LOCUS       sequence-405301-       10089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid of enhanced activity FnCas9 variant 
            en1FnCas9 with T2A-EGFP and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10089)
  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 10089)
  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-en1FnCas9
FEATURES             Location/Qualifiers
     source          1..10089
                     /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..340
                     /label=Fn gRNA scaffold
                     /note="guide RNA scaffold for the Francisella novicida 
                     CRISPR/Cas9 system (Hirano et al., 2016)"
     enhancer        437..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        724..1001
                     /label=chicken beta-actin promoter
     intron          1002..1229
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1241..1250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1256..1336
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1343..1363
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1388..6271
                     /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
                     YNTALGYKKYSKEMEFLRSLAYRSRRVKIKSIDDVKQVLDKDSNFIIGKITLPFKKEWQ
                     RLYREWQNTTIKDDYEFLKSFFNVKSITKLHKKVRKDFSLPISTNEGKFLVKRKTWDNN
                     FIYQILNDSDSRADGTKPFIPAFDISKNEIVEAIIDSFTSKNIFWLPKNIELQKVDNKN
                     IFAIDTSKWFEVETPSDLRDIGIATIQYKIDNNSRPKVRVKLDYVIDDDSKINYFMNHS
                     LLKSRYPDKVLEILKQSTIIEFESSGFNKTIKEMLGMKLAGIYNETSNN"
     CDS             6272..6319
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6335..6388
                     /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(6389..6391,6392..7102)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6389..6391,6392..7102)
                     /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(6431..6452)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6692..6711)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7039..7060
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7130..7147)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7136..7343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7352..7492
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7352..7481
                     /label=AAV2 ITR
     rep_origin      7567..8022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7654..7673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7864..7885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8039..8058)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8158..8180
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8218..8236)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8304..8408
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8409..8477,8478..9269)
                     /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(8627..8646)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9440..10028
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9929..9948
                     /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 tcagttgcgc cgaaaggcgc tctgtaatca
      301 tttaaaagta ttttgaacgg acctctgttt gacacgtctg ttttttgttt tagagctaga
      361 aatagcaagt taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa
      421 tggctctaga ggtacccgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      481 acgacccccg cccattgacg tcaatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattgt gcccagtaca
      661 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc
      781 caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg
      841 gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag
      901 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
      961 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     1021 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     1081 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga
     1141 gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg
     1201 agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg gatcctaccc
     1261 atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt acccatacga
     1321 tgttccagat tacgctatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     1381 agcagccaat tttaagatcc tgcctatcgc aatcgacctg ggcgtcaaga atactggcgt
     1441 gtttagtgct ttttatcaga aggggacctc actggagaga ctggacaata agaacggaaa
     1501 agtgtatgaa ctgtccaagg attcttacac tctgctgatg aacaatagga ccgcacggag
     1561 acaccagagg cgaggaattg acaggaaaca gctggtgaag cgcctgttca aactgatctg
     1621 gacagagcag ctgaacctgg aatgggataa ggacactcag caggccatca gcttcctgtt
     1681 taatcgacgg ggattctctt ttattactga cggctatagt cctgagtacc tgaacatcgt
     1741 gccagaacag gtcaaggcaa tcctgatgga cattttcgac gattataatg gcgaggacga
     1801 tctggattcc tacctgaaac tggccacaga gcaagagagt aagatcagcg aaatctacaa
     1861 caagctgatg cagaagatcc tggagttcaa gctgatgaaa ctgtgcaccg acatcaagga
     1921 cgataaagtg agtaccaaga cactgaaaga gatcacaagc tacgagttcg aactgctggc
     1981 cgattatctg gctaactaca gcgaatccct gaaaacccag aaattttcct acacagacaa
     2041 gcagggcaat ctgaaagagc tgtcttacta ccaccatgat aagtacaaca tccaggagtt
     2101 cctgaagaga cacgccacca tcaatgacag gattctggat acactgctga ctgacgatct
     2161 ggacatctgg aacttcaact tcgagaagtt cgatttcgac aagaacgagg aaaaactgca
     2221 gaatcaggaa gataaggacc acattcaggc tcatctgcac catttcgtgt ttgcagtcaa
     2281 taagatcaaa agcgagatgg catccggcgg gcgccatcga agccagtact tccaggaaat
     2341 caccaacgtg ctggacgaga acaatcacca ggaaggctac ctgaaaaact tctgtgagaa
     2401 tctgcataac aagaagtaca gcaatctgtc cgtgaagaat ctggtcaacc tgattggaaa
     2461 tctgtccaac ctggaactga agcccctgcg caaatacttc aacgacaaga tccacgctaa
     2521 agcagaccat tgggatgagc agaagtttac tgaaacctat tgccactgga ttctgggcga
     2581 gtggcgggtg ggggtcaagg atcaggacaa gaaagacggc gcaaagtatt cttacaagga
     2641 cctgtgtaac gagctgaagc agaaagtgac taaggccggg ctggtggact tcctgctgga
     2701 gctggacccc tgccgaacca ttccacctta cctggacaac aataacagaa agccacccaa
     2761 atgtcagagc ctgatcctga atcccaagtt tctggataat cagtatccta actggcagca
     2821 gtacctgcag gagctgaaga aactgcagtc aatccagaac tacctggaca gcttcgaaac
     2881 cgatctgaag gtgctgaaaa gctccaagga ccagccttac ttcgtcgagt acaagtctag
     2941 taaccagcag atcgcttccg gccagcggga ttacaaggat ctggacgcaa gaatcctgca
     3001 gttcattttt gacagggtga aggcctctga tgagctgctg ctgaacgaaa tctatttcca
     3061 ggcaaagaaa ctgaagcaga aagcctcaag cgagctggaa aagctggagt cctctaagaa
     3121 actggacgaa gtgatcgcta actctcagct gagtcagatt ctgaagtctc agcacacaaa
     3181 tggaatcttc gagcagggca cttttctgca tctggtgtgc aaatactata agcagcgaca
     3241 gagagccagg gacagccgcc tgtacatcat gcctgaatat cgatacgata agaaactgca
     3301 caagtacaac aacaccggcc gctttgacga tgacaaccag ctgctgacat attgtaatca
     3361 taagccccgg cagaaaagat accagctgct gaacgacctg gcaggagtgc tgcaggtctc
     3421 tcctaatttt ctgaaggata aaatcgggtc cgatgacgat ctgttcattt ctaagtggct
     3481 ggtggagcac atccggggct ttaagaaggc ctgcgaagat agcctgaaaa tccagaagga
     3541 taacagggga ctgctgaatc ataagatcaa cattgcacgc aataccaagg gcaaatgcga
     3601 gaaagaaatc ttcaacctga tctgtaagat tgaggggagc gaagataaga aagggaatta
     3661 taagcacgga ctggcctacg agctgggagt gctgctgttc ggagagccaa acgaggccag
     3721 caagcccgaa tttgatagga aaatcaagaa attcaattca atctacagct ttgcccagat
     3781 ccagcagatt gcctttgctg agaggaaggg gaatgcaaac acatgcgccg tgtgtagtgc
     3841 agacaacgcc catcgcatgc agcagatcaa aattactgag ccagtcgaag ataataagga
     3901 taaaatcatt ctgtcagcaa aggcacagcg actgcctgca atcccaaccc gaattgtgga
     3961 tggagctgtc aagaaaatgg ctacaattct ggcaaagaat atcgtggacg ataattggca
     4021 gaacattaag caggtcctga gcgcaaaaca ccagctgcat atcccaatca ttaccgagtc
     4081 caacgccttc gagtttgaac ccgctctggc agacgtgaag ggcaaatctc tgaaggatag
     4141 aaggaagaaa gccctggagc gaattagtcc cgaaaacatc ttcaaggata agaacaacag
     4201 aatcaaggag tttgctaagg ggatttccgc ctactctgga gctaacctga cagatgggga
     4261 cttcgatgga gcaaaggagg aactggatca catcattcct cgcagccata agaaatatgg
     4321 cactctgaac gacgaggcta atctgatttg cgtgacccgg ggcgataata agaacaaagg
     4381 gaaccggatc ttctgtctga gagacctggc cgataattac aagctgaaac agtttgagac
     4441 cacagacgat ctggagatcg aaaagaaaat tgccgacacc atctgggatg ctaataagaa
     4501 ggacttcaag ttcggaaact atcggagctt catcaatctg acacctcagg agcagaaagc
     4561 attcagacac gccctgtttc tggctgatga aaacccaatc aagcaggcag tgatcagagc
     4621 cattaataac cgcaaccgaa ccttcgtgaa tggcacacag aggtattttg ctgaggtcct
     4681 ggcaaataac atctacctgc gcgccaagaa agaaaatctg aacactgaca agatcagctt
     4741 cgattacttt ggaatcccta ccattggaaa cggccgaggg atcgctgaga ttcggcagct
     4801 gtatgaaaag gtggacagtg atatccaggc ctacgctaaa ggcgacaagc cacaggcctc
     4861 ttatagtcac ctgattgatg ctatgctggc attctgcatc gccgctgacg agcatcggaa
     4921 cgatggatct attggcctgg aaatcgacaa aaactatagt ctgtaccctc tggataagaa
     4981 tactggcgag gtgttcacca aagacatctt ttcacagatc aagattaccg acaacgagtt
     5041 cagcgataag aaactggtca gaaagaaagc tattgaaggg tttaacacac acagacagat
     5101 gactagggat ggaatctatg cagagaatta cctgcctatc ctgattcata aggagctgaa
     5161 cgaagtgagg aaggggtaca catggaaaaa ttccgaggaa atcaaaattt tcaagggaaa
     5221 gaaatacgac atccagcagc tgaataacct ggtgtattgt ctgaagtttg tggacaaacc
     5281 aatcagtatt gatatccaga tttcaaccct ggaggaactg agaaacatcc tgactaccaa
     5341 taacattgca gccactgccg agtactatta cattaatctg aaaacccaga agctgcacga
     5401 gtattacatc gaaaattaca acacagccct ggggtataag aaatacagca aggagatgga
     5461 gttcctgagg tccctggctt ataggtctag gcgcgtgaag atcaaaagta ttgacgatgt
     5521 caagcaggtc ctggacaagg attcaaactt catcatcgga aagatcacac tgcccttcaa
     5581 gaaagagtgg cagcgactgt accgggaatg gcagaacaca actatcaaag acgattatga
     5641 gtttctgaag agcttcttta atgtgaagtc cattactaaa ctgcacaaga aagtccggaa
     5701 agacttctct ctgcccatca gtacaaacga gggcaagttt ctggtgaaga gaaaaacttg
     5761 ggataataac ttcatctacc agattctgaa tgactcagat agcagggcag acgggactaa
     5821 acccttcatt cctgcctttg atatcagcaa gaacgagatt gtggaagcca tcattgacag
     5881 tttcacctca aaaaacatct tttggctgcc aaagaatatt gagctgcaga aggtggacaa
     5941 caagaacatc ttcgccattg ataccagcaa gtggtttgag gtcgaaacac catccgacct
     6001 gcgcgatatc ggcattgcta ccattcagta caagatcgac aataactcac gccccaaggt
     6061 gcgagtcaaa ctggattacg tgatcgacga tgacagcaag attaactatt tcatgaatca
     6121 ctcactgctg aagagccggt atcccgacaa agtcctggag atcctgaagc agagcacaat
     6181 cattgagttc gaaagttcag ggtttaacaa aactattaag gagatgctgg gaatgaagct
     6241 ggccggcatc tacaatgaaa cctccaataa caaaaggccg gcggccacga aaaaggccgg
     6301 ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg
     6361 cggtgacgtc gaggagaatc ctggcccagt gagcaagggc gaggagctgt tcaccggggt
     6421 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     6481 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     6541 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     6601 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     6661 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     6721 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     6781 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     6841 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     6901 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     6961 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     7021 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     7081 cggcatggac gagctgtaca aggaattcta actagagctc gctgatcagc ctcgactgtg
     7141 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     7201 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     7261 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     7321 gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact
     7381 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     7441 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     7501 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     7561 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     7621 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     7681 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     7741 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     7801 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     7861 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     7921 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     7981 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     8041 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     8101 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     8161 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     8221 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     8281 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     8341 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     8401 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     8461 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     8521 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     8581 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     8641 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     8701 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     8761 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     8821 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     8881 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     8941 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     9001 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     9061 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     9121 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     9181 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     9241 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     9301 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     9361 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     9421 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     9481 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     9541 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     9601 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     9661 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     9721 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     9781 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     9841 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     9901 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     9961 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
    10021 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
    10081 ctcacatgt
//
