LOCUS       sequence-496394-       13160 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Overexpression of SpCas9-BFP with HBB IVS2 intron in 5' UTR and 
            blasticidin resistance gene. Contains minimal A2UCOE and Cp36 
            recombination site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13160)
  AUTHORS   Arana S, Du PP, Vaughan-Jackson A, Enright N, Spees K, Valbuena R, 
            Garcia CA, Nguyen T, Venida A, Seczynska M, Bintu L, Lehner PJ, 
            Prolo LM, Bassik MC
  TITLE     Reduced Cas9 transgene silencing by incorporation of intron 
            sequences.
  JOURNAL   Nat Commun. 2025 Nov 27;16(1):10656. doi: 
            10.1038/s41467-025-65669-0.
  PUBMED    41309578
REFERENCE   2  (bases 1 to 13160)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-11-27"; volume: "16(1):10656. doi"; pages: " 
            10.1038/s41467-025-65669-0"
COMMENT     Sequence Label: pSA128 A2UCOE-HBB_IVS2-Cas9-BFP
FEATURES             Location/Qualifiers
     source          1..13160
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        801..1979
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1032..1970
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1927..1947
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2961..2970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2967..7070
                     /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             2967..7070
                     /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             7074..7100
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7080..7106
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             7119..7139
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7146..7166
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7206..7904
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    7956..8077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7967..7986)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8021..8040
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      8192..8238
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      8268..8404
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    8449..8520
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(8746..9334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8826..8845)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9834..11043
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     10880..10899
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             11047..11445
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    11472..11520
                     /note="synthetic polyadenylation signal"
     CDS             join(11786..11854,11855..12646)
                     /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     complement(12004..12023)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      12659..12795
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    12947..13018
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(13023..13069)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    13087..13135
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 cttaaccgct tttgaatgtc cgtcttagtt aatccctaat tgaacgctcc aaaaggtgac
       61 ttccaatagg gatttattcc ttttaaaatt aacggcataa tcgtagatcg catgccggga
      121 ggtggtccct gcagttacgc caatgataac ccccgccaga aaaatcttag tagccttccc
      181 tttttgtttt ccgtgcccca actcggcgga ttgactcggc cccttccgga aacacccgaa
      241 tcaacttcta gtcaaattat tgttcacgcc gcaatgaccc acccctggcc cgcgtctgtg
      301 gaactgaccc ctggtgtaca ggagagttcg ctgctgaaag tggtcccaaa ggggtactag
      361 tttttaagct cccaactccc cctcccccag cgtctggagg attccacacc ctcgcaccgc
      421 aggggcgagg aagtgggcgg agtccggttt tggcgccagc cgctgaggct gccaagcaga
      481 aaagccaccg ctgaggagac tccggtcact gtcctcgccc cgcctccccc ttccctcccc
      541 ttggggacca ccgggcgcca cgccgcgaac ggtaagtgcc gcggtcgtcg gcgcctccgc
      601 cctcccccta gggccccaat tcccagcggg cgcggcgcgc ggcccctccc cccgccgggc
      661 gcgcgcccgc tgccccgccc ttcgtggccg cccggcgtgg gcggtgccac ccctcccccc
      721 ggcggccccg cgcgcagctc ccggctccct cccccttcgg atgtggcttg agctgtaggc
      781 gcggaggggc tagctcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      841 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      901 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      961 gagaaccgta tataagtgca ctagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     1021 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
     1081 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
     1141 ttcgggttgg aagtgggtgg gagagttcgt ggccttgcgc ttaaggagcc ccttcgcctc
     1201 gtgcttgagt tgtggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     1261 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     1321 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat cagcacactg
     1381 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     1441 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     1501 cccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     1561 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     1621 cagggagcac aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     1681 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     1741 cgccgtccag gcacctcgat tagttctcca gcttttggag tacgtcgtct ttaggttggg
     1801 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     1861 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     1921 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa
     1981 aactacccct aaaagccaaa agatctttgt cgatcctacc atccactcga cacacccgcc
     2041 agcggccgct gccaagcttg ctaggaattc cgcgtctgca ggatatcaag cttgcggtac
     2101 gtgagtctat gggacgcttg atgttttctt tccccttctt ttctatggtt aagttcatgt
     2161 cataggaagg ggataagtaa cagggtacag tttagaatgg gaaacagacg aatgattgca
     2221 tcagtgtgga agtctcagga tcgttttagt ttcttttatt tgctgttcat aacaattgtt
     2281 ttcttttgtt taattcttgc tttctttttt tttcttctcc gcaattttta ctattatact
     2341 taatgcctta acattgtgta taacaaaagg aaatatctct gagatacatt aagtaactta
     2401 aaaaaaaact ttacacagtc tgcctagtac attactattt ggaatatatg tgtgcttatt
     2461 tgcatattca taatctccct actttatttt cttttatttt taattgatac ataatcatta
     2521 tacatattta tgggttaaag tgtaatgttt taatatgtgt acacatattg accaaatcag
     2581 ggtaattttg catttgtaat tttaaaaaat gctttcttct tttaatatac ttttttgttt
     2641 atcttatttc taatactttc cctaatctct ttctttcagg gcaataatga tacaatgtat
     2701 catgcctctt tgcaccattc taaagaataa cagtgataat ttctgggtta aggcaatagc
     2761 aatatctctg catataaata tttctgcata taaattgtaa ctgatgtaag aggtttcata
     2821 ttgctaatag cagctacaat ccagctacca ttctgctttt attttatggt tgggataagg
     2881 ctggattatt ctgagtccaa gctaggccct tttgctaatc atgttcatac ctcttatctt
     2941 cctcccacag cgcgggcccg gccaccatgg acaagaagta cagcatcggc ctggacatcg
     3001 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     3061 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggcgccctgc
     3121 tgttcgacag cggagaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     3181 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     3241 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     3301 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     3361 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     3421 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     3481 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     3541 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     3601 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     3661 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     3721 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     3781 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     3841 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     3901 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     3961 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     4021 tcgaccagag caagaacggc tacgccggct acatcgatgg cggagccagc caggaagagt
     4081 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     4141 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     4201 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     4261 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     4321 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     4381 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgccagcgcc cagagcttca
     4441 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     4501 gcctgctgta cgagtacttc accgtgtaca acgagctgac caaagtgaaa tacgtgaccg
     4561 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaagccatc gtggacctgc
     4621 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     4681 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     4741 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     4801 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     4861 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     4921 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     4981 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     5041 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     5101 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     5161 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     5221 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     5281 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     5341 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     5401 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     5461 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     5521 tcgataacaa agtgctgact cggagcgaca agaaccgggg caagagcgac aacgtgccct
     5581 ccgaagaggt cgtgaagaag atgaagaact actggcgcca gctgctgaat gccaagctga
     5641 ttacccagag gaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     5701 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     5761 cacagatcct ggactcccgg atgaacacta agtacgacga gaacgacaaa ctgatccggg
     5821 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     5881 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     5941 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     6001 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     6061 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     6121 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa acaggcgaga
     6181 tcgtgtggga taagggccgg gactttgcca ccgtgcggaa agtgctgtct atgccccaag
     6241 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     6301 ccaagaggaa cagcgacaag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     6361 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     6421 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     6481 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     6541 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     6601 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     6661 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     6721 atgagcagaa acagctgttt gtggaacagc acaaacacta cctggacgag atcatcgagc
     6781 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaggtgctga
     6841 gcgcctacaa caagcacaga gacaagccta tcagagagca ggccgagaat atcatccacc
     6901 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     6961 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     7021 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac gcctatccct
     7081 atgacgtgcc cgattatgcc agcctgggca gcggctcccc caagaaaaaa cgcaaggtgg
     7141 aagatcctaa gaaaaagcgg aaagtggacg gcattggtag tgggagcaac ggcagcagcg
     7201 gatccagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
     7261 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
     7321 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
     7381 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
     7441 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
     7501 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
     7561 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
     7621 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
     7681 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
     7741 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
     7801 aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca
     7861 gatactgcga cctccctagc aaactggggc acaagcttaa ttagtaacta gcttgactga
     7921 ctgagataca gcgtaccttc agctcacaga catgataaga tacattgatg agtttggaca
     7981 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     8041 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     8101 tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
     8161 atgtggtatt ggcccatctc tatcggtatc gtagcataac cccttggggc ctctaaacgg
     8221 gtcttgaggg gttttttgtg cccctcgggc cggattgcta tctaccggca ttggcgcaga
     8281 aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag attcagcaac
     8341 ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag aaatttatcc
     8401 ttaaggtcgt cagctatcct gcaggcgatc tctcgatttc gatcaagaca ttcctttaat
     8461 ggtcttttct ggacaccact aggggtcaga agtagttcat caaactttct tccctcccta
     8521 atctcattgg ttaccttggg ctatcgaaac ttaattaacc agtcaagtca gctacttggc
     8581 gagatcgact tgtctgggtt tcgactacgc tcagaattgc gtcagtcaag ttcgatctgg
     8641 tccttgctat tgcacccgtt ctccgattac gagtttcatt taaatcatgt gagcaaaagg
     8701 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     8761 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     8821 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     8881 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     8941 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     9001 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     9061 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     9121 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     9181 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     9241 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     9301 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     9361 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     9421 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     9481 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     9541 gatctgtcta tttcgttcat ccatagttgc atttaaattt ccgaactctc caaggccctc
     9601 gtcggaaaat cttcaaacct ttcgtccgat ccatcttgca ggctacctct cgaacgaact
     9661 atcgcaagtc tcttggccgg ccttgcgcct tggctattgc ttggcagcgc ctatcgccag
     9721 gtattactcc aatcccgaat atccgagatc gggatcaccc gagagaagtt caacctacat
     9781 cctcaatccc gatctatccg agatccgagg aatatcgaaa tcggggcgcg cctggcctcc
     9841 gcgccgggtt ttggcgcctc ccgcgggcgc ccccctcgtc acggcgagcg ctgccacgtc
     9901 agacgaaggg cgcaggagcg tcctgatcct tccgcccgga cgctcaggac agcggcccgc
     9961 tgctcataag actcggcctt agaaccccag tatcagcaga aggacatttt aggacgggac
    10021 ttgggtgact ctagggcact ggttttcttt ccagagagcg gaacaggcga ggaaaagtag
    10081 tcccttctcg gcgattctgc ggagggatct ccgtggggcg gtgaacgccg atgattatat
    10141 aaggacgcgc cgggtgtggc acagctagtt ccgtcgcagc cgggatttgg gtcgcggttc
    10201 ttgtttgtgg atcgctgtga tcgtcacttg gtgagtagcg ggctgctggg ctggccgggg
    10261 ctttcgtggc cgccgggccg ctcggtggga cggaagcgtg tggagagacc gccaagggct
    10321 gtagtctggg tccgcgagca aggttgccct gaactggggg ttggggggag cgcagcaaaa
    10381 tggcggctgt tcccgagtct tgaatggaag acgcttgtga ggcgggctgt gaggtcgttg
    10441 aaacaaggtg gggggcatgg tgggcggcaa gaacccaagg tcttgagccc ttcgctaatg
    10501 cgggaaagct cttattcggg tgagatgggc tgggcaccat ctggggaccc tgacgtgaag
    10561 tttgtcactg actggagaac tcggtttgtc gtctgttgcg ggggcggcag ttatggcggt
    10621 gccgttgggc agtgcacccg tacctttggg agcgcgcgcc ctcgtcgtgt cgtgacgtca
    10681 cccgttctgt tggcttataa tgcagggtgg ggccacctgc cggtaggtgt gcggtaggct
    10741 tttctccgtc gcaggacgca gggttcgggc ctagggtagg ctctcctgaa tcgacaggcg
    10801 ccggacctct ggtgagggga gggataagtg aggcgtcagt ttctttggtc ggttttatgt
    10861 acctatcttc ttaagtagct gaagctccgg ttttgaacta tgcgctcggg gttggcgagt
    10921 gtgttttgtg aagtttttta ggcacctttt gaaatgtaat catttgggtc aatatgtaat
    10981 tttcagtgtt agacttgtaa attgtccgct aaattctggc cgtttttggc ttttttgtta
    11041 gacaacatgg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag agcaacggct
    11101 acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc tctctctagc
    11161 gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa
    11221 ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg tatcgtcgcg
    11281 atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca ggtgcttctc
    11341 gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc gacggcagtt
    11401 gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctaagctag tatgtaagcc
    11461 tagtcttaga taataaaatc gctatccatc gaagatggat gtgtgttggt tttttgtgtg
    11521 tgtaacgcta ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat
    11581 ccatatcgtt gcttggcagt cagccagtcg gaatccagct tgggacccag gaagtccaat
    11641 cgtcagatat tgtactcaag cctggtcacg gcagcgtacc gatctgttta aacctagata
    11701 ttgatagtct gatcggtcaa cgtataatcg agtcctagct tttgcaaaca tctatcaaga
    11761 gacaggatca gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg cccttattcc
    11821 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
    11881 agatgctgaa gatcagttgg gtgcgcgagt gggttacatc gaactggatc tcaacagcgg
    11941 taagatcctt gagagttttc gccccgaaga acgctttcca atgatgagca cttttaaagt
    12001 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
    12061 catacactat tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac
    12121 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
    12181 ggccaactta cttctgacaa cgattggagg accgaaggag ctaaccgctt ttttgcacaa
    12241 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
    12301 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaaccttgc gtaaactatt
    12361 aactggcgaa ctacttactc tagcttcccg gcaacagttg atagactgga tggaggcgga
    12421 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
    12481 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
    12541 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
    12601 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaaccga ttctaggtgc
    12661 attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca
    12721 gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca
    12781 gaaatttatc cttaagatcc cgaatcgttt aaactcgact ctggctctat cgaatctccg
    12841 tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg
    12901 tcagctatcg tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt
    12961 agctccacag gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa
    13021 ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc
    13081 gttacacaca caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta
    13141 actgctgatc gagtgtagcc
//
