lenti-EF1a-dCas9-VPR-Puro

LOCUS       lenti-EF1a-dCas9-VPR-Puro               15702 bp ds-DNA     circular SYN 18-JAN-2019
DEFINITION  3rd generation lenti vector encoding dCas9 (S. pyogenes) fused with 
            VP64-p65-Rta (VPR) and 2A puromycin resistance marker; 
            EF1a-dCas9-VPR-P2A-Puro-WPRE).
ACCESSION   .
VERSION     .
KEYWORDS    lenti-EF1a-dCas9-VPR-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15702)
  AUTHORS   Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I, 
            Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ
  TITLE     Evaluating Synthetic Activation and Repression of 
            Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and 
            Astrocytes.
  JOURNAL   Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi: 
            10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27.
  PUBMED    28757163
REFERENCE   2  (bases 1 to 15702)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 18, 2019 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..15702
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        130..1308
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          361..1299
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1256..1276
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1333..1342
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1339..5442
                     /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             5455..5475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    5512..5536
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5566..5715
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             5740..5760
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6190..6546
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             6565..7134
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             7150..7206
                     /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             7219..7812
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVT
                     ELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ
                     QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAP
                     RNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7709..7729
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7837..8425
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7890..7910)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8308..8319)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8428..8444)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8429..8445)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8950..9130
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9156..9173)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9162..9386
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9432..9860
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9519..9538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9729..9750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9869..9889)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9874..10203
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10054..10189
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10116..10135
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10251..10298
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10317..10691
                     /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    10821..10942
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10858..10877)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10912..10931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10991..11007)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10991..11007)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11004..11026)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11015..11031
                     /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(11039..11069)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11084..11105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11222..11239)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11393..11981)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11473..11492)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12152..13012)
                     /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     12775..12794
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13013..13117)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(13192..13211)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        13383..13762
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        13764..13962
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     13917..13937
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             13980..14160
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    14207..14332
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    14825..15058
                     /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    15585..15702
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgggtttatt acagggacag cagagatcca gtttggttaa ttagctagct gcaaagatgg
       61 ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga
      121 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
      181 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
      241 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
      301 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
      361 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
      421 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
      481 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
      541 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
      601 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
      661 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
      721 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
      781 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
      841 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
      901 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
      961 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     1021 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     1081 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     1141 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     1201 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     1261 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacg tacgctagag
     1321 gatcgaaccc ttgccaccat ggacaagaag tactccattg ggctcgctat cggcacaaac
     1381 agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt
     1441 ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct cctgttcgac
     1501 tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag atatacccgc
     1561 agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc taaggtggat
     1621 gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa aaagcacgag
     1681 cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa gtacccaacc
     1741 atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt gcggttgatc
     1801 tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga gggggacctg
     1861 aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac ttacaatcag
     1921 cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat cctgagcgct
     1981 aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg ggagaagaag
     2041 aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa ctttaaatct
     2101 aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta cgatgatgat
     2161 ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt ggcggcaaag
     2221 aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga gatcaccaaa
     2281 gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga cttgactttg
     2341 ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt cttcgatcag
     2401 tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga attttacaaa
     2461 tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt aaagcttaac
     2521 agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc ccaccagatt
     2581 cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc ctttttgaaa
     2641 gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta tgtaggcccc
     2701 ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga gaccatcact
     2761 ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt catcgaaagg
     2821 atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg
     2881 tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac agaagggatg
     2941 agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct cctcttcaag
     3001 acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa gattgaatgt
     3061 ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct gggaacgtat
     3121 cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga gaacgaggac
     3181 attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat gattgaagaa
     3241 cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct caagaggcgc
     3301 cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg agacaagcag
     3361 agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg gaacttcatg
     3421 cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc acaagtttct
     3481 ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc agctatcaaa
     3541 aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat
     3601 aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca gaagggacag
     3661 aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact ggggtcccaa
     3721 atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct ctacctgtac
     3781 tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa tcggctctcc
     3841 gactacgacg tggctgctat cgtgccccag tcttttctca aagatgattc tattgataat
     3901 aaagtgttga caagatccga taaagctaga gggaagagtg ataacgtccc ctcagaagaa
     3961 gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact gatcacacaa
     4021 cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc
     4081 ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt ggcccaaatt
     4141 ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg agaggtgaaa
     4201 gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca gttttataag
     4261 gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc agtggtaggc
     4321 actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg agactataaa
     4381 gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa ggccaccgct
     4441 aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac actggccaat
     4501 ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga aatcgtgtgg
     4561 gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc
     4621 gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct cccgaaaagg
     4681 aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata cggcggattc
     4741 gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa agggaagtct
     4801 aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg atcaagcttc
     4861 gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa aaaagacctc
     4921 atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa acgaatgctc
     4981 gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa atacgttaat
     5041 ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag
     5101 aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga gcaaataagc
     5161 gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct ttctgcttac
     5221 aataagcaca gggataagcc catcagggag caggcagaaa acattatcca cttgtttact
     5281 ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat agacagaaag
     5341 cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc aattacgggg
     5401 ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc tgaccccaag
     5461 aagaagagga aggtgtcgcc agggatccgt cgacttgacg cgttgatatc aacaagtttg
     5521 tacaaaaaag caggctacaa agaggccagc ggttccggac gggctgacgc attggacgat
     5581 tttgatctgg atatgctggg aagtgacgcc ctcgatgatt ttgaccttga catgcttggt
     5641 tcggatgccc ttgatgactt tgacctcgac atgctcggca gtgacgccct tgatgatttc
     5701 gacctggaca tgctgattaa ctctagaagt tccggatctc cgaaaaagaa acgcaaagtt
     5761 ggtagccagt acctgcccga caccgacgac cggcaccgga tcgaggaaaa gcggaagcgg
     5821 acctacgaga cattcaagag catcatgaag aagtccccct tcagcggccc caccgaccct
     5881 agacctccac ctagaagaat cgccgtgccc agcagatcca gcgccagcgt gccaaaacct
     5941 gccccccagc cttacccctt caccagcagc ctgagcacca tcaactacga cgagttccct
     6001 accatggtgt tccccagcgg ccagatctct caggcctctg ctctggctcc agcccctcct
     6061 caggtgctgc ctcaggctcc tgctcctgca ccagctccag ccatggtgtc tgcactggct
     6121 caggcaccag cacccgtgcc tgtgctggct cctggacctc cacaggctgt ggctccacca
     6181 gcccctaaac ctacacaggc cggcgagggc acactgtctg aagctctgct gcagctgcag
     6241 ttcgacgacg aggatctggg agccctgctg ggaaacagca ccgatcctgc cgtgttcacc
     6301 gacctggcca gcgtggacaa cagcgagttc cagcagctgc tgaaccaggg catccctgtg
     6361 gcccctcaca ccaccgagcc catgctgatg gaataccccg aggccatcac ccggctcgtg
     6421 acaggcgctc agaggcctcc tgatccagct cctgcccctc tgggagcacc aggcctgcct
     6481 aatggactgc tgtctggcga cgaggacttc agctctatcg ccgatatgga tttctcagcc
     6541 ttgctgggct ctggcagcgg cagccgggat tccagggaag ggatgttttt gccgaagcct
     6601 gaggccggct ccgctattag tgacgtgttt gagggccgcg aggtgtgcca gccaaaacga
     6661 atccggccat ttcatcctcc aggaagtcca tgggccaacc gcccactccc cgccagcctc
     6721 gcaccaacac caaccggtcc agtacatgag ccagtcgggt cactgacccc ggcaccagtc
     6781 cctcagccac tggatccagc gcccgcagtg actcccgagg ccagtcacct gttggaggat
     6841 cccgatgaag agacgagcca ggctgtcaaa gcccttcggg agatggccga tactgtgatt
     6901 ccccagaagg aagaggctgc aatctgtggc caaatggacc tttcccatcc gcccccaagg
     6961 ggccatctgg atgagctgac aaccacactt gagtccatga ccgaggatct gaacctggac
     7021 tcacccctga ccccggaatt gaacgagatt ctggatacct tcctgaacga cgagtgcctc
     7081 ttgcatgcca tgcatatcag cacaggactg tccatcttcg acacatctct ctttgaattc
     7141 ggatctggtg ccacgaattt ctcgctactc aagcaggccg gtgatgtcga ggaaaaccct
     7201 ggtcctatgg tgtctagaga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     7261 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     7321 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     7381 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     7441 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     7501 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     7561 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     7621 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     7681 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     7741 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     7801 cccggtgcct gagaattcga tatcaagctt atcggtaatc aacctctgga ttacaaaatt
     7861 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     7921 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     7981 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     8041 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     8101 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     8161 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     8221 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     8281 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     8341 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     8401 atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgagacct agaaaaacat
     8461 ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca
     8521 caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact
     8581 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     8641 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     8701 ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga
     8761 tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa tgaaggagag
     8821 aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagta
     8881 ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg
     8941 gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     9001 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     9061 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     9121 atctctagca gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     9181 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     9241 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     9301 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     9361 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     9421 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     9481 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     9541 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     9601 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     9661 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     9721 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     9781 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     9841 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     9901 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     9961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
    10021 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
    10081 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
    10141 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
    10201 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt
    10261 aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg
    10321 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
    10381 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
    10441 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
    10501 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
    10561 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
    10621 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
    10681 agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg
    10741 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
    10801 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
    10861 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
    10921 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
    10981 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
    11041 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
    11101 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
    11161 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
    11221 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
    11281 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
    11341 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
    11401 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
    11461 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
    11521 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
    11581 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
    11641 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
    11701 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
    11761 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
    11821 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
    11881 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
    11941 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
    12001 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
    12061 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
    12121 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
    12181 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
    12241 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
    12301 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
    12361 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
    12421 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
    12481 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
    12541 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
    12601 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
    12661 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
    12721 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
    12781 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
    12841 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
    12901 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
    12961 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
    13021 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
    13081 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
    13141 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
    13201 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
    13261 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
    13321 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
    13381 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
    13441 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
    13501 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
    13561 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
    13621 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
    13681 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
    13741 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
    13801 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
    13861 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
    13921 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg
    13981 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
    14041 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
    14101 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
    14161 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
    14221 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
    14281 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
    14341 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
    14401 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
    14461 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
    14521 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
    14581 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
    14641 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
    14701 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
    14761 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
    14821 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
    14881 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
    14941 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
    15001 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
    15061 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
    15121 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
    15181 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
    15241 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
    15301 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
    15361 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
    15421 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
    15481 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
    15541 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
    15601 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
    15661 actaaagaat tacaaaaaca aattacaaaa attcaaaatt tt
//