LOCUS       sequence-328123-        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: Ascl1-2)
FEATURES             Location/Qualifiers
     source          1..9826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        151..1333
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          382..1324
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1281..1301
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1339..1348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1348..1413
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1420..1440
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1465..5565
                     /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             1465..5565
                     /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             5566..5613
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5629..5682
                     /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(5683..5685,5686..6396)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5683..5685,5686..6396)
                     /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(5986..6005)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6333..6354
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6424..6441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6430..6637
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6646..6786
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6646..6775
                     /label=AAV2 ITR
     rep_origin      6861..7316
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6948..6967)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7158..7179
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7333..7352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7452..7474
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7512..7530)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7598..7702
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7703..7771,7772..8563)
                     /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(7921..7940)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8734..9322
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9223..9242
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9384..9632
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9384..9404
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9555..9574
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9654..9729
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gtaattcata caaaaggact cgcccctgcc ttggggaatc ccagggaccg tcgttaaact
       61 cccactaacg tagaacccag agatcgctgc gttcccgccc cctcacccgc ccgctctcgt
      121 catcactgag gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg ggcagagcgc
      181 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
      241 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
      301 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
      361 gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt
      421 acgggttatg gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat
      481 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg
      541 agccccttcg cctcgtgctt gagttgaggc ctggcttggg cgctggggcc gccgcgtgcg
      601 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
      661 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca
      721 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc
      781 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg
      841 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc
      901 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
      961 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg
     1021 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc
     1081 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc
     1141 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga
     1201 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
     1261 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt
     1321 tcaggtgtcg tgaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat
     1381 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
     1441 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
     1501 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
     1561 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
     1621 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
     1681 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
     1741 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
     1801 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
     1861 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
     1921 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
     1981 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     2041 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
     2101 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     2161 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     2221 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     2281 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     2341 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     2401 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     2461 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     2521 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     2581 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     2641 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     2701 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     2761 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     2821 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     2881 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     2941 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     3001 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     3061 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     3121 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     3181 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     3241 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     3301 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     3361 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     3421 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     3481 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     3541 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     3601 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     3661 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     3721 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     3781 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     3841 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
     3901 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     3961 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     4021 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     4081 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     4141 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     4201 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     4261 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     4321 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     4381 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     4441 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     4501 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     4561 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     4621 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     4681 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     4741 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     4801 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     4861 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     4921 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     4981 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     5041 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     5101 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     5161 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     5221 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     5281 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     5341 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     5401 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     5461 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     5521 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc
     5581 acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga
     5641 agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag
     5701 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     5761 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     5821 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     5881 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     5941 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     6001 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     6061 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     6121 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     6181 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     6241 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     6301 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     6361 gccgggatca ctctcggcat ggacgagctg tacaaggaat tctaactaga gctcgctgat
     6421 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     6481 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     6541 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     6601 gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga
     6661 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     6721 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     6781 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6841 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6901 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     6961 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     7021 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     7081 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     7141 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     7201 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     7261 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     7321 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     7381 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     7441 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7501 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     7561 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7621 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     7681 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     7741 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7801 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7861 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7921 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     7981 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     8041 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     8101 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     8161 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     8221 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     8281 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     8341 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     8401 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     8461 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     8521 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     8581 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     8641 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8701 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     8761 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     8821 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     8881 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     8941 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     9001 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     9061 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     9121 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     9181 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     9241 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     9301 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     9361 ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata
     9421 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     9481 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     9541 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
     9601 ctttatatat cttgtggaaa ggacgaaaca ccgagggggc ggtcacaagt caggttttag
     9661 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     9721 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
     9781 ttagcgcgtg cgccaattct gcagacaaat ggctctagag gtacca
//
