LOCUS       sequence-272585-        9214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA_A to knockout human AMPK alpha 1 using Cas9n.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9214)
  AUTHORS   Toyama EQ, Herzig S, Courchet J, Lewis TL Jr, Loson OC, Hellberg K, 
            Young NP, Chen H, Polleux F, Chan DC, Shaw RJ
  TITLE     Metabolism. AMP-activated protein kinase mediates mitochondrial 
            fission in response to energy stress.
  JOURNAL   Science. 2016 Jan 15;351(6270):275-81. doi: 10.1126/science.aab4138.
  PUBMED    26816379
REFERENCE   2  (bases 1 to 9214)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2016-01-15- 15"; volume: "351"; 
            issue: "6270"; pages: "275-81"
COMMENT     Sequence Label: pX462-hPRKAA1-gRNA_A
FEATURES             Location/Qualifiers
     source          1..9214
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1045..1272
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1284..1293
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1293..1358
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1365..1385
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1410..1433,1434..1436,1437..5510)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1410..5510
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5511..5558
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5574..5627
                     /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             5628..6227
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5628..5647)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(6255..6272)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6261..6468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6477..6617
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6477..6606
                     /label=AAV2 ITR
     rep_origin      6692..7147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6779..6798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6989..7010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7164..7183)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7283..7305
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7343..7361)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7429..7533
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7534..7602,7603..8394)
                     /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(7752..7771)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8565..9153
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9054..9073
                     /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 gctgtcgcca tctttctccg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 ggggattttt taattatttt gtgcagcgat gggggcgggg ggggggggcg cgcgccaggc
      901 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
      961 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
     1021 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     1081 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     1141 agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt
     1201 aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc
     1261 actttttttc aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca
     1321 aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga
     1381 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggccatcg
     1441 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     1501 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     1561 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     1621 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     1681 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     1741 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     1801 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     1861 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     1921 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     1981 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     2041 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     2101 agaagaagaa tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact
     2161 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     2221 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     2281 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     2341 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     2401 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     2461 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     2521 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     2581 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     2641 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     2701 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     2761 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     2821 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     2881 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     2941 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     3001 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     3061 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     3121 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     3181 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     3241 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     3301 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     3361 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     3421 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     3481 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     3541 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     3601 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     3661 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     3721 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3781 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     3841 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3901 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     3961 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     4021 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     4081 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     4141 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     4201 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     4261 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     4321 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     4381 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     4441 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     4501 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     4561 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     4621 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     4681 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     4741 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     4801 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4861 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4921 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4981 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     5041 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     5101 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     5161 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     5221 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     5281 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     5341 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     5401 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     5461 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     5521 cggccacgaa aaaggccggc caggcaaaaa agaaaaagga attcggcagt ggagagggca
     5581 gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaatg accgagtaca
     5641 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     5701 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     5761 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     5821 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     5881 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     5941 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     6001 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     6061 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     6121 cccacaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     6181 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagaa ttctaactag
     6241 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     6301 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     6361 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     6421 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     6481 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     6541 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     6601 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     6661 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     6721 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     6781 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6841 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6901 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6961 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     7021 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     7081 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     7141 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     7201 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     7261 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     7321 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     7381 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     7441 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     7501 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     7561 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     7621 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     7681 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     7741 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     7801 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     7861 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     7921 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     7981 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     8041 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     8101 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     8161 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     8221 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     8281 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     8341 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     8401 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     8461 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     8521 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     8581 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     8641 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     8701 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     8761 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     8821 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     8881 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     8941 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     9001 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     9061 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     9121 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     9181 ttcctggcct tttgctggcc ttttgctcac atgt
//
