pHAGE TRE dCas9-KRAB

LOCUS       pHAGE TRE dCas9-KRAB               14645 bp ds-DNA     circular SYN 10-JAN-2018
DEFINITION  Tet-regulatable dCas9-KRAB lentiviral expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    pHAGE TRE dCas9-KRAB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14645)
  AUTHORS   Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R
  TITLE     Cas9 effector-mediated regulation of transcription and 
            differentiation in human pluripotent stem cells.
  JOURNAL   Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341.
  PUBMED    24346702
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|biofeng.com|
COMMENT     VNTNAME|pHAGE TRE dCas9-KRAB|
FEATURES             Location/Qualifiers
     source          1..14645
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             67..93
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        179..1390
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1227..1246
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             1449..2195
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    2237..2805
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2399..2416)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2626..2645
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2821..3615
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2875..2894)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3485..3504
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3792..4380
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3845..3865)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4263..4274)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4455..4688
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4807..4825)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4893..4997
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4998..5858
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5216..5235)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6029..6617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6518..6537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6771..6788
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6880..6899
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    7028..7049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7064..7094
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7102..7118
                     /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)."
     primer_bind     7107..7129
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7126..7142
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7126..7142
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7192..7211
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             7403..8036
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    8083..8208
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    8705..8938
                     /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    9460..9577
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    9632..9902
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        9935..9973
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9970..9994
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    10103..10127
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      10131..10140
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             10137..14240
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             14253..14273
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             14274..14363
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             14400..14594
                     /codon_start=1
                     /gene="ZNF10"
                     /gene_synonym="KOX1"
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     protein_bind    complement(14635..14)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 ttgtacaaag tggttgatat ccagcacagt ggcggccgct cgagtctaga gggcccgcgg
       61 ttcgaatacc catacgacgt cccagactac gcttagtaat gattaattaa actagaaatt
      121 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gtaacccacc caagatctgg
      181 cctccgcgcc gggttttggc gcctcccgcg ggcgcccccc tcctcacggc gagcgctgcc
      241 acgtcagacg aagggcgcag cgagcgtcct gatccttccg cccggacgct caggacagcg
      301 gcccgctgct cataagactc ggccttagaa ccccagtatc agcagaagga cattttagga
      361 cgggacttgg gtgactctag ggcactggtt ttctttccag agagcggaac aggcgaggaa
      421 aagtagtccc ttctcggcga ttctgcggag ggatctccgt ggggcggtga acgccgatga
      481 ttatataagg acgcgccggg tgtggcacag ctagttccgt cgcagccggg atttgggtcg
      541 cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga gtagcgggct gctgggctgg
      601 ccggggcttt cgtggccgcc gggccgctcg gtgggacgga agcgtgtgga gagaccgcca
      661 agggctgtag tctgggtccg cgagcaaggt tgccctgaac tgggggttgg ggggagcgca
      721 gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc ttgtgaggcg ggctgtgagg
      781 tcgttgaaac aaggtggggg gcatggtggg cggcaagaac ccaaggtctt gaggccttcg
      841 ctaatgcggg aaagctctta ttcgggtgag atgggctggg gcaccatctg gggaccctga
      901 cgtgaagttt gtcactgact ggagaactcg gtttgtcgtc tgttgcgggg gcggcagtta
      961 tggcggtgcc gttgggcagt gcacccgtac ctttgggagc gcgcgccctc gtcgtgtcgt
     1021 gacgtcaccc gttctgttgg cttataatgc agggtggggc cacctgccgg taggtgtgcg
     1081 gtaggctttt ctccgtcgca ggacgcaggg ttcgggccta gggtaggctc tcctgaatcg
     1141 acaggcgccg gacctctggt gaggggaggg ataagtgagg cgtcagtttc tttggtcggt
     1201 tttatgtacc tatcttctta agtagctgaa gctccggttt tgaactatgc gctcggggtt
     1261 ggcgagtgtg ttttgtgaag ttttttaggc accttttgaa atgtaatcat ttgggtcaat
     1321 atgtaatttt cagtgttaga ctagtaaatt gtccgctaaa ttctggccgt ttttggcttt
     1381 tttgttagac gaagcttggt accgagctcg gatctccacc ccgtaccggt cctgcagtcg
     1441 aattcaccat gtctagactg gacaagagca aagtcataaa cggagctctg gaattactca
     1501 atggtgtcgg tatcgaaggc ctgacgacaa ggaaactcgc tcaaaagctg ggagttgagc
     1561 agcctaccct gtactggcac gtgaagaaca agcgggccct gctcgatgcc ctgccaatcg
     1621 agatgctgga caggcatcat acccacttct gccccctgga aggcgagtca tggcaagact
     1681 ttctgcggaa caacgccaag tcataccgct gtgctctcct ctcacatcgc gacggggcta
     1741 aagtgcatct cggcacccgc ccaacagaga aacagtacga aaccctggaa aatcagctcg
     1801 cgttcctgtg tcagcaaggc ttctccctgg agaacgcact gtacgctctg tccgccgtgg
     1861 gccactttac actgggctgc gtattggagg aacaggagca tcaagtagca aaagaggaaa
     1921 gagagacacc taccaccgat tctatgcccc cacttctgag acaagcaatt gagctgttcg
     1981 accggcaggg agccgaacct gccttccttt tcggcctgga actaatcata tgtggcctgg
     2041 agaaacagct aaagtgcgaa agcggcgggc cgaccgacgc ccttgacgat tttgacttag
     2101 acatgctccc agccgatgcc cttgacgact ttgaccttga tatgctgcct gctgacgctc
     2161 ttgacgattt tgaccttgac atgctccccg ggtaactaag taaggatccg cggccgcact
     2221 agaggaattc cgcccctccc cccccccccc cctaacgtta ctggccgaag ccgcttggaa
     2281 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat
     2341 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct
     2401 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct
     2461 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc
     2521 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa
     2581 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc
     2641 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg
     2701 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc
     2761 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagctta ccggtccacc
     2821 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2881 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     2941 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3001 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3061 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3121 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3181 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3241 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3301 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     3361 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3421 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3481 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3541 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3601 gacgagttct tctgatgtac aagtaaagcg gccgcgactc tagatcataa tcagccatac
     3661 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3721 acataaaatg aatgcaattg ttgttgttta gtccctccca attcgatatc aagcttatcg
     3781 cgatagatcc taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3841 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3901 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3961 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4021 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     4081 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     4141 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     4201 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     4261 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     4321 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     4381 ctgagatcct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     4441 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     4501 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4561 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4621 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4681 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4741 aatatcagag agtgagaggc ccgggttaat taaggaaagg gctagatcat tcttgaagac
     4801 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4861 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4921 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4981 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5041 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5101 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5161 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5221 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     5281 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5341 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5401 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5461 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5521 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5581 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5641 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5701 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5761 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5821 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5881 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5941 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6001 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6061 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6121 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6181 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6241 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6301 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6361 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6421 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6481 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6541 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6601 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6661 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6721 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6781 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6841 ttcattaatg cagcaagctc atggctgact aatttttttt atttatgcag aggccgaggc
     6901 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     6961 ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7021 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7081 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7141 acatgattac gaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     7201 ccaaactcat caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa
     7261 atcatcccaa acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt
     7321 tactctaaac ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa
     7381 tatgtactac aaacttagta gttggaaggg ctaattcact cccaaagaag acaagatatc
     7441 cttgatctgt ggatctacca cacacaaggc tacttccctg attagcagaa ctacacacca
     7501 gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag
     7561 ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca ccctgtgagc
     7621 ctgcatggga tggatgaccc ggagagagaa gtgttagagt ggaggtttga cagccgccta
     7681 gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg ctgatatcga
     7741 gcttgctaca agggactttc cgctggggac tttccaggga ggcgtggcct gggcgggact
     7801 ggggagtggc gagccctcag atcctgcata taagcagctg ctttttgcct gtactgggtc
     7861 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     7921 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     7981 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     8041 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     8101 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     8161 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     8221 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     8281 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     8341 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     8401 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     8461 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     8521 aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag
     8581 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     8641 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     8701 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     8761 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     8821 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     8881 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     8941 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     9001 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     9061 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     9121 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     9181 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     9241 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     9301 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     9361 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
     9421 ggtatcgccg aattcacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     9481 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     9541 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     9601 agatccagtt tggactagga tcctttacca ctccctatca gtgatagaga aaagtgaaag
     9661 tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct
     9721 atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag
     9781 tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca
     9841 ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga
     9901 gaaaagtgaa agtcgagctc ggtacccggg tcgaggtagg cgtgtacggt gggaggccta
     9961 tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc cacgctgttt
    10021 tgacctccat agaagacacc gggaccgatc cagcctccgc ggccccgaac tagtccagtg
    10081 tggtggaatt ctgcagatat caacaagttt gtacaaaaaa gcaggctggc gccaccatgg
    10141 acaagaagta ctccattggg ctcgctatcg gtaccaacag cgtcggctgg gccgtcatta
    10201 cggacgagta caaggtgccg agcaaaaaat tcaaagttct gggcaatacc gatcgccaca
    10261 gcataaagaa gaacctcatt ggagccctcc tgttcgactc cggggagacg gccgaagcca
    10321 cgcggctcaa aagaacagca cggcgcagat atacccgcag aaagaatcgg atctgctacc
    10381 tgcaggagat ctttagtaat gagatggcta aggtggatga ctctttcttc cataggctgg
    10441 aggagtcctt tttggtggag gaggataaaa agcacgagcg ccacccaatc tttggcaata
    10501 tcgtggacga ggtggcgtac catgaaaagt acccaaccat atatcatctg aggaagaagc
    10561 tggtagacag tactgataag gctgacttgc ggttgatcta tctcgcgctg gcgcacatga
    10621 tcaaatttcg gggacacttc ctcatcgagg gggacctgaa cccagacaac agcgatgtcg
    10681 acaaactctt tatccaactg gttcagactt acaatcagct tttcgaggag aacccgatca
    10741 acgcatccgg cgttgacgcc aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc
    10801 tcgaaaacct catcgcacag ctccctgggg agaagaagaa cggcctgttt ggtaatctta
    10861 tcgccctgtc actcgggctg acccccaact ttaaatctaa cttcgacctg gccgaagatg
    10921 ccaagctgca actgagcaaa gacacctacg atgatgatct cgacaatctg ctggcccaga
    10981 tcggcgacca gtacgcagac ctttttttgg cggcaaagaa cctgtcagac gccattctgc
    11041 tgagtgatat tctgcgagtg aacacggaga tcaccaaagc tccgctgagc gctagtatga
    11101 tcaagcgcta tgatgagcac caccaagact tgactttgct gaaggccctt gtcagacagc
    11161 aactgcctga gaagtacaag gaaattttct tcgatcagtc taaaaatggc tacgccggat
    11221 acattgacgg cggagcaagc caggaggaat tttacaaatt tattaagccc atcttggaaa
    11281 aaatggacgg caccgaggag ctgctggtaa agctgaacag agaagatctg ttgcgcaaac
    11341 agcgcacttt cgacaatgga agcatccccc accagattca cctgggcgaa ctgcacgcta
    11401 tcctcaggcg gcaagaggat ttctacccct ttttgaaaga taacagggaa aagattgaga
    11461 aaatcctcac atttcggata ccctactatg taggccccct cgctcgggga aattccagat
    11521 tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc ctggaacttc gaggaagtcg
    11581 tggataaggg ggcctctgcc cagtccttca tcgaaaggat gactaacttt gataaaaatc
    11641 tgcctaacga aaaggtgctt cctaaacact ctctgctgta cgagtacttc acagtttata
    11701 acgagctcac caaggtcaaa tacgtcacag aagggatgag aaagccagca ttcctgtctg
    11761 gagagcagaa gaaagctatc gtggacctcc tcttcaagac gaaccggaaa gttaccgtga
    11821 aacagctcaa agaagactat ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg
    11881 gagtggagga tcgcttcaac gcatccctgg gaacgtatca cgatctcctg aaaatcatta
    11941 aagacaagga cttcctggac aatgaggaga acgaggacat tcttgaggac attgtcctca
    12001 cccttacgtt gtttgaagat agggagatga ttgaagaacg cttgaaaact tacgctcatc
    12061 tcttcgacga caaagtcatg aaacagctca agagacgccg atatacagga tgggggcggc
    12121 tgtcaagaaa actgatcaat ggcatccgag acaagcagag tggaaagaca atcctggatt
    12181 ttcttaagtc cgatggattt gccaaccgga acttcatgca gttgatccat gatgactctc
    12241 tcacctttaa ggaggacatc cagaaagcac aagtttctgg ccagggggac agtcttcacg
    12301 agcacatcgc taatcttgca ggtagcccag ctatcaaaaa gggaatactg cagaccgtta
    12361 aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa gcccgagaat atcgttatcg
    12421 agatggcccg agagaaccaa actacccaga agggacagaa gaacagtagg gaaaggatga
    12481 agaggattga agagggtata aaagaactgg ggtcccaaat ccttaaggaa cacccagttg
    12541 aaaacaccca gcttcagaat gagaagctct acctgtacta cctgcagaac ggcagggaca
    12601 tgtacgtgga tcaggaactg gacatcaacc ggttgtccga ctacgacgtg gatgctatcg
    12661 tgccccaaag ctttctcaaa gatgattcta ttgataataa agtgttgaca agatccgata
    12721 aaaatagagg gaagagtgat aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt
    12781 attggcggca gctgctgaac gccaaactga tcacacaacg gaagttcgat aatctgacta
    12841 aggctgaacg aggtggcctg tctgagttgg ataaagccgg cttcatcaaa aggcagcttg
    12901 ttgagacacg ccagatcacc aagcacgtgg cccaaattct cgattcacgc atgaacacca
    12961 agtacgatga aaatgacaaa ctgattcgag aggtgaaagt tattactctg aagtctaagc
    13021 tggtctcaga tttcagaaag gactttcagt tttataaggt gagagagatc aacaattacc
    13081 accatgcgca tgatgcctac ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc
    13141 ccaagctgga atctgaattt gtttacggag actataaagt gtacgatgtt aggaaaatga
    13201 tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa gtacttcttt tacagcaata
    13261 ttatgaattt tttcaagacc gagattacac tggccaatgg agagattcgg aagcgaccac
    13321 ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga caagggtagg gatttcgcga
    13381 cagtccgcaa ggtcctgtcc atgccgcagg tgaacatcgt taaaaagacc gaagtacaga
    13441 ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac
    13501 gcaaaaaaga ttgggacccc aagaaatacg gcggattcga ttctcctaca gtcgcttaca
    13561 gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg
    13621 aactgctggg catcacaatc atggagcgat ccagcttcga gaaaaacccc atcgactttc
    13681 tcgaagcgaa aggatataaa gaggtcaaaa aagacctcat cattaagctg cccaagtact
    13741 ctctctttga gcttgaaaac ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga
    13801 aaggtaacga gctggcactg ccctctaaat acgttaattt cttgtatctg gccagccact
    13861 atgaaaagct caaagggtct cccgaagata atgagcagaa gcagctgttc gtggaacaac
    13921 acaaacacta ccttgatgag atcatcgagc aaataagcga gttctccaaa agagtgatcc
    13981 tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg gataagccca
    14041 tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg ggcgcgcctg
    14101 cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct acaaaggagg
    14161 tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca agaatcgacc
    14221 tctctcagct cggtggagac ggcaccggcg ggcccaagaa gaagaggaag gtatacccat
    14281 acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca ggatcgtatc
    14341 cttatgacgt tccagattac gctggatcca tggacgcgaa atcacttacg gcatggtcga
    14401 gaacactggt tacgttcaag gacgtgtttg tggactttac acgtgaggag tggaaattgc
    14461 tggatactgc gcaacaaatt gtgtatcgaa atgtcatgct tgagaattac aagaacctcg
    14521 tcagtctcgg ataccagttg acgaaaccgg atgtgatcct taggctcgaa aagggggaag
    14581 aaccttggct ggtataattc gaatccggat aagcggccgc ctcgagatat ctagacccag
    14641 ctttc
//