LOCUS       sequence-311925-       10163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For generate double-strand DNA break near the stop codon of the 
            human SLC12A5 gene that encodes neuronal chloride transporter KCC2 
            protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10163)
  AUTHORS   Tang X, Drotar J, Li K, Clairmont CD, Brumm AS, Sullins AJ, Wu H, 
            Liu XS, Wang J, Gray NS, Sur M, Jaenisch R
  TITLE     Pharmacological enhancement of KCC2 gene expression exerts 
            therapeutic effects on human Rett syndrome neurons and Mecp2 mutant 
            mice.
  JOURNAL   Sci Transl Med. 2019 Jul 31;11(503). pii: 11/503/eaau0164. doi: 
            10.1126/scitranslmed.aau0164.
  PUBMED    31366578
REFERENCE   2  (bases 1 to 10163)
  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: "Sci Transl 
            Med."; date: "2019-07-31"; volume: "11(503). pii: 11/503/eaau0164. 
            doi"; pages: " 10.1126/scitranslmed.aau0164"
COMMENT     Sequence Label: sgRNA targeting human SLC12A5 gene stop codon
FEATURES             Location/Qualifiers
     source          1..10163
                     /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        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1247..1256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1256..1321
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1328..1348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1373..5473
                     /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             1373..5473
                     /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             5474..5521
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5549..5566)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5555..5762
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5831..6134
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6135..6338
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6289..6309
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(6367..6369,6370..6372,6373..7086)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6367..6369,6370..6372,6373..7083)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6673..6692)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7020..7041
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      7641..8096
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7728..7747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7938..7959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8113..8132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8232..8254
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8292..8310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8378..8482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8483..8551,8552..9343)
                     /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(8701..8720)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9514..10102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10003..10022
                     /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 accatctact cctgagaacc gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattrtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgccg gggggggggg gggggggggg gggggggggs ggggsgrggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
     1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5761 gagcggccgc tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     5821 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     5881 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     5941 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     6001 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     6061 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     6121 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
     6181 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
     6241 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
     6301 gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
     6361 gctagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     6421 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     6481 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     6541 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     6601 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     6661 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     6721 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     6781 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     6841 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     6901 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     6961 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     7021 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     7081 aagtaaggat ccgggatgca gaaattgatg atctattaaa caataaagat gtccactaaa
     7141 atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac ctacattttg
     7201 aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa tgcctgctct
     7261 ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat atcataattt
     7321 aaacaagcaa aaccaaatta agggccagct cattcctccc actcatgatc tatagatcta
     7381 tagatctctc gtgggatcat tgtttttctc ttgattccca ctttgtggtt ctaagtactg
     7441 tggtttccaa atgtgtcagt ttcatagcct gaagaacgag atcagcagcc tctgttccac
     7501 atacacttca ttctcagtat tgttttgcca agttctaatt ccatcagaag ctggtcgacc
     7561 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     7621 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     7681 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     7741 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     7801 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     7861 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     7921 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     7981 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     8041 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     8101 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     8161 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     8221 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     8281 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     8341 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     8401 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     8461 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     8521 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     8581 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     8641 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     8701 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     8761 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     8821 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     8881 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     8941 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     9001 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     9061 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     9121 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     9181 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     9241 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     9301 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     9361 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     9421 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     9481 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     9541 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     9601 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     9661 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     9721 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     9781 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     9841 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     9901 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     9961 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
    10021 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
    10081 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
    10141 ttgctggcct tttgctcaca tgt
//
