LOCUS       sequence-336110-        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-3)
FEATURES             Location/Qualifiers
     source          1..9825
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        145..1327
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          376..1318
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1275..1295
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1333..1342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1342..1407
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1414..1434
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1459..5559
                     /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             1459..5559
                     /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             5560..5607
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5623..5676
                     /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(5677..5679,5680..6390)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5677..5679,5680..6390)
                     /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(5980..5999)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6327..6348
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6418..6435)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6424..6631
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6640..6780
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6640..6769
                     /label=AAV2 ITR
     rep_origin      6855..7310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6942..6961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7152..7173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7327..7346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7446..7468
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7506..7524)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7592..7696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7697..7765,7766..8557)
                     /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(7915..7934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8728..9316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9217..9236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9378..9626
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9378..9398
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9549..9568
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9647..9722
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 catacaaaag gactcgcccc tgccttgggg aatcccaggg accgtcgtta aactcccact
       61 aacgtagaac ccagagatcg ctgcgttccc gccccctcac ccgcccgctc tcgtcatcac
      121 tgaggtggag aagagcatgc gtgaggctcc ggtgcccgtc agtgggcaga gcgcacatcg
      181 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
      241 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
      301 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
      361 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
      421 tatggccctt gcgtgccttg aattacttcc acgcccctgg ctgcagtacg tgattcttga
      481 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
      541 ttcgcctcgt gcttgagttg aggcctggct tgggcgctgg ggccgccgcg tgcgaatctg
      601 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
      661 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
      721 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
      781 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
      841 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
      901 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
      961 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     1021 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     1081 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     1141 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     1201 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     1261 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     1321 gtcgtgaccg gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat
     1381 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
     1441 cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct
     1501 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     1561 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     1621 ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg
     1681 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     1741 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     1801 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     1861 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     1921 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     1981 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     2041 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     2101 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     2161 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     2221 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     2281 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     2341 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     2401 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     2461 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     2521 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     2581 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     2641 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     2701 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     2761 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     2821 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     2881 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     2941 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     3001 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     3061 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     3121 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     3181 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     3241 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     3301 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     3361 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     3421 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     3481 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     3541 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     3601 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     3661 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     3721 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     3781 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     3841 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     3901 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     3961 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     4021 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     4081 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     4141 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     4201 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     4261 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     4321 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     4381 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     4441 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     4501 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     4561 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     4621 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     4681 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     4741 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac
     4801 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac
     4861 agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     4921 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     4981 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     5041 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     5101 tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     5161 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     5221 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     5281 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     5341 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     5401 accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg
     5461 tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     5521 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa
     5581 aaggccggcc aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg
     5641 ctaacatgcg gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc
     5701 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     5761 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     5821 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     5881 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     5941 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     6001 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     6061 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     6121 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     6181 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     6241 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     6301 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     6361 atcactctcg gcatggacga gctgtacaag gaattctaac tagagctcgc tgatcagcct
     6421 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     6481 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     6541 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     6601 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     6661 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     6721 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     6781 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     6841 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     6901 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     6961 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     7021 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     7081 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     7141 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     7201 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     7261 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     7321 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     7381 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     7441 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     7501 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     7561 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     7621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     7681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     7741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     7801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     7861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     7921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     7981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     8041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     8101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     8161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     8221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     8281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     8341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     8401 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     8461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     8521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     8581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     8641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     8701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     8761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     8821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     8881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     8941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     9001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     9061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     9121 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     9181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     9241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     9301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     9361 gccttttgct cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     9421 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     9481 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     9541 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     9601 atatcttgtg gaaaggacga aacaccggtg gcacgtgcag tgtacagttt tagagctaga
     9661 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     9721 gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc
     9781 gtgcgccaat tctgcagaca aatggctcta gaggtaccag taatt
//
