LOCUS       sequence-496387-       12813 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Overexpression of SpCas9-BFP with PFDN2 IVS3 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 12813)
  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 12813)
  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: pSA093 PFDN2_IVS3-Cas9-BFP
FEATURES             Location/Qualifiers
     source          1..12813
                     /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      6440..6449
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6446..10549
                     /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             6446..10549
                     /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             10553..10579
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             10559..10585
                     /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             10598..10618
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10625..10645
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10685..11383
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    11435..11556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11446..11465)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     11500..11519
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      11671..11717
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      11747..11883
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    11928..11999
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(12225..12813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12305..12324)
                     /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 ccgcgtctgc
     5221 aggatatcaa gcttgcggta cgtgagcaag aatcattgaa taggatcggg tacagtggct
     5281 gtaaacccag cactttggga ggccaaggca ggtggactct ttgagtccgg gagttcgaga
     5341 ccagcctggg caacatggtg aaaccatatc actacaaaaa atacaaaaga ttagctgggt
     5401 atggtggtat gtgcctgtag tcccagctac tcacctgagc ctaaaatgtc gaggcttcag
     5461 tgagctgtga tcacctcact gcactccagc ctgggtgaca gagtgagacc ctgtctcaga
     5521 taaaaggagg gcagcataaa atagcctatg ctcattatgc tatgctaagt gcaactctta
     5581 gacaagagga attgctgact gctgtgggct ctgtgacttg gctgaattgg ggtgggagga
     5641 ttggggttag agtaatgttt ctcatagtgt gtgtttcacg aatacatgaa tcaaaatcac
     5701 ctgaaggact aatttataaa tgcagcttcc cagacccact ggattagaac ctttgggagt
     5761 gccatgaagt gacattttaa aaataagcta ggcaggttgc agtgactcac acctgtaatc
     5821 ccagcacttt gggaggccga ggcgggcgga tcacgaggtc aggaggtcaa gaccatcctg
     5881 gctagcatgg tgaaacccca tctctactaa gaatacgaaa aattggctgg gcgttgtggt
     5941 gggcgcctgt ggtcctagct actcgggagg gcaaggcagg agaatggcgt gaacctgcga
     6001 ggcggagctt gcagtgagct gagattgcac cactgcactc cagcctgggc gacacagcga
     6061 tgctgcaact caaaaaaaaa atattaaaaa taaaaaatga aaaaataaaa ataagctcat
     6121 gcctgtattc ctagcgcttt gggagaccga ggcaggagga ctgcttgagg cagaagtttg
     6181 agaccagcct gggtaacata gtaggaactt gtctctgtaa aaaataaaaa taagcagccc
     6241 aggtgattct aagtacatta aagtgtataa accactgtgt tcaagaaagg aataatttta
     6301 atgtctgggc tcctctactt ggttaaaggt aaggttaaat gtgttaacac ttcctccaac
     6361 catagtccat tccaggtggg ggaaaggagt cctgaattcc ttatataatc cctgtcactt
     6421 gtcctctagc gcgggcccgg ccaccatgga caagaagtac agcatcggcc tggacatcgg
     6481 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     6541 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gcgccctgct
     6601 gttcgacagc ggagaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     6661 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     6721 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     6781 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     6841 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     6901 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     6961 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     7021 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     7081 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     7141 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     7201 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     7261 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     7321 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     7381 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     7441 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     7501 cgaccagagc aagaacggct acgccggcta catcgatggc ggagccagcc aggaagagtt
     7561 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     7621 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     7681 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     7741 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     7801 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     7861 catcaccccc tggaacttcg aggaagtggt ggacaagggc gccagcgccc agagcttcat
     7921 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     7981 cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaagtgaaat acgtgaccga
     8041 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaagccatcg tggacctgct
     8101 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     8161 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     8221 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     8281 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     8341 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     8401 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     8461 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     8521 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     8581 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     8641 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     8701 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     8761 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     8821 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     8881 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     8941 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     9001 cgataacaaa gtgctgactc ggagcgacaa gaaccggggc aagagcgaca acgtgccctc
     9061 cgaagaggtc gtgaagaaga tgaagaacta ctggcgccag ctgctgaatg ccaagctgat
     9121 tacccagagg aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     9181 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     9241 acagatcctg gactcccgga tgaacactaa gtacgacgag aacgacaaac tgatccggga
     9301 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     9361 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     9421 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     9481 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     9541 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     9601 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa caggcgagat
     9661 cgtgtgggat aagggccggg actttgccac cgtgcggaaa gtgctgtcta tgccccaagt
     9721 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     9781 caagaggaac agcgacaagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     9841 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     9901 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     9961 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
    10021 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
    10081 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
    10141 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
    10201 tgagcagaaa cagctgtttg tggaacagca caaacactac ctggacgaga tcatcgagca
    10261 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aggtgctgag
    10321 cgcctacaac aagcacagag acaagcctat cagagagcag gccgagaata tcatccacct
    10381 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
    10441 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
    10501 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgacg cctatcccta
    10561 tgacgtgccc gattatgcca gcctgggcag cggctccccc aagaaaaaac gcaaggtgga
    10621 agatcctaag aaaaagcgga aagtggacgg cattggtagt gggagcaacg gcagcagcgg
    10681 atccagcgag ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga
    10741 caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac
    10801 catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac
    10861 tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt
    10921 caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg
    10981 cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa
    11041 gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg
    11101 ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat
    11161 ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc
    11221 caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga
    11281 aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag
    11341 atactgcgac ctccctagca aactggggca caagcttaat tagtaactag cttgactgac
    11401 tgagatacag cgtaccttca gctcacagac atgataagat acattgatga gtttggacaa
    11461 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
    11521 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
    11581 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
    11641 tgtggtattg gcccatctct atcggtatcg tagcataacc ccttggggcc tctaaacggg
    11701 tcttgagggg ttttttgtgc ccctcgggcc ggattgctat ctaccggcat tggcgcagaa
    11761 aaaaatgcct gatgcgacgc tgcgcgtctt atactcccac atatgccaga ttcagcaacg
    11821 gatacggctt ccccaacttg cccacttcca tacgtgtcct ccttaccaga aatttatcct
    11881 taaggtcgtc agctatcctg caggcgatct ctcgatttcg atcaagacat tcctttaatg
    11941 gtcttttctg gacaccacta ggggtcagaa gtagttcatc aaactttctt ccctccctaa
    12001 tctcattggt taccttgggc tatcgaaact taattaacca gtcaagtcag ctacttggcg
    12061 agatcgactt gtctgggttt cgactacgct cagaattgcg tcagtcaagt tcgatctggt
    12121 ccttgctatt gcacccgttc tccgattacg agtttcattt aaatcatgtg agcaaaaggc
    12181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    12241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    12301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    12361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    12421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    12481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    12541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    12601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    12661 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    12721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    12781 cagcagatta cgcgcagaaa aaaaggatct caa
//
