LOCUS       sequence-496516-       12557 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Overexpression of SpCas9-BFP with HBB IVS2 intron within BFP coding 
            sequence and blasticidin resistance gene. Contains Cp36 
            recombination site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12557)
  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 12557)
  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: pSA074 Cas9-BFP/HBB_IVS2/BFP
FEATURES             Location/Qualifiers
     source          1..12557
                     /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      5337..5346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5343..9446
                     /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             5343..9446
                     /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             9450..9476
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             9456..9482
                     /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             9495..9515
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9522..9542
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    11179..11300
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11190..11209)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     11244..11263
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      11415..11461
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      11491..11627
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    11672..11743
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(11969..12557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12049..12068)
                     /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 gtgttgccct 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 cggtaccgcg ggcccggcca
     5341 ccatggacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg
     5401 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     5461 ggcacagcat caagaagaac ctgatcggcg ccctgctgtt cgacagcgga gaaacagccg
     5521 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     5581 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     5641 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     5701 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     5761 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     5821 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     5881 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     5941 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     6001 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca
     6061 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     6121 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     6181 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     6241 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     6301 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     6361 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     6421 ccggctacat cgatggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     6481 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     6541 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     6601 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     6661 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     6721 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     6781 aagtggtgga caagggcgcc agcgcccaga gcttcatcga gcggatgacc aacttcgata
     6841 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     6901 tgtacaacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     6961 tgagcggcga gcagaaaaaa gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     7021 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     7081 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     7141 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     7201 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     7261 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     7321 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     7381 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     7441 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     7501 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     7561 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     7621 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     7681 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     7741 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     7801 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     7861 atatcgtgcc tcagagcttt ctgaaggacg actccatcga taacaaagtg ctgactcgga
     7921 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     7981 agaactactg gcgccagctg ctgaatgcca agctgattac ccagaggaag ttcgacaatc
     8041 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     8101 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     8161 acactaagta cgacgagaac gacaaactga tccgggaagt gaaagtgatc accctgaagt
     8221 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     8281 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     8341 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
     8401 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
     8461 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
     8521 ggcctctgat cgagacaaac ggcgaaacag gcgagatcgt gtgggataag ggccgggact
     8581 ttgccaccgt gcggaaagtg ctgtctatgc cccaagtgaa tatcgtgaaa aagaccgagg
     8641 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gacaagctga
     8701 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
     8761 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
     8821 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
     8881 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
     8941 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
     9001 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
     9061 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
     9121 aacagcacaa acactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
     9181 tgatcctggc cgacgctaat ctggacaagg tgctgagcgc ctacaacaag cacagagaca
     9241 agcctatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
     9301 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
     9361 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
     9421 tcgacctgtc tcagctggga ggcgacgcct atccctatga cgtgcccgat tatgccagcc
     9481 tgggcagcgg ctcccccaag aaaaaacgca aggtggaaga tcctaagaaa aagcggaaag
     9541 tggacggcat tggtagtggg agcaacggca gcagcggatc cagcgagctg attaaggaga
     9601 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     9661 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     9721 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     9781 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc ccgtgagtct
     9841 atgggacgct tgatgttttc tttccccttc ttttctatgg ttaagttcat gtcataggaa
     9901 ggggataagt aacagggtac agtttagaat gggaaacaga cgaatgattg catcagtgtg
     9961 gaagtctcag gatcgtttta gtttctttta tttgctgttc ataacaattg ttttcttttg
    10021 tttaattctt gctttctttt tttttcttct ccgcaatttt tactattata cttaatgcct
    10081 taacattgtg tataacaaaa ggaaatatct ctgagataca ttaagtaact taaaaaaaaa
    10141 ctttacacag tctgcctagt acattactat ttggaatata tgtgtgctta tttgcatatt
    10201 cataatctcc ctactttatt ttcttttatt tttaattgat acataatcat tatacatatt
    10261 tatgggttaa agtgtaatgt tttaatatgt gtacacatat tgaccaaatc agggtaattt
    10321 tgcatttgta attttaaaaa atgctttctt cttttaatat acttttttgt ttatcttatt
    10381 tctaatactt tccctaatct ctttctttca gggcaataat gatacaatgt atcatgcctc
    10441 tttgcaccat tctaaagaat aacagtgata atttctgggt taaggcaata gcaatatctc
    10501 tgcatataaa tatttctgca tataaattgt aactgatgta agaggtttca tattgctaat
    10561 agcagctaca atccagctac cattctgctt ttattttatg gttgggataa ggctggatta
    10621 ttctgagtcc aagctaggcc cttttgctaa tcatgttcat acctcttatc ttcctcccac
    10681 agtgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
    10741 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
    10801 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
    10861 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
    10921 gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct
    10981 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
    11041 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
    11101 cctagcaaac tggggcacaa gcttaattag ctagcttgac tgactgagat acagcgtacc
    11161 ttcagctcac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
    11221 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
    11281 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
    11341 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt attggcccat
    11401 ctctatcggt atcgtagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
    11461 gtgcccctcg ggccggattg ctatctaccg gcattggcgc agaaaaaaat gcctgatgcg
    11521 acgctgcgcg tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa
    11581 cttgcccact tccatacgtg tcctccttac cagaaattta tccttaaggt cgtcagctat
    11641 cctgcaggcg atctctcgat ttcgatcaag acattccttt aatggtcttt tctggacacc
    11701 actaggggtc agaagtagtt catcaaactt tcttccctcc ctaatctcat tggttacctt
    11761 gggctatcga aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg
    11821 gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc
    11881 gttctccgat tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga
    11941 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
    12001 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
    12061 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
    12121 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
    12181 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
    12241 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
    12301 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
    12361 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
    12421 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
    12481 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
    12541 gaaaaaaagg atctcaa
//
