LOCUS       PJY109_AAVS1_PJY       15566 bp DNA     circular SYN 15-JUL-2024
DEFINITION  PEmax_AAVS1_knockin_HDR_donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15566)
  AUTHORS   Yan J, Oyler-Castrillo P, Ravisankar P, Ward CC, Levesque S, Jing Y,
            Simpson D, Zhao A, Li H, Yan W, Goudy L, Schmidt R, Solley SC, 
            Gilbert LA, Chan MM, Bauer DE, Marson A, Parsons LR, Adamson B.
  TITLE     Improving prime editing with an endogenous small RNA-binding 
            protein.
  JOURNAL   Nature 2024 Apr;628:639-647
  PUBMED    38570691
REFERENCE   2  (bases 1 to 15566)
  AUTHORS   Jun Yan
  TITLE     Direct Submission
  JOURNAL   Exported Jul 15, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-024-07259-6"; journalName: "Nature"; date: 
            "2024-04"; volume: "628"; pages: "639-647"
COMMENT     Sequence Label: PJY109_AAVS1_PJY015_NeoR_bGH_EF1a_PEmax_IRES2_...
FEATURES             Location/Qualifiers
     source          1..15566
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..816
                     /label=New HA-L
     misc_feature    1..804
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    823..848
                     /label=SA
                     /note="splice acceptor site"
     CDS             872..925
                     /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             935..1735
                     /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="GTMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    1761..1985
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2088..2299
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2300..3238
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     promoter        3260..3278
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3292..3348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simianvirus 
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="MKRTADGSEFESPKKKRKV"
     CDS             3349..7449
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from 
                     theStreptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(R221K N394K H840A)
                     /note="generates RNA-guided single strand nicks in DNAdueto
                     the H840A mutation in the HNH catalytic domain"
                     /note="/Original_Translation=DKKYSIGLDIGTNSVGWAVITDEYKVPSKK
                     FKVLG 
                     NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD 
                     DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR 
                     LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA 
                     ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 
                     DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD 
                     EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD 
                     GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                     ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                     NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
                     VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                     EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ 
                     SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                     IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE 
                     LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK 
                     DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                     GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS 
                     DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI 
                     AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF 
                     ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT 
                     VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII 
                     KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ 
                     KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL 
                     FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /note="/modified_by=chenp"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7450..7551
                     /label=SGGSx2-sv40NLS-SGGSx2
                     /note="/created_by=chenp"
     CDS             7552..9582
                     /label=MMLV-RT codon optimized Genscript
                     /label=RT codon optimized Genscript
                     /note="/created_by=jessiedavis"
     CDS             9595..9648
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simianvirus 
                     40) large T antigen"
                     /label=Bipartite SV40 NLS
                     /label=SV40 NLS
                     /note="/Original_Translation=KRTADGSEFEPKKKRKV"
                     /translation="KRTADGSEFESPKKKRKV"
     CDS             9649..9684
                     /codon_start=1
                     /label=GSG-cmycNLS
                     /note="/created_by=chenp"
                     /note="/modified_by=chenp"
                     /translation="GSGPAAKRVKLD"
     misc_feature    join(9741..10315,10316..10318,10319..10327)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             join(10328..10330,10331..10333,10334..11044)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    complement(11067..11100)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    11156..11744
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     polyA_signal    11776..11831
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     misc_feature    11882..12578
                     /label=New HA-R
     misc_feature    11888..12578
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(12725..12743)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(12750..12766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      12908..13363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        13389..13493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13494..13562,13563..14354)
                     /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"
     rep_origin      14525..15113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    15401..15422
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(15437..15442,15443..15460,15461..15467)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15475..15491
                     /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     15499..15515
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        15536..15554
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgctttctct gaccagcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt
       61 ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt
      121 cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc
      181 cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg
      241 tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct
      301 gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct
      361 tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc
      421 gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc
      481 ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt
      541 catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc
      601 ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc
      661 ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct
      721 gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct
      781 gtcccctcca ccccacagtg gggccactag ggacagaagc ttctgacctc ttctcttcct
      841 cccacagggc ctcgagagat ctggcagcgg agagggcaga ggaagtcttc taacatgcgg
      901 tgacgtggag gagaatcccg gccctaggct cgagggtacc atgattgaac aagatggatt
      961 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     1021 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     1081 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     1141 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     1201 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     1261 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     1321 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     1381 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     1441 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     1501 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     1561 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     1621 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     1681 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagttta
     1741 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1801 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1861 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1921 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1981 tatggactag tattaattaa atcactcacg cgtctaggat ccatgccgat agcgttggtt
     2041 gagtggataa ccgtattacc gccaagctta tgcatgtgcc cgtcagtggg cagagcgcac
     2101 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     2161 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2221 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2281 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     2341 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     2401 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     2461 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     2521 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     2581 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     2641 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     2701 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     2761 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     2821 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     2881 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     2941 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     3001 gtaccgggcg ccgtccaggc acctcgatta gttctcgcgc ttttggagta cgtcgtcttt
     3061 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     3121 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     3181 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     3241 gtcgtgagct agcggccgct aatacgactc actataggga gagccgccac catgaaacgg
     3301 acagccgacg gaagcgagtt cgagtcacca aagaagaagc ggaaagtcga caagaagtac
     3361 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     3421 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     3481 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     3541 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     3601 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     3661 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     3721 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     3781 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     3841 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     3901 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     3961 gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagaaagct ggaaaatctg
     4021 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     4081 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     4141 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     4201 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     4261 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     4321 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     4381 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     4441 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     4501 accgaggaac tgctcgtgaa gctgaagaga gaggacctgc tgcggaagca gcggaccttc
     4561 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     4621 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     4681 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     4741 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     4801 gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     4861 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     4921 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     4981 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     5041 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     5101 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     5161 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     5221 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     5281 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     5341 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     5401 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     5461 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     5521 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     5581 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     5641 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     5701 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     5761 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     5821 caggaactgg acatcaaccg gctgtccgac tacgatgtgg acgctatcgt gcctcagagc
     5881 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     5941 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     6001 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     6061 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     6121 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     6181 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     6241 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     6301 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     6361 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     6421 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     6481 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     6541 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     6601 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     6661 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     6721 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     6781 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     6841 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     6901 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     6961 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     7021 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     7081 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     7141 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     7201 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     7261 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     7321 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     7381 accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg
     7441 ggaggtgact ccggcggaag ctctggtggc agcaagcgga ccgccgacgg ctctgaattc
     7501 gagagcccta agaagaaaag aaaggtgagc ggaggctcta gcggcggaag caccctgaac
     7561 attgaagacg agtatagact gcatgaaaca agcaaggaac ccgacgtgtc cctgggctcc
     7621 acctggctgt ccgactttcc ccaggcctgg gccgagacag gaggaatggg cctggccgtg
     7681 cggcaggcac ccctgatcat ccctctgaag gccacctcta cacccgtgag catcaagcag
     7741 taccctatgt ctcaggaggc cagactgggc atcaagcctc acatccagag gctgctggac
     7801 cagggcatcc tggtgccatg ccagagcccc tggaacacac cactgctgcc cgtgaagaag
     7861 ccaggcacca atgactatag acccgtgcag gatctgagag aggtgaacaa gagggtggag
     7921 gatatccacc ccaccgtgcc caacccttac aatctgctgt ccggcctgcc cccttctcac
     7981 cagtggtata cagtgctgga cctgaaggat gccttctttt gtctgagact gcaccctacc
     8041 agccagccac tgttcgcctt tgagtggagg gaccctgaga tgggcatctc tggccagctg
     8101 acctggacac gcctgcctca gggcttcaag aatagcccaa cactgtttaa cgaggccctg
     8161 caccgcgacc tggcagattt ccggatccag cacccagatc tgatcctgct gcagtacgtg
     8221 gacgatctgc tgctggccgc caccagcgag ctggattgcc agcagggaac acgcgccctg
     8281 ctgcagaccc tgggaaacct gggatatagg gcatccgcca agaaggccca gatctgtcag
     8341 aagcaggtga agtacctggg ctatctgctg aaggagggcc agagatggct gacagaggcc
     8401 aggaaggaga cagtgatggg ccagccaaca cccaagaccc caagacagct gagggagttc
     8461 ctgggcaaag caggattttg caggctgttc atcccaggat tcgcagagat ggcagcacct
     8521 ctgtacccac tgaccaagcc gggcaccctg tttaattggg gccctgacca gcagaaggcc
     8581 tatcaggaga tcaagcaggc cctgctgaca gcaccagccc tgggcctgcc agacctgacc
     8641 aagcctttcg agctgtttgt ggatgagaag cagggctacg ccaagggcgt gctgacccag
     8701 aagctgggac catggagacg gcccgtggcc tatctgtcca agaagctgga cccagtggca
     8761 gcaggatggc caccatgcct gaggatggtg gcagcaatcg ccgtgctgac aaaggatgcc
     8821 ggcaagctga ccatgggaca gccactggtc atcctggcac cacacgcagt ggaggccctg
     8881 gtgaagcagc ctccagatcg ctggctgtct aacgcccgga tgacacacta ccaggccctg
     8941 ctgctggaca ccgatcgcgt gcagtttggc cctgtggtgg ccctgaatcc agccaccctg
     9001 ctgcctctgc cagaggaggg cctgcagcac aactgtctgg acatcctggc agaggcacac
     9061 ggaacaaggc cagacctgac cgatcagccc ctgcctgacg ccgatcacac atggtatacc
     9121 gatggaagct ccctgctgca ggagggccag aggaaggcag gagcagcagt gaccacagag
     9181 acagaagtga tctgggccaa ggccctgcca gcaggcacat ccgcccagcg ggccgagctg
     9241 atcgccctga cccaggccct gaagatggcc gagggcaaga agctgaacgt gtacacagac
     9301 tccagatatg ccttcgccac cgcacacatc cacggagaga tctacaggcg ccggggctgg
     9361 ctgacctctg agggcaagga gatcaagaac aaggatgaga tcctggccct gctgaaggcc
     9421 ctgtttctgc ccaagcggct gagcatcatc cactgtcctg gacaccagaa gggacactcc
     9481 gccgaggcaa ggggcaatcg gatggccgac caggccgcca gaaaggctgc tattactgaa
     9541 actcccgaca cttccactct gctgattgaa aactcctccc cttctggcgg ctcaaaaaga
     9601 accgccgacg gcagcgaatt cgagtctccc aagaagaaga ggaaagtcgg ctctggccct
     9661 gccgctaaga gagtgaagct ggactaaccg gtagcgctcg gccaattctg cagtcgacgg
     9721 taccgcgggc ccgggatccg cccctctccc tccccccccc ctaacgttac tggccgaagc
     9781 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     9841 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     9901 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     9961 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
    10021 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
    10081 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
    10141 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
    10201 tctgatctgg ggcctcggta cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
    10261 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc
    10321 cacaaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
    10381 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
    10441 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
    10501 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
    10561 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
    10621 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
    10681 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
    10741 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
    10801 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
    10861 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
    10921 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
    10981 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
    11041 caagtaagaa ttcgtcgagg gacctaataa cttcgtatag catacattat acgaagttat
    11101 acatgtttaa gggttccggt tccactaggt acaattcgat atcaagctta tcgataatca
    11161 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
    11221 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
    11281 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
    11341 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
    11401 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
    11461 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
    11521 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
    11581 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
    11641 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
    11701 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatgaag ccccttgagc
    11761 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
    11821 tgtgtctctc actcggaagg acatatggga gggcaaatca taagtcgaca gtactaagct
    11881 tgattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc ctgatattgg
    11941 gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc ccatttcctg
    12001 gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc cagagaggat
    12061 cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg acctgcccgg
    12121 ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg acgcggctgt
    12181 ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag aggagaagca
    12241 gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc tcttcacctt
    12301 tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag ataaggccag
    12361 tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt ggaaaactcc
    12421 ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct actccctttc
    12481 tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca tattcctccg
    12541 cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt tgagtccttg
    12601 gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac catctcatgc
    12661 ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctagc gatcgccaat
    12721 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
    12781 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
    12841 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
    12901 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
    12961 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
    13021 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
    13081 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
    13141 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
    13201 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
    13261 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
    13321 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
    13381 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
    13441 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
    13501 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
    13561 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
    13621 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
    13681 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
    13741 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
    13801 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
    13861 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
    13921 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
    13981 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
    14041 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
    14101 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
    14161 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
    14221 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
    14281 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
    14341 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
    14401 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
    14461 tcccttaacg tgagttttcg ttccactgag cgtcaaaccc cgtagaaaag atcaaaggat
    14521 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
    14581 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
    14641 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
    14701 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
    14761 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
    14821 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
    14881 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
    14941 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
    15001 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
    15061 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    15121 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
    15181 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
    15241 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
    15301 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
    15361 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
    15421 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
    15481 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
    15541 accctcacta aagggaacaa aagctg
//
