LOCUS       sequence-446879-        9178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 and gRNA for disruption of Rab7A gene in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9178)
  AUTHORS   Talaia G, Bentley-DeSousa A, Ferguson SM
  TITLE     Lysosomal TBK1 responds to amino acid availability to relieve 
            Rab7-dependent mTORC1 inhibition.
  JOURNAL   EMBO J. 2024 Sep;43(18):3948-3967. doi: 10.1038/s44318-024-00180-8. 
            Epub 2024 Aug 5.
  PUBMED    39103493
REFERENCE   2  (bases 1 to 9178)
  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: 
            "10.1038/s44318-024-00180-8"; journalName: "EMBO J"; date: 
            "2024-09"; volume: "43"; issue: "18"; pages: "3948-3967"
COMMENT     Sequence Label: pSpCas9-2A-Puro-RAB7A-gRNA1 (PX459)
FEATURES             Location/Qualifiers
     source          1..9178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     490..510
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(621..638)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    627..834
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   843..983
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   843..972
                     /label=AAV2 ITR
     rep_origin      1058..1513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1145..1164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1355..1376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1530..1549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1649..1671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1709..1727)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1795..1899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1900..1968,1969..2760)
                     /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(2118..2137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2931..3519
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3420..3439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3581..3829
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3581..3601
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3752..3771
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3851..3926
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        4023..4308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4589..4816
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      4828..4837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4837..4902
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4909..4929
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4954..9054
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4954..9054
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             9055..9102
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9118..9171
                     /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(9172..9178,1..593)
                     /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(join(9172..9178,1..13))
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
       61 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
      121 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
      181 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
      241 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
      301 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
      361 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg
      421 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
      481 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
      541 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgagaattct
      601 aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
      661 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
      721 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
      781 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
      841 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
      901 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
      961 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     1021 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     1081 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     1141 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     1201 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1261 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1321 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1381 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     1441 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1501 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     1561 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     1621 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     1681 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     1741 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     1801 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     1861 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     1921 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     1981 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2041 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2101 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2161 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2221 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2281 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2341 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2401 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     2461 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2521 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2581 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     2641 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     2701 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     2761 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     2821 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     2881 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     2941 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3001 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3061 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3121 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3181 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3241 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3301 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3361 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3421 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3481 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3541 ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat
     3601 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     3661 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     3721 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     3781 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtcatccacc
     3841 atcacctcct gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     3901 ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa
     3961 ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc tctagaggta
     4021 cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     4081 ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     4141 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     4201 gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac
     4261 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc
     4321 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
     4381 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg cgcgcgccag
     4441 gcggggcggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggcgc
     4501 cagggggggg gggggggggc gcgggcctct ttttctggcc ggggggcggg gcggggcccc
     4561 ccctacaaca cacagcgccc ggggcggggg agtcgctgcg cgctgccttc gccccgtgcc
     4621 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     4681 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
     4741 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     4801 aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac
     4861 tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag
     4921 cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac
     4981 atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag
     5041 aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc
     5101 ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga
     5161 agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg
     5221 gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat
     5281 aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag
     5341 aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac
     5401 ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc
     5461 gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag
     5521 acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc
     5581 atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc
     5641 ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc
     5701 aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc
     5761 tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt
     5821 ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc
     5881 gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag
     5941 gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt
     6001 ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa
     6061 gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc
     6121 gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc
     6181 ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac
     6241 ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac
     6301 tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag
     6361 gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc
     6421 ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag
     6481 cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg
     6541 accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac
     6601 ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag
     6661 aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc
     6721 ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag
     6781 gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag
     6841 atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag
     6901 ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc
     6961 cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac
     7021 agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa
     7081 gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc
     7141 cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg
     7201 atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc
     7261 cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag
     7321 ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag
     7381 ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc
     7441 aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac
     7501 tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg
     7561 ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag
     7621 ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa
     7681 ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac
     7741 gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc
     7801 cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc
     7861 cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac
     7921 gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac
     7981 ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc
     8041 aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt
     8101 accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg
     8161 gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc
     8221 caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc
     8281 ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag
     8341 tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa
     8401 aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa
     8461 agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg
     8521 aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg
     8581 aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc
     8641 aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag
     8701 gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc
     8761 gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg
     8821 ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc
     8881 cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc
     8941 atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag
     9001 agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg
     9061 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag
     9121 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc aatgaccg
//
