctga gagccctcac gtcacggggg aacgagctgg
     3421 agtccatcgc tttccgaagt gaagacatta cgggccaact tgggaataat gatgacctca
     3481 tcttgtcctg ctgcctgcac tactgcaagg acaaggctaa ggacttcatg cctgcctcca
     3541 aggaggagcc tatccgattg ttgagggaag tagtactttt gacggacgac cgcaacctcc
     3601 gggtaaaggc gctgactcga aatgtcccag taagggatat accggcgttc cttacatggg
     3661 ctcaagtagg gagtggaagt gagacaccgg gaacctcaga gagcgccacg ccagaaagcg
     3721 acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca
     3781 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     3841 gcatcaagaa gaacctgatc ggcgccctgc tgttcgacag cggagaaaca gccgaggcca
     3901 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     3961 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     4021 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     4081 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     4141 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     4201 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     4261 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     4321 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     4381 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga
     4441 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     4501 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     4561 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     4621 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     4681 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     4741 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     4801 acatcgatgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     4861 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     4921 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     4981 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     5041 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     5101 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     5161 tggacaaggg cgccagcgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     5221 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtaca
     5281 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     5341 gcgagcagaa aaaagccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     5401 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     5461 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     5521 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     5581 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     5641 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     5701 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     5761 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     5821 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     5881 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     5941 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     6001 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     6061 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     6121 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     6181 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgctatcg
     6241 tgcctcagag ctttctgaag gacgactcca tcgataacaa agtgctgact cggagcgaca
     6301 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     6361 actggcgcca gctgctgaat gccaagctga ttacccagag gaagttcgac aatctgacca
     6421 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     6481 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     6541 agtacgacga gaacgacaaa ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     6601 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     6661 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     6721 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     6781 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     6841 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     6901 tgatcgagac aaacggcgaa acaggcgaga tcgtgtggga taagggccgg gactttgcca
     6961 ccgtgcggaa agtgctgtct atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     7021 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgacaag ctgatcgcca
     7081 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     7141 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     7201 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     7261 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     7321 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     7381 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     7441 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     7501 acaaacacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     7561 tggccgacgc taatctggac aaggtgctga gcgcctacaa caagcacaga gacaagccta
     7621 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     7681 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     7741 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     7801 tgtctcagct gggaggcgac gcctatccct atgacgtgcc cgattatgcc agcctgggca
     7861 gcggctcccc caagaaaaaa cgcaaggtgg aagatcctaa gaaaaagcgg aaagtggacg
     7921 tgttcgaagg gtccggcgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag
     7981 agaatcctgg accgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     8041 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     8101 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     8161 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     8221 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     8281 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     8341 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca
     8401 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     8461 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     8521 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     8581 ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     8641 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     8701 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     8761 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     8821 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     8881 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     8941 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     9001 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     9061 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     9121 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     9181 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     9241 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     9301 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     9361 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     9421 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     9481 agtgtggaaa atctctagca gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     9541 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     9601 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     9661 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     9721 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     9781 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     9841 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     9901 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     9961 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
    10021 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
    10081 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
    10141 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
    10201 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
    10261 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
    10321 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
    10381 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
    10441 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
    10501 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
    10561 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
    10621 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
    10681 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
    10741 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
    10801 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
    10861 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
    10921 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
    10981 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
    11041 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
    11101 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
    11161 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
    11221 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
    11281 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
    11341 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
    11401 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
    11461 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
    11521 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
    11581 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
    11641 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
    11701 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
    11761 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
    11821 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
    11881 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
    11941 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
    12001 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
    12061 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
    12121 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
    12181 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
    12241 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
    12301 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
    12361 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
    12421 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
    12481 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
    12541 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
    12601 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
    12661 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
    12721 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
    12781 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
    12841 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
    12901 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
    12961 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
    13021 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
    13081 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
    13141 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
    13201 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
    13261 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
    13321 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
    13381 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
    13441 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
    13501 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
    13561 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
    13621 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
    13681 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
    13741 gatatacgcg tt
//