PB-TRE-dCas9-VPR

LOCUS       PB-TRE-dCas9-VPR              13808 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  SP-dCas9-VPR with doxycycline-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    PB-TRE-dCas9-VPR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13808)
  AUTHORS   Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin 
            S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, 
            Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, 
            Church GM
  TITLE     Highly efficient Cas9-mediated transcriptional programming.
  JOURNAL   Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312.
  PUBMED    25730490
REFERENCE   2  (bases 1 to 13808)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..13808
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(14..469)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(151..172)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     363..382
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     596..618
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     610..627
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     611..627
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   757..819
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1268..1646
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    1276..1294
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1312..1330
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1348..1366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1384..1402
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1420..1438
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1456..1474
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1492..1510
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1555..1579
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1673..1682
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1679..5782
                     /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             5795..5815
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5840..5989
                     /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             6014..6034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6464..6820
                     /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             6839..7408
                     /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"
     primer_bind     complement(7463..7482)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7507..7555
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        7786..7997
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             8010..8278
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             8312..9055
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             9056..9109
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9131..10150
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(10207..10227)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    10261..10382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10298..10317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10352..10371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   11145..11179
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11670..11686)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11670..11686)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11683..11705)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11694..11710
                     /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(11718..11748)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11763..11784
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11901..11918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12072..12660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12152..12171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(12831..13691)
                     /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     13454..13473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13692..13796)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
       61 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
      121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
      181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
      241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
      301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
      361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
      421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
      481 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      541 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      601 tcacgacgtt gtaaaacgac ggccagtgag cgcgcctcgt tcattcacgt ttttgaaccc
      661 gtggaggacg ggcagactcg cggtgcaaat gtgttttaca gcgtgatgga gcagatgaag
      721 atgctcgaca cgctgcagaa cacgcagcta gattaaccct agaaagataa tcatattgtg
      781 acgtacgtta aagataatca tgtgtaaaat tgacgcatgt gttttatcgg tctgtatatc
      841 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
      901 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aacaaaaact
      961 caaaatttct tctataaagt aacaaaactt ttatgaggga cagccccccc ccaaagcccc
     1021 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
     1081 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
     1141 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
     1201 agacacctgg ggggatacgg ggaaaaggcc tccacggcca ctagtctttc gtcttcaaga
     1261 attcctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     1321 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     1381 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     1441 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     1501 gtgatagaga acgtataagc tttaggcgtg tacggtgggc gcctataaaa gcagagctcg
     1561 tttagtgaac cgtcagatcg cctggagcaa ttccacaaca cttttgtctt ataccaactt
     1621 tccgtaccac ttcctaccct cgtaaaggtc tagagctagc gaattcgaat ttgccaccat
     1681 ggacaagaag tactccattg ggctcgctat cggcacaaac agcgtcggct gggccgtcat
     1741 tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca
     1801 cagcataaag aagaacctca ttggcgccct cctgttcgac tccggggaga cggccgaagc
     1861 cacgcggctc aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta
     1921 cctgcaggag atctttagta atgagatggc taaggtggat gactctttct tccataggct
     1981 ggaggagtcc tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa
     2041 tatcgtggac gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa
     2101 gcttgtagac agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat
     2161 gatcaaattt cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt
     2221 cgacaaactc tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat
     2281 caacgcatcc ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg
     2341 gctcgaaaac ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct
     2401 tatcgccctg tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga
     2461 tgccaagctt caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca
     2521 gatcggcgac cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct
     2581 gctgagtgat attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat
     2641 gatcaagcgc tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca
     2701 gcaactgcct gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg
     2761 atacattgac ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga
     2821 aaaaatggac ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa
     2881 acagcgcact ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc
     2941 tatcctcagg cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga
     3001 gaaaatcctc acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag
     3061 attcgcgtgg atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt
     3121 cgtggataag ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa
     3181 tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta
     3241 taacgagctc accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc
     3301 tggagagcag aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt
     3361 gaaacagctc aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag
     3421 cggagtggag gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat
     3481 taaagacaag gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct
     3541 cacccttacg ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca
     3601 tctcttcgac gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg
     3661 gctgtcaaga aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga
     3721 ttttcttaag tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc
     3781 tctcaccttt aaggaggaca tccagaaagc acaagtttct ggccaggggg acagtcttca
     3841 cgagcacatc gctaatcttg caggtagccc agctatcaaa aagggaatac tgcagaccgt
     3901 taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat aagcccgaga atatcgttat
     3961 cgagatggcc cgagagaacc aaactaccca gaagggacag aagaacagta gggaaaggat
     4021 gaagaggatt gaagagggta taaaagaact ggggtcccaa atccttaagg aacacccagt
     4081 tgaaaacacc cagcttcaga atgagaagct ctacctgtac tacctgcaga acggcaggga
     4141 catgtacgtg gatcaggaac tggacatcaa tcggctctcc gactacgacg tggctgctat
     4201 cgtgccccag tcttttctca aagatgattc tattgataat aaagtgttga caagatccga
     4261 taaagctaga gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa
     4321 ttattggcgg cagctgctga acgccaaact gatcacacaa cggaagttcg ataatctgac
     4381 taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca aaaggcagct
     4441 tgttgagaca cgccagatca ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac
     4501 caagtacgat gaaaatgaca aactgattcg agaggtgaaa gttattactc tgaagtctaa
     4561 gctggtctca gatttcagaa aggactttca gttttataag gtgagagaga tcaacaatta
     4621 ccaccatgcg catgatgcct acctgaatgc agtggtaggc actgcactta tcaaaaaata
     4681 tcccaagctt gaatctgaat ttgtttacgg agactataaa gtgtacgatg ttaggaaaat
     4741 gatcgcaaag tctgagcagg aaataggcaa ggccaccgct aagtacttct tttacagcaa
     4801 tattatgaat tttttcaaga ccgagattac actggccaat ggagagattc ggaagcgacc
     4861 acttatcgaa acaaacggag aaacaggaga aatcgtgtgg gacaagggta gggatttcgc
     4921 gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca
     4981 gaccggaggc ttctccaagg aaagtatcct cccgaaaagg aacagcgaca agctgatcgc
     5041 acgcaaaaaa gattgggacc ccaagaaata cggcggattc gattctccta cagtcgctta
     5101 cagtgtactg gttgtggcca aagtggagaa agggaagtct aaaaaactca aaagcgtcaa
     5161 ggaactgctg ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt
     5221 tctcgaggcg aaaggatata aagaggtcaa aaaagacctc atcattaagc ttcccaagta
     5281 ctctctcttt gagcttgaaa acggccggaa acgaatgctc gctagtgcgg gcgagctgca
     5341 gaaaggtaac gagctggcac tgccctctaa atacgttaat ttcttgtatc tggccagcca
     5401 ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag aagcagctgt tcgtggaaca
     5461 acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat
     5521 cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc
     5581 catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc
     5641 tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga
     5701 ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga
     5761 cctctctcag ctcggtggag acagcagggc tgaccccaag aagaagagga aggtggaggc
     5821 cagcggttcc ggacgggctg acgcattgga cgattttgat ctggatatgc tgggaagtga
     5881 cgccctcgat gattttgacc ttgacatgct tggttcggat gcccttgatg actttgacct
     5941 cgacatgctc ggcagtgacg cccttgatga tttcgacctg gacatgctga ttaactctag
     6001 aagttccgga tctccgaaaa agaaacgcaa agttggtagc cagtacctgc ccgacaccga
     6061 cgaccggcac cggatcgagg aaaagcggaa gcggacctac gagacattca agagcatcat
     6121 gaagaagtcc cccttcagcg gccccaccga ccctagacct ccacctagaa gaatcgccgt
     6181 gcccagcaga tccagcgcca gcgtgccaaa acctgccccc cagccttacc ccttcaccag
     6241 cagcctgagc accatcaact acgacgagtt ccctaccatg gtgttcccca gcggccagat
     6301 ctctcaggcc tctgctctgg ctccagcccc tcctcaggtg ctgcctcagg ctcctgctcc
     6361 tgcaccagct ccagccatgg tgtctgcact ggctcaggca ccagcacccg tgcctgtgct
     6421 ggctcctgga cctccacagg ctgtggctcc accagcccct aaacctacac aggccggcga
     6481 gggcacactg tctgaagctc tgctgcagct gcagttcgac gacgaggatc tgggagccct
     6541 gctgggaaac agcaccgatc ctgccgtgtt caccgacctg gccagcgtgg acaacagcga
     6601 gttccagcag ctgctgaacc agggcatccc tgtggcccct cacaccaccg agcccatgct
     6661 gatggaatac cccgaggcca tcacccggct cgtgacaggc gctcagaggc ctcctgatcc
     6721 agctcctgcc cctctgggag caccaggcct gcctaatgga ctgctgtctg gcgacgagga
     6781 cttcagctct atcgccgaca tggacttctc cgcactgctg ggtagcggat cgggatctcg
     6841 ggattccagg gaagggatgt ttttgccgaa gcctgaggcc ggctccgcta ttagtgacgt
     6901 gtttgagggc cgcgaggtgt gccagccaaa acgaatccgg ccatttcatc ctccaggaag
     6961 tccatgggcc aaccgcccac tccccgccag cctcgcacca acaccaaccg gtccagtaca
     7021 tgagccagtc gggtcactga ccccggcacc agtccctcag ccactggatc cagcgcccgc
     7081 agtgactccc gaggccagtc acctgttgga ggatcccgat gaagagacga gccaggctgt
     7141 caaagccctt cgggagatgg ccgatactgt gattccccag aaggaagagg ctgcaatctg
     7201 tggccaaatg gacctttccc atccgccccc aaggggccat ctggatgagc tgacaaccac
     7261 acttgagtcc atgaccgagg atctgaacct ggactcaccc ctgaccccgg aattgaacga
     7321 gattctggat accttcctga acgacgagtg cctcttgcat gccatgcata tcagcacagg
     7381 actgtccatc ttcgacacat ctctgttttg aaagggttcg atccctaccg gttagtaatg
     7441 agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     7501 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     7561 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     7621 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     7681 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg aaatcggatc
     7741 cagctaaatc taagtggatc tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat
     7801 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaacgggt gcctagagaa
     7861 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
     7921 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
     7981 ttgccgccag aacacagctg aagcttcgag gggctcgcat ctctccttca cgcgcccgcc
     8041 gccctacctg aggccgccat ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg
     8101 gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa gctcaggtcg agaccgggcc
     8161 tttgtccggc gctcccttgg agcctaccta gactcagccg gctctccacg ctttgcctga
     8221 ccctgcttgc tcaactctac gtctttgttt cgttttctgt tctgcgccgt tacagatcta
     8281 agctgtgacc ggcgcctact ctagagccgc catgtctaga ctggacaaga gcaaagtcat
     8341 aaactctgct ctggaattac tcaatggagt cggtatcgaa ggcctgacga caaggaaact
     8401 cgctcaaaag ctgggagttg agcagcctac cctgtactgg cacgtgaaga acaagcgggc
     8461 cctgctcgat gccctgccaa tcgagatgct ggacaggcat catacccact cctgccccct
     8521 ggaaggcgag tcatggcaag actttctgcg gaacaacgcc aagtcatacc gctgtgctct
     8581 cctctcacat cgcgacgggg ctaaagtgca tctcggcacc cgcccaacag agaaacagta
     8641 cgaaaccctg gaaaatcagc tcgcgttcct gtgtcagcaa ggcttctccc tggagaacgc
     8701 actgtacgct ctgtccgccg tgggccactt tacactgggc tgcgtattgg aggaacagga
     8761 gcatcaagta gcaaaagagg aaagagagac acctaccacc gattctatgc ccccacttct
     8821 gaaacaagca attgagctgt tcgaccggca gggagccgaa cctgccttcc ttttcggcct
     8881 ggaactaatc atatgtggcc tggagaaaca gctaaagtgc gaaagcggcg ggccgaccga
     8941 cgcccttgac gattttgact tagacatgct cccagccgat gcccttgacg actttgacct
     9001 tgatatgctg cctgctgacg ctcttgacga ttttgacctt gacatgctcc ccggggaggg
     9061 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg aattcatgga
     9121 tagatccgga aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
     9181 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
     9241 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
     9301 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
     9361 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
     9421 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
     9481 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     9541 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     9601 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     9661 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     9721 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     9781 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     9841 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     9901 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     9961 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
    10021 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
    10081 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
    10141 aaaggaataa gccaaggaac ctctggatta caaaatttgt gaaagattga ctggtattct
    10201 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcagtt
    10261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
    10321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
    10381 tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct catctggtct
    10441 cccttccggg ggacaagaca tccctgttta atatttaaac agcagtgttc ccaaactggg
    10501 ttcttatatc ccttgctctg gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga
    10561 agtccctaaa gaaacagtgg cagccaggtt tagccccgga attgactgga ttcctttttt
    10621 agggcccatt ggtatggctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
    10681 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
    10741 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
    10801 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
    10861 ggctgtccct gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga
    10921 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
    10981 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
    11041 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
    11101 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
    11161 gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt
    11221 ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc
    11281 cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat
    11341 tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc
    11401 ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg
    11461 gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag
    11521 ccggaactgc cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt
    11581 cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt
    11641 acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
    11701 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
    11761 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
    11821 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
    11881 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
    11941 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
    12001 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
    12061 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
    12121 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
    12181 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
    12241 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
    12301 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
    12361 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
    12421 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
    12481 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
    12541 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
    12601 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
    12661 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
    12721 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
    12781 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
    12841 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
    12901 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
    12961 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
    13021 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
    13081 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
    13141 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
    13201 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
    13261 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
    13321 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
    13381 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
    13441 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
    13501 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
    13561 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
    13621 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
    13681 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
    13741 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
    13801 tttccccg
//