LOCUS       sequence-204359-       10123 bp    DNA     circular SYN 15-APR-2025
DEFINITION  transient expression of dCas9-dJMJD2A fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10123)
  AUTHORS   Stephan Beck
  TITLE     Beck lab Cas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10123)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: p-dCas9-JMJD2A-S288G-T289G-Hygro
FEATURES             Location/Qualifiers
     source          1..10123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(57..556)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(105..124)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     498..521
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     enhancer        561..940
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        941..1144
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1095..1115
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1186..1205
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1186..1204
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1212..1221
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1218..4619,4620..4622,4623..5219,5220,5221..5223,
                     5224..5225,5226..5228,5229..5321)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /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             join(1218..1244,1245..1247,1248..3734,3735..3737,
                     3738..5321)
                     /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             5328..5348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5355..5420
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(6507..6524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6513..6737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6808..6824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6808..6824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6821..6843)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6832..6848
                     /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(join(6856..6862,6863..6880,6881..6886))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6901..6922
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7039..7056)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7210..7798)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7290..7309)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7969..8760,8761..8829))
                     /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     8592..8611
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8830..8934)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8985..9106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8996..9015)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     9050..9069
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(9137..10123,1..39))
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(join(9137..10123,1..39))
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
ORIGIN
        1 cttctcgaca gacgtcgcgg tgagttcagg ctttttcatg gtggcgggat gcaggtcgaa
       61 aggcccggag atgaggaaga ggagaacagc gcggcagacg tgcgcttttg aagcgtgcag
      121 aatgccgggc ctccggagga ccttcgggcg cccgccccgc cccnnnnnnn ncccctgagc
      181 ccgcccccgg acccacccct tcccagcctc tgagcccaga aagcgaagga gcaaagctgc
      241 tattggccgc tgccccaaag gcctacccgc ttccattgct cagcggtgct gtccatctgc
      301 acgagactag tgagacgtgc tacttccatt tgtcacgtcc tgcacgacgc gagctgcggg
      361 gcggggggga acttcctgac taggggagga gtagaaggtg gcgcgaaggg gccaccaaag
      421 aacggagccg gttggcgcct accggtggat gtggaatgtg tgcgaggcca gaggccactt
      481 gtgtagcgcc aagtgcccag cggggctgct aaagcgcatg ctccagactg ccttgggaaa
      541 agcgcctccc ctacccggtt gacattgatt attgactagt tattaatagt aatcaattac
      601 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      661 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      721 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      781 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      841 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      901 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      961 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     1021 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     1081 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     1141 agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta
     1201 tagggagagc cgccaccatg gataaaaagt attctattgg tttagccatc ggcactaatt
     1261 ccgttggatg ggctgtcata accgatgaat acaaagtacc ttcaaagaaa tttaaggtgt
     1321 tggggaacac agaccgtcat tcgattaaaa agaatcttat cggtgccctc ctattcgata
     1381 gtggcgaaac ggcagaggcg actcgcctga aacgaaccgc tcggagaagg tatacacgtc
     1441 gcaagaaccg aatatgttac ttacaagaaa tttttagcaa tgagatggcc aaagttgacg
     1501 attctttctt tcaccgtttg gaagagtcct tccttgtcga agaggacaag aaacatgaac
     1561 ggcaccccat ctttggaaac atagtagatg aggtggcata tcatgaaaag tacccaacga
     1621 tttatcacct cagaaaaaag ctagttgact caactgataa agcggacctg aggttaatct
     1681 acttggctct tgcccatatg ataaagttcc gtgggcactt tctcattgag ggtgatctaa
     1741 atccggacaa ctcggatgtc gacaaactgt tcatccagtt agtacaaacc tataatcagt
     1801 tgtttgaaga gaaccctata aatgcaagtg gcgtggatgc gaaggctatt cttagcgccc
     1861 gcctctctaa atcccgacgg ctagaaaacc tgatcgcaca attacccgga gagaagaaaa
     1921 atgggttgtt cggtaacctt atagcgctct cactaggcct gacaccaaat tttaagtcga
     1981 acttcgactt agctgaagat gccaaattgc agcttagtaa ggacacgtac gatgacgatc
     2041 tcgacaatct actggcacaa attggagatc agtatgcgga cttatttttg gctgccaaaa
     2101 accttagcga tgcaatcctc ctatctgaca tactgagagt taatactgag attaccaagg
     2161 cgccgttatc cgcttcaatg atcaaaaggt acgatgaaca tcaccaagac ttgacacttc
     2221 tcaaggccct agtccgtcag caactgcctg agaaatataa ggaaatattc tttgatcagt
     2281 cgaaaaacgg gtacgcaggt tatattgacg gcggagcgag tcaagaggaa ttctacaagt
     2341 ttatcaaacc catattagag aagatggatg ggacggaaga gttgcttgta aaactcaatc
     2401 gcgaagatct actgcgaaag cagcggactt tcgacaacgg tagcattcca catcaaatcc
     2461 acttaggcga attgcatgct atacttagaa ggcaggagga tttttatccg ttcctcaaag
     2521 acaatcgtga aaagattgag aaaatcctaa cctttcgcat accttactat gtgggacccc
     2581 tggcccgagg gaactctcgg ttcgcatgga tgacaagaaa gtccgaagaa acgattactc
     2641 catggaattt tgaggaagtt gtcgataaag gtgcgtcagc tcaatcgttc atcgagagga
     2701 tgaccaactt tgacaagaat ttaccgaacg aaaaagtatt gcctaagcac agtttacttt
     2761 acgagtattt cacagtgtac aatgaactca cgaaagttaa gtatgtcact gagggcatgc
     2821 gtaaacccgc ctttctaagc ggagaacaga agaaagcaat agtagatctg ttattcaaga
     2881 ccaaccgcaa agtgacagtt aagcaattga aagaggacta ctttaagaaa attgaatgct
     2941 tcgattctgt cgagatctcc ggggtagaag atcgatttaa tgcgtcactt ggtacgtatc
     3001 atgacctcct aaagataatt aaagataagg acttcctgga taacgaagag aatgaagata
     3061 tcttagaaga tatagtgttg actcttaccc tctttgaaga tcgggaaatg attgaggaaa
     3121 gactaaaaac atacgctcac ctgttcgacg ataaggttat gaaacagtta aagaggcgtc
     3181 gctatacggg ctggggacga ttgtcgcgga aacttatcaa cgggataaga gacaagcaaa
     3241 gtggtaaaac tattctcgat tttctaaaga gcgacggctt cgccaatagg aactttatgc
     3301 agctgatcca tgatgactct ttaaccttca aagaggatat acaaaaggca caggtttccg
     3361 gacaagggga ctcattgcac gaacatattg cgaatcttgc tggttcgcca gccatcaaaa
     3421 agggcatact ccagacagtc aaagtagtgg atgagctagt taaggtcatg ggacgtcaca
     3481 aaccggaaaa cattgtaatc gagatggcac gcgaaaatca aacgactcag aaggggcaaa
     3541 aaaacagtcg agagcggatg aagagaatag aagagggtat taaagaactg ggcagccaga
     3601 tcttaaagga gcatcctgtg gaaaataccc aattgcagaa cgagaaactt tacctctatt
     3661 acctacaaaa tggaagggac atgtatgttg atcaggaact ggacataaac cgtttatctg
     3721 attacgacgt cgatgccatt gtaccccaat cctttttgaa ggacgattca atcgacaata
     3781 aagtgcttac acgctcggat aagaaccgag ggaaaagtga caatgttcca agcgaggaag
     3841 tcgtaaagaa aatgaagaac tattggcggc agctcctaaa tgcgaaactg ataacgcaaa
     3901 gaaagttcga taacttaact aaagctgaga ggggtggctt gtctgaactt gacaaggccg
     3961 gatttattaa acgtcagctc gtggaaaccc gccaaatcac aaagcatgtt gcacagatac
     4021 tagattcccg aatgaatacg aaatacgacg agaacgataa gctgattcgg gaagtcaaag
     4081 taatcacttt aaagtcaaaa ttggtgtcgg acttcagaaa ggattttcaa ttctataaag
     4141 ttagggagat aaataactac caccatgcgc acgacgctta tcttaatgcc gtcgtaggga
     4201 ccgcactcat taagaaatac ccgaagctag aaagtgagtt tgtgtatggt gattacaaag
     4261 tttatgacgt ccgtaagatg atcgcgaaaa gcgaacagga gataggcaag gctacagcca
     4321 aatacttctt ttattctaac attatgaatt tctttaagac ggaaatcact ctggcaaacg
     4381 gagagatacg caaacgacct ttaattgaaa ccaatgggga gacaggtgaa atcgtatggg
     4441 ataagggccg ggacttcgcg acggtgagaa aagttttgtc catgccccaa gtcaacatag
     4501 taaagaaaac tgaggtgcag accggagggt tttcaaagga atcgattctt ccaaaaagga
     4561 atagtgataa gctcatcgct cgtaaaaagg actgggaccc gaaaaagtac ggtggcttcg
     4621 atagccctac agttgcctat tctgtcctag tagtggcaaa agttgagaag ggaaaatcca
     4681 agaaactgaa gtcagtcaaa gaattattgg ggataacgat tatggagcgc tcgtcttttg
     4741 aaaagaaccc catcgacttc cttgaggcga aaggttacaa ggaagtaaaa aaggatctca
     4801 taattaaact accaaagtat agtctgtttg agttagaaaa tggccgaaaa cggatgttgg
     4861 ctagcgccgg agagcttcaa aaggggaacg aactcgcact accgtctaaa tacgtgaatt
     4921 tcctgtattt agcgtcccat tacgagaagt tgaaaggttc acctgaagat aacgaacaga
     4981 agcaactttt tgttgagcag cacaaacatt atctcgacga aatcatagag caaatttcgg
     5041 aattcagtaa gagagtcatc ctagctgatg ccaatctgga caaagtatta agcgcataca
     5101 acaagcacag ggataaaccc atacgtgagc aggcggaaaa tattatccat ttgtttactc
     5161 ttaccaacct cggcgctcca gccgcattca agtattttga cacaacgata gatcgcaaac
     5221 gatacacttc taccaaggag gtgctagacg cgacactgat tcaccaatcc atcacgggat
     5281 tatatgaaac tcggatagat ttgtcacagc ttgggggtga cggatccccc aagaagaaga
     5341 ggaaagtctc gagcgactac aaagaccatg acggtgatta taaagatcat gacatcgatt
     5401 acaaggatga cgatgacaag gctgcaggag gcggaggtag cgcttctgag tctgaaactc
     5461 tgaatcccag tgctaggata atgacctttt atccaactat ggaagagttc cgaaacttca
     5521 gtagatacat tgcctacatt gaatcccaag gagctcatcg ggcagggcta gccaaggttg
     5581 ttcctccaaa agagtggaag ccacgagcat cctatgatga cattgatgat ttggtcattc
     5641 ctgcccccat tcaacagctg gtgacggggc agtctggcct ctttactcag tacaacatac
     5701 agaagaaagc catgactgtt cgagagttcc gcaagatagc caatagcgat aagtactgta
     5761 ccccacgcta tagtgagttt gaagagctcg agcggaaata ctggaaaaat cttacattca
     5821 atcctccaat ctatggtgca gatgtgaatg gtaccctcta tgaaaagcat gttgatgagt
     5881 ggaatattgg ccggctgaga acaatcctgg acttggtgga aaaggagagt gggatcacca
     5941 ttgagggtgt gaacacccca tacctgtact ttggcatgtg gaagacatcc tttgcttggc
     6001 acactgaaga catggacctc tacagcatca actacctgca ctttggagaa ccaaagtcct
     6061 ggtactctgt tccacctgag catggaaagc ggttggaacg cctcgccaaa ggctttttcc
     6121 caggaagtgc tcaaagctgt gaggcatttc tccgccacaa gatgaccctg atttccccgt
     6181 taatgctgaa gaaatatgga attccctttg acaaggtgac tcaagaggct ggagagttta
     6241 tgatcacttt cccttatggt taccatgccg gctttaacca tggttttaac tgtgcggagg
     6301 gtggcaattt tgctacccgt cggtggattg agtacggcaa gcaagctgtg ctgtgctcct
     6361 gtagaaagga catggtgaag atctccatgg atgtgtttgt gagaaagttc cagccagaaa
     6421 ggtacaaact ttggaaagct gggaaggaca acacagttat tgaccatact ctgcccacgc
     6481 cagaagcatg aaaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     6541 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     6601 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     6661 tggggtgggg taggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     6721 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga taccgtcgac
     6781 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     6841 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct agggtgccta
     6901 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6961 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     7021 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     7081 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     7141 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     7201 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     7261 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     7321 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     7381 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     7441 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     7501 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     7561 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     7621 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     7681 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     7741 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     7801 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     7861 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     7921 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     7981 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     8041 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     8101 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     8161 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     8221 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     8281 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     8341 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     8401 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     8461 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     8521 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8581 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8641 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8701 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8761 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8821 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     8881 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     8941 tccccgaaaa gtgccacctg acgtcgacgg gtatacagac atgataagat acattgatga
     9001 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     9061 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
     9121 actctcggca tctactctat tcctttgccc tcggacgagt gctggggcgt cggtttccac
     9181 tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt
     9241 gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc
     9301 aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc
     9361 ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
     9421 agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct
     9481 cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat
     9541 tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc
     9601 agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt
     9661 cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac
     9721 gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc
     9781 taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca
     9841 gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg
     9901 tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg
     9961 caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca
    10021 ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga
    10081 gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaa
//
