LOCUS       sequence-334622-        8947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cas9 and 2 guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8947)
  AUTHORS   Fang H, Bygrave AM, Roth RH, Johnson RC, Huganir RL
  TITLE     An optimized CRISPR/Cas9 approach for precise genome editing in 
            neurons.
  JOURNAL   Elife. 2021 Mar 10;10. pii: 65202. doi: 10.7554/eLife.65202.
  PUBMED    33689678
REFERENCE   2  (bases 1 to 8947)
  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: "Elife."; 
            date: "2021-03-10"; volume: "10. pii: 65202. doi"; pages: " 
            10.7554/eLife.65202"
COMMENT     Sequence Label: GluA2 (px330:2-guideCas9)
FEATURES             Location/Qualifiers
     source          1..8947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          178..405
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      417..426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             426..491
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             498..518
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             543..4643
                     /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             543..4643
                     /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             4644..4691
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4719..4736)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4725..4932
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4941..5081
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4941..5070
                     /label=AAV2 ITR
     rep_origin      5156..5611
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5243..5262)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5453..5474
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5628..5647)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5747..5769
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5807..5825)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5893..5997
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5998..6066,6067..6858)
                     /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(6216..6235)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7029..7617
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7518..7537
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7679..7927
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        7949..8024
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8115..8363
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8385..8460
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8560..8845
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        join(8847..8947,1..177)
                     /label=chicken beta-actin promoter
ORIGIN
        1 ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
       61 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      121 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      181 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      241 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      301 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
      361 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca
      421 ccatggacta taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg
      481 atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
      541 ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
      601 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
      661 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
      721 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
      781 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
      841 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
      901 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
      961 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     1021 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1081 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     1141 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     1201 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     1261 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     1321 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     1381 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     1441 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     1501 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     1561 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     1621 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     1681 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     1741 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     1801 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     1861 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     1921 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     1981 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     2041 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     2101 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     2161 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     2221 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     2281 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     2341 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     2401 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     2461 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     2521 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     2581 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     2641 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     2701 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     2761 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     2821 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     2881 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     2941 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     3001 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     3061 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     3121 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     3181 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     3241 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     3301 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     3361 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     3421 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     3481 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     3541 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     3601 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     3661 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     3721 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     3781 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     3841 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     3901 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     3961 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     4021 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4081 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     4141 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     4201 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     4261 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     4321 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     4381 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     4441 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     4501 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     4561 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     4621 acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
     4681 aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     4741 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4801 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4861 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     4921 gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4981 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     5041 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     5101 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     5161 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5221 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5281 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5341 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     5401 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5461 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5521 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5581 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5641 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5701 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5761 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5821 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5881 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5941 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6001 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6061 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6121 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6181 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6241 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6301 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6361 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6421 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6481 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6541 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6601 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6661 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     6721 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6781 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6841 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6901 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6961 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7021 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7081 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7141 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     7201 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7261 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7321 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7381 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7441 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7501 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7561 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7621 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtga
     7681 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     7741 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     7801 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     7861 gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg
     7921 aaacaccgaa cagccaccag ctaaacctgt tttagagcta gaaatagcaa gttaaaataa
     7981 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc
     8041 tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga
     8101 caaatggctc tagcgagggc ctatttccca tgattccttc atatttgcat atacgataca
     8161 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     8221 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     8281 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     8341 tcttgtggaa aggacgaaac accgtatgcc ttttgacaca ataggtttta gagctagaaa
     8401 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     8461 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
     8521 gcgccaattc tgcagacaaa tggctctaga ggtggtaccc gttacataac ttacggtaaa
     8581 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
     8641 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     8701 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     8761 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     8821 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
     8881 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
     8941 agcgatg
//
