LOCUS       sequence-496493-       12615 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Overexpression of SpCas9-BFP with HBB IVS2 intron in 5' UTR and 
            blasticidin resistance gene. Contains Cp36 recombination site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12615)
  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 12615)
  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: pSA073 HBB_IVS2-Cas9-BFP
FEATURES             Location/Qualifiers
     source          1..12615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        500..1709
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1546..1565
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             1713..2111
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2138..2186
                     /note="synthetic polyadenylation signal"
     CDS             join(2452..2520,2521..3312)
                     /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(2670..2689)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      3325..3461
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    3613..3684
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(3689..3735)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3753..3801
                     /note="synthetic polyadenylation signal"
     promoter        3942..5120
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4173..5111
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5068..5088
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      6242..6251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6248..10351
                     /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             6248..10351
                     /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             10355..10381
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10361..10387
                     /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             10400..10420
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10427..10447
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10487..11185
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    11237..11358
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11248..11267)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     11302..11321
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      11473..11519
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      11549..11685
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    11730..11801
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(12027..12615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12107..12126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt
      241 aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat
      301 cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc
      361 tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga
      421 tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata
      481 tcgaaatcgg ggcgcgcctg gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc
      541 ctcgtcacgg cgagcgctgc cacgtcagac gaagggcgca ggagcgtcct gatccttccg
      601 cccggacgct caggacagcg gcccgctgct cataagactc ggccttagaa ccccagtatc
      661 agcagaagga cattttagga cgggacttgg gtgactctag ggcactggtt ttctttccag
      721 agagcggaac aggcgaggaa aagtagtccc ttctcggcga ttctgcggag ggatctccgt
      781 ggggcggtga acgccgatga ttatataagg acgcgccggg tgtggcacag ctagttccgt
      841 cgcagccggg atttgggtcg cggttcttgt ttgtggatcg ctgtgatcgt cacttggtga
      901 gtagcgggct gctgggctgg ccggggcttt cgtggccgcc gggccgctcg gtgggacgga
      961 agcgtgtgga gagaccgcca agggctgtag tctgggtccg cgagcaaggt tgccctgaac
     1021 tgggggttgg ggggagcgca gcaaaatggc ggctgttccc gagtcttgaa tggaagacgc
     1081 ttgtgaggcg ggctgtgagg tcgttgaaac aaggtggggg gcatggtggg cggcaagaac
     1141 ccaaggtctt gagcccttcg ctaatgcggg aaagctctta ttcgggtgag atgggctggg
     1201 caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg tttgtcgtct
     1261 gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc tttgggagcg
     1321 cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca gggtggggcc
     1381 acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt tcgggcctag
     1441 ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga taagtgaggc
     1501 gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag ctccggtttt
     1561 gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca ccttttgaaa
     1621 tgtaatcatt tgggtcaata tgtaattttc agtgttagac ttgtaaattg tccgctaaat
     1681 tctggccgtt tttggctttt ttgttagaca acatggccaa gcctttgtct caagaagaat
     1741 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     1801 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     1861 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     1921 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     1981 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     2041 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     2101 gggagggcta agctagtatg taagcctagt cttagataat aaaatcgcta tccatcgaag
     2161 atggatgtgt gttggttttt tgtgtgtgta acgctaggcg cgcctggtgt accgagaacg
     2221 atcctctcag tgcgagtctc gacgatccat atcgttgctt ggcagtcagc cagtcggaat
     2281 ccagcttggg acccaggaag tccaatcgtc agatattgta ctcaagcctg gtcacggcag
     2341 cgtaccgatc tgtttaaacc tagatattga tagtctgatc ggtcaacgta taatcgagtc
     2401 ctagcttttg caaacatcta tcaagagaca ggatcagcag gaggctttcg catgagtatt
     2461 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2521 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc gcgagtgggt
     2581 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgc
     2641 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2701 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtat
     2761 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2821 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat tggaggaccg
     2881 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2941 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3001 atggcaacaa ccttgcgtaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3061 cagttgatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3121 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3181 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3241 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3301 aagcattggt aaccgattct aggtgcattg gcgcagaaaa aaatgcctga tgcgacgctg
     3361 cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc ccaacttgcc
     3421 cacttccata cgtgtcctcc ttaccagaaa tttatcctta agatcccgaa tcgtttaaac
     3481 tcgactctgg ctctatcgaa tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc
     3541 atttgctcgt cgggcatcga atctcgtcag ctatcgtcag cttacctttt tggcagcgat
     3601 cgcggctccc gacatcttgg accattagct ccacaggtat cttcttccct ctagtggtca
     3661 taacagcagc ttcagctacc tctcaattca aaaaacccct caagacccgt ttagaggccc
     3721 caaggggtta tgctatcaat cgttgcgtta cacacacaaa aaaccaacac acatccatct
     3781 tcgatggata gcgattttat tatctaactg ctgatcgagt gtagccctta accgcttttg
     3841 aatgtccgtc ttagttaatc cctaattgaa cgctccaaaa ggtgacttcc aatagggatt
     3901 tattcctttt aaaattaacg gcataatcgt agatctcgtg aggctccggt gcccgtcagt
     3961 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     4021 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     4081 gcctttttcc cgagggtggg ggagaaccgt atataagtgc actagtcgcc gtgaacgttc
     4141 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     4201 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     4261 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg tggccttgcg
     4321 cttaaggagc cccttcgcct cgtgcttgag ttgtggcctg gcctgggcgc tggggccgcc
     4381 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     4441 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     4501 cgggccaaga tcagcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     4561 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     4621 gacgggggta gtctcaagct gcccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     4681 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     4741 ggccgcttcc cggccctgct gcagggagca caaaatggag gacgcggcgc tcgggagagc
     4801 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     4861 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcc agcttttgga
     4921 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     4981 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     5041 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     5101 ttccatttca ggtgtcgtga aaactacccc taaaagccaa aagatctttg tcgatcctac
     5161 catccactcg acacacccgc cagcggccgc tgccaagctt gctaggaatt cgtaacgctc
     5221 ttcgagaaag cagattctca tatccatctt gagtcttctt tctcgcaaga caacacgaaa
     5281 tagacacagt ctcttcccta gctgtacact gagccgaatt ccgcgtctgc aggatatcaa
     5341 gcttggaatt cgtgagtcta tgggacgctt gatgttttct ttccccttct tttctatggt
     5401 taagttcatg tcataggaag gggataagta acagggtaca gtttagaatg ggaaacagac
     5461 gaatgattgc atcagtgtgg aagtctcagg atcgttttag tttcttttat ttgctgttca
     5521 taacaattgt tttcttttgt ttaattcttg ctttcttttt ttttcttctc cgcaattttt
     5581 actattatac ttaatgcctt aacattgtgt ataacaaaag gaaatatctc tgagatacat
     5641 taagtaactt aaaaaaaaac tttacacagt ctgcctagta cattactatt tggaatatat
     5701 gtgtgcttat ttgcatattc ataatctccc tactttattt tcttttattt ttaattgata
     5761 cataatcatt atacatattt atgggttaaa gtgtaatgtt ttaatatgtg tacacatatt
     5821 gaccaaatca gggtaatttt gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata
     5881 cttttttgtt tatcttattt ctaatacttt ccctaatctc tttctttcag ggcaataatg
     5941 atacaatgta tcatgcctct ttgcaccatt ctaaagaata acagtgataa tttctgggtt
     6001 aaggcaatag caatatctct gcatataaat atttctgcat ataaattgta actgatgtaa
     6061 gaggtttcat attgctaata gcagctacaa tccagctacc attctgcttt tattttatgg
     6121 ttgggataag gctggattat tctgagtcca agctaggccc ttttgctaat catgttcata
     6181 cctcttatct tcctcccaca gcgcgtctgc aggatatcaa gcttgcggta ccgcgggccc
     6241 ggccaccatg gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     6301 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     6361 cgaccggcac agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggagaaac
     6421 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     6481 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     6541 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     6601 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     6661 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     6721 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     6781 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     6841 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     6901 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     6961 cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     7021 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     7081 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     7141 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     7201 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     7261 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     7321 ctacgccggc tacatcgatg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     7381 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     7441 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     7501 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     7561 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     7621 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     7681 cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc atcgagcgga tgaccaactt
     7741 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     7801 caccgtgtac aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     7861 cttcctgagc ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa
     7921 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     7981 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     8041 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     8101 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     8161 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     8221 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     8281 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     8341 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     8401 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     8461 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     8521 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     8581 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     8641 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     8701 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     8761 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgataaca aagtgctgac
     8821 tcggagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     8881 gatgaagaac tactggcgcc agctgctgaa tgccaagctg attacccaga ggaagttcga
     8941 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     9001 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     9061 gatgaacact aagtacgacg agaacgacaa actgatccgg gaagtgaaag tgatcaccct
     9121 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     9181 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     9241 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     9301 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     9361 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     9421 gaagcggcct ctgatcgaga caaacggcga aacaggcgag atcgtgtggg ataagggccg
     9481 ggactttgcc accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac
     9541 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgacaa
     9601 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     9661 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     9721 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     9781 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     9841 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     9901 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     9961 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
    10021 tgtggaacag cacaaacact acctggacga gatcatcgag cagatcagcg agttctccaa
    10081 gagagtgatc ctggccgacg ctaatctgga caaggtgctg agcgcctaca acaagcacag
    10141 agacaagcct atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
    10201 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
    10261 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
    10321 acggatcgac ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc ccgattatgc
    10381 cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg
    10441 gaaagtggac ggcattggta gtgggagcaa cggcagcagc ggatccagcg agctgattaa
    10501 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
    10561 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
    10621 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
    10681 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
    10741 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
    10801 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
    10861 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac
    10921 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
    10981 cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa
    11041 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
    11101 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
    11161 caaactgggg cacaagctta attagtaact agcttgactg actgagatac agcgtacctt
    11221 cagctcacag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
    11281 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
    11341 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
    11401 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat tggcccatct
    11461 ctatcggtat cgtagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgt
    11521 gcccctcggg ccggattgct atctaccggc attggcgcag aaaaaaatgc ctgatgcgac
    11581 gctgcgcgtc ttatactccc acatatgcca gattcagcaa cggatacggc ttccccaact
    11641 tgcccacttc catacgtgtc ctccttacca gaaatttatc cttaaggtcg tcagctatcc
    11701 tgcaggcgat ctctcgattt cgatcaagac attcctttaa tggtcttttc tggacaccac
    11761 taggggtcag aagtagttca tcaaactttc ttccctccct aatctcattg gttaccttgg
    11821 gctatcgaaa cttaattaac cagtcaagtc agctacttgg cgagatcgac ttgtctgggt
    11881 ttcgactacg ctcagaattg cgtcagtcaa gttcgatctg gtccttgcta ttgcacccgt
    11941 tctccgatta cgagtttcat ttaaatcatg tgagcaaaag gccagcaaaa ggccaggaac
    12001 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
    12061 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
    12121 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
    12181 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
    12241 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
    12301 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
    12361 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
    12421 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
    12481 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
    12541 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
    12601 aaaaaaggat ctcaa
//
