LOCUS       sequence-328129-        9825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing the gRNA targeting an 
            enhancer of mouse Otx2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9825)
  AUTHORS   Kaufman ML, Goodson NB, Park KU, Schwanke M, Office E, Schneider SR,
            Abraham J, Hensley A, Jones KL, Brzezinski JA
  TITLE     Initiation of Otx2 expression in the developing mouse retina 
            requires a unique enhancer and either Ascl1 or Neurog2 activity.
  JOURNAL   Development. 2021 Jun 15;148(12). pii: 269198. doi: 
            10.1242/dev.199399. Epub 2021 Jun 18.
  PUBMED    34143204
REFERENCE   2  (bases 1 to 9825)
  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: 
            "Development."; date: "2021-06-15"; volume: "148(12). pii: 269198. 
            doi"; pages: " 10.1242/dev.199399. Epub 2021 Jun 18"
COMMENT     Sequence Label: pX458-Ef1a-Cas9-GFP (gRNA: DHS-4D-2)
FEATURES             Location/Qualifiers
     source          1..9825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        76..1258
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          307..1249
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1206..1226
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1264..1273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1273..1338
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1345..1365
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1390..5490
                     /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             1390..5490
                     /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             5491..5538
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5554..5607
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5608..5610,5611..6321)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5608..5610,5611..6321)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5911..5930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6258..6279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6349..6366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6355..6562
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6571..6711
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6571..6700
                     /label=AAV2 ITR
     rep_origin      6786..7241
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6873..6892)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7083..7104
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7258..7277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7377..7399
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7437..7455)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7523..7627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7628..7696,7697..8488)
                     /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(7846..7865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8659..9247
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9148..9167
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9309..9557
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9309..9329
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9480..9499
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9578..9653
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cccagagatc gctgcgttcc cgccccctca cccgcccgct ctcgtcatca ctgaggtgga
       61 gaagagcatg cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
      121 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
      181 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
      241 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
      301 acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct
      361 tgcgtgcctt gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct
      421 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg
      481 tgcttgagtt gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct
      541 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc
      601 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg
      661 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc
      721 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg
      781 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag
      841 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc
      901 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca
      961 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc
     1021 gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg
     1081 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc
     1141 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt
     1201 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc
     1261 ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
     1321 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
     1381 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     1441 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     1501 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     1561 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     1621 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     1681 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     1741 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     1801 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1861 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1921 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1981 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     2041 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     2101 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     2161 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     2221 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     2281 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     2341 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     2401 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     2461 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     2521 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     2581 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     2641 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     2701 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     2761 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     2821 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     2881 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2941 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     3001 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     3061 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     3121 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     3181 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     3241 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     3301 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     3361 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     3421 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     3481 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     3541 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     3601 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     3661 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     3721 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     3781 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     3841 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3901 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     3961 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     4021 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     4081 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     4141 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     4201 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     4261 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     4321 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     4381 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     4441 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     4501 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     4561 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     4621 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     4681 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     4741 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     4801 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4861 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4921 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4981 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     5041 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     5101 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     5161 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     5221 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     5281 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     5341 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     5401 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     5461 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     5521 caggcaaaaa agaaaaagga attcggcagt ggagagggca gaggaagtct gctaacatgc
     5581 ggtgacgtcg aggagaatcc tggcccagtg agcaagggcg aggagctgtt caccggggtg
     5641 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     5701 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     5761 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     5821 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     5881 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     5941 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     6001 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     6061 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     6121 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     6181 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     6241 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     6301 ggcatggacg agctgtacaa ggaattctaa ctagagctcg ctgatcagcc tcgactgtgc
     6361 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     6421 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     6481 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     6541 agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc
     6601 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     6661 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     6721 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     6781 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6841 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     6901 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6961 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     7021 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7081 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     7141 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     7201 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     7261 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     7321 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     7381 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     7441 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     7501 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     7561 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     7621 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     7681 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     7741 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     7801 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     7861 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     7921 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     7981 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     8041 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     8101 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     8161 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     8221 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     8281 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     8341 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     8401 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     8461 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     8521 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     8581 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     8641 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     8701 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     8761 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     8821 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     8881 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     8941 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     9001 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     9061 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     9121 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     9181 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     9241 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     9301 tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     9361 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     9421 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     9481 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     9541 ggaaaggacg aaacaccgtt tatgaagctc ggcagaggtt ttagagctag aaatagcaag
     9601 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     9661 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
     9721 ttctgcagac aaatggctct agaggtacca gtaattcata caaaaggact cgcccctgcc
     9781 ttggggaatc ccagggaccg tcgttaaact cccactaacg tagaa
//
