LOCUS       sequence-360243-       10034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP knock-in for beta3-tubulin-GFP (amino acid position: stop 
            codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10034)
  AUTHORS   Droogers WJ, Willems J, MacGillavry HD, de Jong APH
  TITLE     Duplex Labeling and Manipulation of Neuronal Proteins Using 
            Sequential CRISPR/Cas9 Gene Editing.
  JOURNAL   eNeuro. 2022 Jul 18;9(4):ENEURO.0056-22.2022. doi: 
            10.1523/ENEURO.0056-22.2022.
  PUBMED    35851300
REFERENCE   2  (bases 1 to 10034)
  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: 
            "10.1523/ENEURO.0056-22"; journalName: "eNeuro"; date: "2022-07-18- 
            18"; volume: "9"; issue: "4"; pages: "ENEURO.0056-22.2022"
COMMENT     Sequence Label: pORANGE Tubb3-2xGFP KI
FEATURES             Location/Qualifiers
     source          1..10034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        39..287
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     39..59
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     210..229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(509..511,512..1225)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(509..511,512..1222)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1302..1304,1305..2018)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1302..1304,1305..2015)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     enhancer        2076..2361
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2641..2868
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             2889..2915
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2919..2939
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2949..7049
                     /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             2949..7049
                     /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             7053..7073
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7113..7130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7119..7326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7335..7475
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7335..7464
                     /label=AAV2 ITR
     rep_origin      7550..8005
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7637..7656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7847..7868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8022..8041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8141..8163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8201..8219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8287..8391
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8392..8460,8461..9252)
                     /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(8610..8629)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9423..10011
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9912..9931
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc
       61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgct gcgagcaact
      301 tcacttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa
      421 ggctagtccg tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaagcttgct
      481 gcgagcaact tcacttgggg gatcaggcgt gagcaagggc gaggagctgt tcaccggggt
      541 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      601 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      661 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      721 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      781 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      841 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      901 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
      961 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1021 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1081 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1141 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1201 cggcatggac gagctgtaca agtaatgtgc tgcgagcaac ttcacttggg ctcgaggatc
     1261 ctagaagctt gctgcgagca acttcacttg ggggatcagg cgtgagcaag ggcgaggagc
     1321 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1381 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1441 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1501 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1561 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1621 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1681 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     1741 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     1801 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     1861 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     1921 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     1981 ccgggatcac tctcggcatg gacgagctgt acaagtaatg tgctgcgagc aacttcactt
     2041 gggctcgagg atcctaggac gcgtccagag gtacccgtta cataacttac ggtaaatggc
     2101 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg
     2161 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     2221 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     2281 tggcattgtg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     2341 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
     2401 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
     2461 atgggggcgg gggggggggg ggggcgcgcg ccrggsgggg sggggsgggg sgrggggsgg
     2521 ggsggggsgr ggcggrgagg kgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     2581 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     2641 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     2701 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     2761 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     2821 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     2881 ccaccatgta cccatacgat gttccagatt acgcttcgcc gaagaaaaag cgcaaggtcg
     2941 aagcgtccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     3001 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     3061 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     3121 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     3181 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     3241 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     3301 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     3361 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     3421 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     3481 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     3541 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     3601 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     3661 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     3721 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     3781 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     3841 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     3901 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     3961 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     4021 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     4081 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     4141 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     4201 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     4261 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     4321 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     4381 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     4441 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     4501 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     4561 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     4621 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     4681 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     4741 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     4801 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     4861 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     4921 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     4981 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     5041 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     5101 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     5161 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     5221 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     5281 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     5341 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     5401 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     5461 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     5521 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     5581 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     5641 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     5701 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     5761 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     5821 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     5881 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     5941 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     6001 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     6061 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     6121 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     6181 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     6241 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     6301 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     6361 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     6421 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     6481 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     6541 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     6601 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     6661 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     6721 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     6781 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     6841 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     6901 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     6961 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     7021 ggatcgacct gtctcagctg ggaggcgaca gccccaagaa gaagagaaag gtggaggcca
     7081 gctaagaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     7141 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     7201 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     7261 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     7321 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     7381 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     7441 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     7501 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     7561 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     7621 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     7681 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     7741 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     7801 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     7861 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     7921 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     7981 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     8041 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     8101 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     8161 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     8221 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     8281 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     8341 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     8401 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     8461 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     8521 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     8581 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     8641 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     8701 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     8761 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     8821 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     8881 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     8941 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     9001 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     9061 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     9121 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     9181 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     9241 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     9301 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     9361 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     9421 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     9481 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     9541 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     9601 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     9661 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     9721 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     9781 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     9841 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     9901 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     9961 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
    10021 aacgcggcct tttt
//
