LOCUS       sequence-354837-        9174 bp    DNA     circular SYN 15-APR-2025
DEFINITION  High-fidelity SpCas9-HF1 with 2A-Puro, and a golden gate cloning 
            backbone for sgRNA. sgRNA scaffold seqeuence has been modified for 
            increased sgRNA expression (v3)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9174)
  AUTHORS   Chey YCJ, Gierus L, Lushington C, Arudkumar JC, B Geiger A, Staker 
            LG, Robertson LJ, Pfitzner C, Kennedy JG, Lee RHB, Godahewa GI, 
            Adikusuma F, Thomas PQ
  TITLE     Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA 
            transcript levels through scaffold poly-T tract reduction.
  JOURNAL   BMC Genomics. 2025 Feb 12;26(1):138. doi: 
            10.1186/s12864-025-11317-2.
  PUBMED    39939860
REFERENCE   2  (bases 1 to 9174)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Genomics."; date: "2025-02-12"; volume: "26(1):138. doi"; pages: " 
            10.1186/s12864-025-11317-2"
COMMENT     Sequence Label: PX459v3-SpCas9-HF1
FEATURES             Location/Qualifiers
     source          1..9174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1370..2854,2855..2857,2858..3346,3347..3349,3350..3448,
                     3449..3451,3452..4141,4142..4144,4145..5470)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1370..5470
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5471..5518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5534..5587
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5588..6187
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5588..5607)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6084..6104
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6215..6232)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6221..6428
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6437..6577
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6437..6566
                     /label=AAV2 ITR
     rep_origin      6652..7107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6739..6758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6949..6970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7124..7143)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7243..7265
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7303..7321)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7389..7493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7494..7562,7563..8354)
                     /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(7712..7731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8525..9113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9014..9033
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt tcagagctag aaatagcaag ttgaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccrggs ggggsggggs ggggsgrggg gsggggsggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     1381 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2821 cgcttccgcc cagagcttca tcgagcggat gaccgccttc gataagaacc tgcccaacga
     2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcgcgc tgagccggaa
     3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     3421 cgacggcttc gccaacagaa acttcatggc gctgatccac gacgacagcc tgacctttaa
     3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     4141 ggcgatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga
     5521 attcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     5581 tggcccaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     5641 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     5701 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     5761 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     5821 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     5881 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     5941 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     6001 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     6061 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     6121 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     6181 tgcctgagaa ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     6241 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     6301 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     6361 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     6421 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     6481 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     6541 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     6601 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     6661 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     6721 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     6781 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     6841 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     6901 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6961 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     7021 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     7081 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     7141 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     7201 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     7261 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     7321 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     7381 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     7441 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     7501 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     7561 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     7621 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     7681 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     7741 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     7801 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     7861 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     7921 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     7981 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     8041 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     8101 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     8161 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     8221 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     8281 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     8341 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     8401 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     8461 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     8521 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     8581 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     8641 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     8701 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     8761 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     8821 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     8881 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     8941 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     9001 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     9061 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     9121 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
