LOCUS       sequence-328124-        9826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing the gRNA targeting mouse 
            Ascl1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9826)
  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 9826)
  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: Ascl1-3)
FEATURES             Location/Qualifiers
     source          1..9826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        129..1311
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          360..1302
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1259..1279
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1317..1326
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1326..1391
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1398..1418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1443..5543
                     /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             1443..5543
                     /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             5544..5591
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5607..5660
                     /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(5661..5663,5664..6374)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5661..5663,5664..6374)
                     /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(5964..5983)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6311..6332
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6402..6419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6408..6615
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6624..6764
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6624..6753
                     /label=AAV2 ITR
     rep_origin      6839..7294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6926..6945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7136..7157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7311..7330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7430..7452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7490..7508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7576..7680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7681..7749,7750..8541)
                     /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(7899..7918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8712..9300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9201..9220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9362..9610
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9362..9382
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9533..9552
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9632..9707
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cccctgcctt ggggaatccc agggaccgtc gttaaactcc cactaacgta gaacccagag
       61 atcgctgcgt tcccgccccc tcacccgccc gctctcgtca tcactgaggt ggagaagagc
      121 atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
      181 agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact
      241 gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata
      301 taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg
      361 taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc
      421 cttgaattac ttccacgccc ctggctgcag tacgtgattc ttgatcccga gcttcgggtt
      481 ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga
      541 gttgaggcct ggcttgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
      601 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
      661 ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg
      721 gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
      781 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
      841 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
      901 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc
      961 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     1021 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     1081 aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     1141 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     1201 cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     1261 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg accggtgcca
     1321 ccatggacta taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg
     1381 atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
     1441 ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
     1501 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     1561 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     1621 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     1681 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     1741 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     1801 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     1861 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     1921 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1981 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     2041 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     2101 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     2161 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     2221 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     2281 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     2341 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     2401 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     2461 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     2521 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     2581 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     2641 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     2701 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     2761 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     2821 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     2881 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     2941 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     3001 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     3061 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     3121 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     3181 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     3241 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     3301 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     3361 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     3421 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     3481 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     3541 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     3601 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     3661 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     3721 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     3781 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     3841 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     3901 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     3961 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     4021 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     4081 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     4141 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     4201 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     4261 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     4321 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     4381 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     4441 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     4501 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     4561 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     4621 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     4681 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     4741 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     4801 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     4861 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     4921 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4981 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     5041 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     5101 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     5161 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     5221 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     5281 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     5341 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     5401 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     5461 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     5521 acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
     5581 aaaagaaaaa ggaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
     5641 tcgaggagaa tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     5701 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     5761 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     5821 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     5881 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     5941 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     6001 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     6061 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     6121 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     6181 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     6241 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     6301 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     6361 acgagctgta caaggaattc taactagagc tcgctgatca gcctcgactg tgccttctag
     6421 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     6481 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     6541 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     6601 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     6661 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     6721 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     6781 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     6841 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     6901 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     6961 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     7021 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     7081 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     7141 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     7201 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     7261 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     7321 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     7381 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     7441 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     7501 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     7561 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     7621 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     7681 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     7741 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     7801 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     7861 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     7921 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     7981 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     8041 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     8101 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     8161 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     8221 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     8281 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     8341 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc
     8401 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     8461 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     8521 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     8581 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     8641 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     8701 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     8761 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     8821 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     8881 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     8941 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     9001 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     9061 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     9121 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     9181 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     9241 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     9301 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     9361 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     9421 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     9481 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     9541 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     9601 acgaaacacc gcgagcgcaa ccgggtcaag tgttttagag ctagaaatag caagttaaaa
     9661 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
     9721 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
     9781 agacaaatgg ctctagaggt accagtaatt catacaaaag gactcg
//
