LOCUS       sequence-405302-       10754 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid of enhanced activity FnCas9 variant 
            en15FnCas9 with T2A-EGFP and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10754)
  AUTHORS   Acharya S, Ansari AH, Kumar Das P, Hirano S, Aich M, Rauthan R, 
            Mahato S, Maddileti S, Sarkar S, Kumar M, Phutela R, Gulati S, 
            Rahman A, Goel A, Afzal C, Paul D, Agrawal T, Pulimamidi VK, Jalali 
            S, Nishimasu H, Mariappan I, Nureki O, Maiti S, Chakraborty D
  TITLE     PAM-flexible Engineered FnCas9 variants for robust and ultra-precise
            genome editing and diagnostics.
  JOURNAL   Nat Commun. 2024 Jun 28;15(1):5471. doi: 10.1038/s41467-024-49233-w.
  PUBMED    38942756
REFERENCE   2  (bases 1 to 10754)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-06-28"; volume: "15(1):5471. doi"; pages: " 
            10.1038/s41467-024-49233-w"
COMMENT     Sequence Label: PX458-3xHA-en15FnCas9
FEATURES             Location/Qualifiers
     source          1..10754
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..543
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        562..634
                     /label=Fn gRNA scaffold
                     /note="guide RNA scaffold for the Francisella novicida 
                     CRISPR/Cas9 system (Hirano et al., 2016)"
     enhancer        731..1016
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1018..1295
                     /label=chicken beta-actin promoter
     intron          1296..1523
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1535..1544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1550..1630
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1637..1657
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1682..6565
                     /codon_start=1
                     /product="Cas9 endonuclease from the Francisella novicida 
                     Type II CRISPR/Cas system (Hirano et al., 2016)"
                     /label=FnCas9
                     /translation="NFKILPIAIDLGVKNTGVFSAFYQKGTSLERLDNKNGKVYELSKD
                     SYTLLMNNRTARRHQRRGIDRKQLVKRLFKLIWTEQLNLEWDKDTQQAISFLFNRRGFS
                     FITDGYSPEYLNIVPEQVKAILMDIFDDYNGEDDLDSYLKLATEQESKISEIYNKLMQK
                     ILEFKLMKLCTDIKDDKVSTKTLKEITSYEFELLADYLANYSESLKTQKFSYTDKQGNL
                     KELSYYHHDKYNIQEFLKRHATINDRILDTLLTDDLDIWNFNFEKFDFDKNEEKLQNQE
                     DKDHIQAHLHHFVFAVNKIKSEMASGGRHRSQYFQEITNVLDENNHQEGYLKNFCENLH
                     NKKYSNLSVKNLVNLIGNLSNLELKPLRKYFNDKIHAKADHWDEQKFTETYCHWILGEW
                     RVGVKDQDKKDGAKYSYKDLCNELKQKVTKAGLVDFLLELDPCRTIPPYLDNNNRKPPK
                     CQSLILNPKFLDNQYPNWQQYLQELKKLQSIQNYLDSFETDLKVLKSSKDQPYFVEYKS
                     SNQQIASGQRDYKDLDARILQFIFDRVKASDELLLNEIYFQAKKLKQKASSELEKLESS
                     KKLDEVIANSQLSQILKSQHTNGIFEQGTFLHLVCKYYKQRQRARDSRLYIMPEYRYDK
                     KLHKYNNTGRFDDDNQLLTYCNHKPRQKRYQLLNDLAGVLQVSPNFLKDKIGSDDDLFI
                     SKWLVEHIRGFKKACEDSLKIQKDNRGLLNHKINIARNTKGKCEKEIFNLICKIEGSED
                     KKGNYKHGLAYELGVLLFGEPNEASKPEFDRKIKKFNSIYSFAQIQQIAFAERKGNANT
                     CAVCSADNAHRMQQIKITEPVEDNKDKIILSAKAQRLPAIPTRIVDGAVKKMATILAKN
                     IVDDNWQNIKQVLSAKHQLHIPIITESNAFEFEPALADVKGKSLKDRRKKALERISPEN
                     IFKDKNNRIKEFAKGISAYSGANLTDGDFDGAKEELDHIIPRSHKKYGTLNDEANLICV
                     TRGDNKNKGNRIFCLRDLADNYKLKQFETTDDLEIEKKIADTIWDANKKDFKFGNYRSF
                     INLTPQEQKAFRHALFLADENPIKQAVIRAINNRNRTFVNGTQRYFAEVLANNIYLRAK
                     KENLNTDKISFDYFGIPTIGNGRGIAEIRQLYEKVDSDIQAYAKGDKPQASYSHLIDAM
                     LAFCIAADEHRNDGSIGLEIDKNYSLYPLDKNTGEVFTKDIFSQIKITDNEFSDKKLVR
                     KKAIEGFNTHRQMTRDGIYAENYLPILIHKELNEVRKGYTWKNSEEIKIFKGKKYDIQQ
                     LNNLVYCLKFVDKPISIDIQISTLEELRNILTTNNIAATAEYYYINLKTQKLHEYYIEN
                     YNTALGYKKYSKEMEFLRSLAYRSERVKIKSIDDVKQVLDKDSNFIIGKITLPFKKEWQ
                     RLYREWQNTTIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEGKFLVKRKTWDNN
                     FIYQILNDSDSRADGTKPFIPAFDISKNEIVEAIIDSFTSKNIFWLPKNIELQKVDNKN
                     IFAIDTSKWFEVETPSDLRDIGIATIQYKIDNNSRPKVRVKLDYVIDDDSKINYFMNHS
                     LLKSRYPDKVLEILKQSTIIEFHSSGFNKTIKEMLGMKLAGIYNETSNN"
     CDS             6566..6613
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6629..6682
                     /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             join(6683..6685,6686..7396)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6683..6685,6686..7396)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6725..6746)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6986..7005)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7333..7354
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7424..7441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7430..7637
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7646..7786
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7646..7775
                     /label=AAV2 ITR
     rep_origin      7861..8316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7948..7967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8158..8179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8333..8352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8452..8474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8512..8530)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8598..8702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8703..8771,8772..9563)
                     /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(8921..8940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9734..10322
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
       61 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      121 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      181 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      241 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtgagggc
      301 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      361 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      421 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      481 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
      541 accgggtctt cgagaagacc tgtttcagtt gcgccgaaag gcgctctgta atcatttaaa
      601 agtattttga acggacctct gtttgacacg tctgtttttt gttttagagc tagaaatagc
      661 aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga caaatggctc
      721 tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      781 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      841 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      901 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
      961 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1021 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1081 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc
     1141 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
     1201 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     1261 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     1321 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     1381 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
     1441 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
     1501 tgcctgaaat cacttttttt caggttggac cggtgccacc atgggatcct acccatacga
     1561 tgttccagat tacgcttacc catacgatgt tccagattac gcttacccat acgatgttcc
     1621 agattacgct atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
     1681 caattttaag atcctgccta tcgcaatcga cctgggcgtc aagaatactg gcgtgtttag
     1741 tgctttttat cagaagggga cctcactgga gagactggac aataagaacg gaaaagtgta
     1801 tgaactgtcc aaggattctt acactctgct gatgaacaat aggaccgcac ggagacacca
     1861 gaggcgagga attgacagga aacagctggt gaagcgcctg ttcaaactga tctggacaga
     1921 gcagctgaac ctggaatggg ataaggacac tcagcaggcc atcagcttcc tgtttaatcg
     1981 acggggattc tcttttatta ctgacggcta tagtcctgag tacctgaaca tcgtgccaga
     2041 acaggtcaag gcaatcctga tggacatttt cgacgattat aatggcgagg acgatctgga
     2101 ttcctacctg aaactggcca cagagcaaga gagtaagatc agcgaaatct acaacaagct
     2161 gatgcagaag atcctggagt tcaagctgat gaaactgtgc accgacatca aggacgataa
     2221 agtgagtacc aagacactga aagagatcac aagctacgag ttcgaactgc tggccgatta
     2281 tctggctaac tacagcgaat ccctgaaaac ccagaaattt tcctacacag acaagcaggg
     2341 caatctgaaa gagctgtctt actaccacca tgataagtac aacatccagg agttcctgaa
     2401 gagacacgcc accatcaatg acaggattct ggatacactg ctgactgacg atctggacat
     2461 ctggaacttc aacttcgaga agttcgattt cgacaagaac gaggaaaaac tgcagaatca
     2521 ggaagataag gaccacattc aggctcatct gcaccatttc gtgtttgcag tcaataagat
     2581 caaaagcgag atggcatccg gcgggcgcca tcgaagccag tacttccagg aaatcaccaa
     2641 cgtgctggac gagaacaatc accaggaagg ctacctgaaa aacttctgtg agaatctgca
     2701 taacaagaag tacagcaatc tgtccgtgaa gaatctggtc aacctgattg gaaatctgtc
     2761 caacctggaa ctgaagcccc tgcgcaaata cttcaacgac aagatccacg ctaaagcaga
     2821 ccattgggat gagcagaagt ttactgaaac ctattgccac tggattctgg gcgagtggcg
     2881 ggtgggggtc aaggatcagg acaagaaaga cggcgcaaag tattcttaca aggacctgtg
     2941 taacgagctg aagcagaaag tgactaaggc cgggctggtg gacttcctgc tggagctgga
     3001 cccctgccga accattccac cttacctgga caacaataac agaaagccac ccaaatgtca
     3061 gagcctgatc ctgaatccca agtttctgga taatcagtat cctaactggc agcagtacct
     3121 gcaggagctg aagaaactgc agtcaatcca gaactacctg gacagcttcg aaaccgatct
     3181 gaaggtgctg aaaagctcca aggaccagcc ttacttcgtc gagtacaagt ctagtaacca
     3241 gcagatcgct tccggccagc gggattacaa ggatctggac gcaagaatcc tgcagttcat
     3301 ttttgacagg gtgaaggcct ctgatgagct gctgctgaac gaaatctatt tccaggcaaa
     3361 gaaactgaag cagaaagcct caagcgagct ggaaaagctg gagtcctcta agaaactgga
     3421 cgaagtgatc gctaactctc agctgagtca gattctgaag tctcagcaca caaatggaat
     3481 cttcgagcag ggcacttttc tgcatctggt gtgcaaatac tataagcagc gacagagagc
     3541 cagggacagc cgcctgtaca tcatgcctga atatcgatac gataagaaac tgcacaagta
     3601 caacaacacc ggccgctttg acgatgacaa ccagctgctg acatattgta atcataagcc
     3661 ccggcagaaa agataccagc tgctgaacga cctggcagga gtgctgcagg tctctcctaa
     3721 ttttctgaag gataaaatcg ggtccgatga cgatctgttc atttctaagt ggctggtgga
     3781 gcacatccgg ggctttaaga aggcctgcga agatagcctg aaaatccaga aggataacag
     3841 gggactgctg aatcataaga tcaacattgc acgcaatacc aagggcaaat gcgagaaaga
     3901 aatcttcaac ctgatctgta agattgaggg gagcgaagat aagaaaggga attataagca
     3961 cggactggcc tacgagctgg gagtgctgct gttcggagag ccaaacgagg ccagcaagcc
     4021 cgaatttgat aggaaaatca agaaattcaa ttcaatctac agctttgccc agatccagca
     4081 gattgccttt gctgagagga aggggaatgc aaacacatgc gccgtgtgta gtgcagacaa
     4141 cgcccatcgc atgcagcaga tcaaaattac tgagccagtc gaagataata aggataaaat
     4201 cattctgtca gcaaaggcac agcgactgcc tgcaatccca acccgaattg tggatggagc
     4261 tgtcaagaaa atggctacaa ttctggcaaa gaatatcgtg gacgataatt ggcagaacat
     4321 taagcaggtc ctgagcgcaa aacaccagct gcatatccca atcattaccg agtccaacgc
     4381 cttcgagttt gaacccgctc tggcagacgt gaagggcaaa tctctgaagg atagaaggaa
     4441 gaaagccctg gagcgaatta gtcccgaaaa catcttcaag gataagaaca acagaatcaa
     4501 ggagtttgct aaggggattt ccgcctactc tggagctaac ctgacagatg gggacttcga
     4561 tggagcaaag gaggaactgg atcacatcat tcctcgcagc cataagaaat atggcactct
     4621 gaacgacgag gctaatctga tttgcgtgac ccggggcgat aataagaaca aagggaaccg
     4681 gatcttctgt ctgagagacc tggccgataa ttacaagctg aaacagtttg agaccacaga
     4741 cgatctggag atcgaaaaga aaattgccga caccatctgg gatgctaata agaaggactt
     4801 caagttcgga aactatcgga gcttcatcaa tctgacacct caggagcaga aagcattcag
     4861 acacgccctg tttctggctg atgaaaaccc aatcaagcag gcagtgatca gagccattaa
     4921 taaccgcaac cgaaccttcg tgaatggcac acagaggtat tttgctgagg tcctggcaaa
     4981 taacatctac ctgcgcgcca agaaagaaaa tctgaacact gacaagatca gcttcgatta
     5041 ctttggaatc cctaccattg gaaacggccg agggatcgct gagattcggc agctgtatga
     5101 aaaggtggac agtgatatcc aggcctacgc taaaggcgac aagccacagg cctcttatag
     5161 tcacctgatt gatgctatgc tggcattctg catcgccgct gacgagcatc ggaacgatgg
     5221 atctattggc ctggaaatcg acaaaaacta tagtctgtac cctctggata agaatactgg
     5281 cgaggtgttc accaaagaca tcttttcaca gatcaagatt accgacaacg agttcagcga
     5341 taagaaactg gtcagaaaga aagctattga agggtttaac acacacagac agatgactag
     5401 ggatggaatc tatgcagaga attacctgcc tatcctgatt cataaggagc tgaacgaagt
     5461 gaggaagggg tacacatgga aaaattccga ggaaatcaaa attttcaagg gaaagaaata
     5521 cgacatccag cagctgaata acctggtgta ttgtctgaag tttgtggaca aaccaatcag
     5581 tattgatatc cagatttcaa ccctggagga actgagaaac atcctgacta ccaataacat
     5641 tgcagccact gccgagtact attacattaa tctgaaaacc cagaagctgc acgagtatta
     5701 catcgaaaat tacaacacag ccctggggta taagaaatac agcaaggaga tggagttcct
     5761 gaggtccctg gcttataggt ctgagcgcgt gaagatcaaa agtattgacg atgtcaagca
     5821 ggtcctggac aaggattcaa acttcatcat cggaaagatc acactgccct tcaagaaaga
     5881 gtggcagcga ctgtaccggg aatggcagaa cacaactatc aaagacgatt atgagtttct
     5941 gaagagcttc tttaatgtga agtccattac taaactgcac aagaaagtcc ggaaagactt
     6001 ctctctgccc atcagtacaa acgagggcaa gtttctggtg aagagaaaaa cttgggataa
     6061 taacttcatc taccagattc tgaatgactc agatagcagg gcagacggga ctaaaccctt
     6121 cattcctgcc tttgatatca gcaagaacga gattgtggaa gccatcattg acagtttcac
     6181 ctcaaaaaac atcttttggc tgccaaagaa tattgagctg cagaaggtgg acaacaagaa
     6241 catcttcgcc attgatacca gcaagtggtt tgaggtcgaa acaccatccg acctgcgcga
     6301 tatcggcatt gctaccattc agtacaagat cgacaataac tcacgcccca aggtgcgagt
     6361 caaactggat tacgtgatcg acgatgacag caagattaac tatttcatga atcactcact
     6421 gctgaagagc cggtatcccg acaaagtcct ggagatcctg aagcagagca caatcattga
     6481 gttccacagt tcagggttta acaaaactat taaggagatg ctgggaatga agctggccgg
     6541 catctacaat gaaacctcca ataacaaaag gccggcggcc acgaaaaagg ccggccaggc
     6601 aaaaaagaaa aaggaattcg gcagtggaga gggcagagga agtctgctaa catgcggtga
     6661 cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     6721 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     6781 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     6841 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     6901 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     6961 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     7021 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     7081 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     7141 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     7201 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     7261 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     7321 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     7381 ggacgagctg tacaaggaat tctaactaga gctcgctgat cagcctcgac tgtgccttct
     7441 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     7501 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     7561 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     7621 agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     7681 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     7741 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     7801 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     7861 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     7921 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     7981 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     8041 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     8101 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     8161 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     8221 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     8281 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     8341 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     8401 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     8461 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     8521 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     8581 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     8641 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     8701 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     8761 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     8821 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     8881 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     8941 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     9001 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     9061 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     9121 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     9181 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     9241 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     9301 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     9361 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     9421 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     9481 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     9541 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     9601 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     9661 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     9721 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     9781 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     9841 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     9901 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     9961 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
    10021 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
    10081 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
    10141 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
    10201 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
    10261 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
    10321 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
    10381 tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
    10441 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
    10501 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
    10561 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat ctgsgkgmgw
    10621 gcacgaavya mggswscggg kmtttaatcw ytatgcttag agctcgtata ttaactgcaa
    10681 ktgtgmaagg ataayaaaac ygcaagagrg wctrctgtga mcatggktac scgargctct
    10741 aggtmgaggk sgac
//
