LOCUS       sequence-360078-       11620 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 11620)
  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 11620)
  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 Tubb3-4xGFP KI
FEATURES             Location/Qualifiers
     source          1..11620
                     /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"
     CDS             join(2095..2097,2098..2811)
                     /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(2095..2097,2098..2808)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2888..2890,2891..3604)
                     /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(2888..2890,2891..3601)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     enhancer        3662..3947
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          4227..4454
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             4475..4501
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4505..4525
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4535..8635
                     /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             4535..8635
                     /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             8639..8659
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(8699..8716)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8705..8912
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8921..9061
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8921..9050
                     /label=AAV2 ITR
     rep_origin      9136..9591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9223..9242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9433..9454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9608..9627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9727..9749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9787..9805)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9873..9977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9978..10046,10047..10838)
                     /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(10196..10215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11009..11597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11498..11517
                     /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 atcctagaag cttgctgcga gcaacttcac ttgggggatc aggcgtgagc
     2101 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2161 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2221 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2281 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2341 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2401 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2461 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2521 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2581 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2641 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2701 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2761 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta atgtgctgcg
     2821 agcaacttca cttgggctcg aggatcctag aagcttgctg cgagcaactt cacttggggg
     2881 atcaggcgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2941 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     3001 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     3061 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     3121 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     3181 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     3241 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3301 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     3361 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     3421 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     3481 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     3541 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     3601 gtaatgtgct gcgagcaact tcacttgggc tcgaggatcc taggacgcgt ccagaggtac
     3661 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     3721 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     3781 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     3841 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
     3901 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     3961 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     4021 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccrg
     4081 gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg grgaggkgcg gcggcagcca
     4141 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
     4201 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
     4261 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
     4321 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
     4381 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
     4441 tcactttttt tcaggttgga ccggtgccac catgtaccca tacgatgttc cagattacgc
     4501 ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag aagtacagca tcggcctgga
     4561 catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa
     4621 gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc
     4681 cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag
     4741 aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat
     4801 ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga
     4861 taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga
     4921 gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga
     4981 cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat
     5041 cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca
     5101 gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc
     5161 catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc
     5221 cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc
     5281 caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac
     5341 ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt
     5401 tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac
     5461 cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca
     5521 ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat
     5581 tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga
     5641 agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct
     5701 cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat
     5761 cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta
     5821 cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta
     5881 ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga
     5941 ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag
     6001 cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa
     6061 gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt
     6121 gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga
     6181 cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa
     6241 gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc
     6301 cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga
     6361 ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga
     6421 gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca
     6481 gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat
     6541 ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa
     6601 cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa
     6661 agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag
     6721 ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt
     6781 gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac
     6841 ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga
     6901 gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa
     6961 gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat
     7021 caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga
     7081 ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt
     7141 gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa
     7201 gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga
     7261 actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca
     7321 cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat
     7381 ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt
     7441 ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa
     7501 cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta
     7561 cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg
     7621 caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat
     7681 taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg
     7741 ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc
     7801 ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat
     7861 cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa
     7921 gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga
     7981 aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga
     8041 aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt
     8101 gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg
     8161 gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc
     8221 caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga
     8281 ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat
     8341 cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt
     8401 gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat
     8461 ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac
     8521 catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca
     8581 gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacagccc
     8641 caagaagaag agaaaggtgg aggccagcta agaattctaa ctagagctcg ctgatcagcc
     8701 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     8761 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     8821 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     8881 gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag
     8941 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     9001 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     9061 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     9121 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     9181 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     9241 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     9301 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     9361 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     9421 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     9481 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     9541 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     9601 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     9661 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     9721 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     9781 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     9841 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     9901 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     9961 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
    10021 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
    10081 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
    10141 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
    10201 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
    10261 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
    10321 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
    10381 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
    10441 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
    10501 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
    10561 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
    10621 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
    10681 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
    10741 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
    10801 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
    10861 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
    10921 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
    10981 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
    11041 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
    11101 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
    11161 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
    11221 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
    11281 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
    11341 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
    11401 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
    11461 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
    11521 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
    11581 ggcggagcct atggaaaaac gccagcaacg cggccttttt
//
