hCas9-VPR

LOCUS       hCas9-VPR               11319 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  nuclease competent SP-Cas9 fused to VPR.
ACCESSION   .
VERSION     .
KEYWORDS    hCas9-VPR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11319)
  AUTHORS   Kiani S, Chavez A, Tuttle M, Hall RN, Chari R, Ter-Ovanesyan D, Qian
            J, Pruitt BW, Beal J, Vora S, Buchthal J, Kowal EJ, Ebrahimkhani MR,
            Collins JJ, Weiss R, Church G
  TITLE     Cas9 gRNA engineering for genome editing, activation and repression.
  JOURNAL   Nat Methods. 2015 Sep 7. doi: 10.1038/nmeth.3580.
  PUBMED    26344044
REFERENCE   2  (bases 1 to 11319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..11319
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      742..751
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             748..4851
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4921..4945
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4975..5124
                     /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             5149..5169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5599..5955
                     /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             5974..6543
                     /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"
     protein_bind    complement(6548..6572)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(6622..6639)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6705..6724)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6749..6797
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6999..7427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7086..7105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7296..7317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7436..7456)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7441..7770
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7621..7756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7683..7702
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             7837..8631
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7891..7910)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8501..8520
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8807..8928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8844..8863)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8898..8917
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8977..8993)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8977..8993)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8990..9012)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9001..9017
                     /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(9025..9055)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9070..9091
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9208..9225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9379..9967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9459..9478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10138..10998)
                     /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     10761..10780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10999..11103)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(11178..11197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgatatc
      781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
      961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1261 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3241 cggctctccg actacgacgt ggatcatatc gtgccccagt cttttctcaa agatgattct
     3301 attgataata aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc
     3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4861 gaccccaaga agaagaggaa ggtgtcgcca gggatccgtc gacttgacgc gttaatatca
     4921 acaagtttgt acaaaaaagc aggctacaaa gaggccagcg gttccggacg ggctgacgca
     4981 ttggacgatt ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac
     5041 atgcttggtt cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt
     5101 gatgatttcg acctggacat gctgattaac tctagaagtt ccggatctcc gaaaaagaaa
     5161 cgcaaagttg gtagccagta cctgcccgac accgacgacc ggcaccggat cgaggaaaag
     5221 cggaagcgga cctacgagac attcaagagc atcatgaaga agtccccctt cagcggcccc
     5281 accgacccta gacctccacc tagaagaatc gccgtgccca gcagatccag cgccagcgtg
     5341 ccaaaacctg ccccccagcc ttaccccttc accagcagcc tgagcaccat caactacgac
     5401 gagttcccta ccatggtgtt ccccagcggc cagatctctc aggcctctgc tctggctcca
     5461 gcccctcctc aggtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct
     5521 gcactggctc aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg
     5581 gctccaccag cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg
     5641 cagctgcagt tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc
     5701 gtgttcaccg acctggccag cgtggacaac agcgagttcc agcagctgct gaaccagggc
     5761 atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga ggccatcacc
     5821 cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct gggagcacca
     5881 ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc cgatatggat
     5941 ttctcagcct tgctgggctc tggcagcggc agccgggatt ccagggaagg gatgtttttg
     6001 ccgaagcctg aggccggctc cgctattagt gacgtgtttg agggccgcga ggtgtgccag
     6061 ccaaaacgaa tccggccatt tcatcctcca ggaagtccat gggccaaccg cccactcccc
     6121 gccagcctcg caccaacacc aaccggtcca gtacatgagc cagtcgggtc actgaccccg
     6181 gcaccagtcc ctcagccact ggatccagcg cccgcagtga ctcccgaggc cagtcacctg
     6241 ttggaggatc ccgatgaaga gacgagccag gctgtcaaag cccttcggga gatggccgat
     6301 actgtgattc cccagaagga agaggctgca atctgtggcc aaatggacct ttcccatccg
     6361 cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg
     6421 aacctggact cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac
     6481 gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg
     6541 ttttgaaacc cagctttctt gtacaaagtg gttgatatca tctagagcgg ccgcaggtac
     6601 ctgaataact aaggccgctt ccctttagtg agggttaatg cttcgagcag acaaagggtt
     6661 cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag
     6721 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
     6781 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     6841 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     6901 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     6961 agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     7021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7141 agctctaaat cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7201 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     7321 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc
     7381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     7441 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     7501 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     7561 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     7621 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     7681 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     7741 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     7801 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     7861 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     7921 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     7981 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     8041 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     8101 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     8161 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     8221 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     8281 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     8341 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     8401 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     8461 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     8521 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     8581 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     8641 tggggttcgc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     8701 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     8761 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     8821 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     8881 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     8941 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     9001 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     9061 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     9121 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     9181 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     9241 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     9301 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     9361 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     9421 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     9481 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     9541 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     9601 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     9661 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     9721 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     9781 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     9841 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     9901 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     9961 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
    10021 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
    10081 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
    10141 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
    10201 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
    10261 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
    10321 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
    10381 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
    10441 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
    10501 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
    10561 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
    10621 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
    10681 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
    10741 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
    10801 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
    10861 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
    10921 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
    10981 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
    11041 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
    11101 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
    11161 ctatggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
    11221 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
    11281 caaggcttga ccgacaattg catgaagaat ctgcttagg
//