LOCUS       sequence-336123-       10657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing two gRNAs targeting mouse 
            Neurog2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10657)
  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 10657)
  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-H2B-mCherry (dual gRNA: Neurog...
FEATURES             Location/Qualifiers
     source          1..10657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        28..276
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        298..373
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        621..1803
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          852..1794
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1751..1771
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1809..1818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1818..1883
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1890..1910
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1935..6035
                     /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             1935..6035
                     /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             6036..6083
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6099..6152
                     /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             6153..6530
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6543..6552
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6549..7256
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6694..6712)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7284..7301)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7290..7497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7506..7646
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7506..7635
                     /label=AAV2 ITR
     rep_origin      7721..8176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7808..7827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8018..8039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8193..8212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8312..8334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8372..8390)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8458..8562
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8563..8631,8632..9423)
                     /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(8781..8800)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9594..10182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10083..10102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        10244..10492
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        10514..10589
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tctgcagaca aatggctcta gacatgtgag ggcctatttc ccatgattcc ttcatatttg
       61 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
      121 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
      181 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
      241 ttggctttat atatcttgtg gaaaggacga aacaccgctc ccgaggtgcc aagacgggtt
      301 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      361 accgagtcgg tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc
      421 gtttttagcg cgtgcgccaa ttctgcagac aaatggctct acaggtacca gtaattcata
      481 caaaaggact cgcccctgcc ttggggaatc ccagggaccg tcgttaaact cccactaacg
      541 tagaacccag agatcgctgc gttcccgccc cctcacccgc ccgctctcgt catcactgag
      601 gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      661 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      721 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      781 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      841 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      901 gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc
      961 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     1021 cctcgtgctt gagttgaggc ctggcttggg cgctggggcc gccgcgtgcg aatctggtgg
     1081 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     1141 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     1201 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     1261 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     1321 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     1381 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     1441 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     1501 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     1561 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     1621 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     1681 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     1741 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     1801 tgaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat catgatattg
     1861 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
     1921 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     1981 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     2041 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     2101 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     2161 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     2221 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     2281 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     2341 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     2401 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     2461 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     2521 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     2581 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     2641 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     2701 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     2761 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     2821 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     2881 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     2941 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     3001 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     3061 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     3121 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     3181 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     3241 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     3301 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     3361 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     3421 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     3481 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     3541 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     3601 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     3661 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     3721 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     3781 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     3841 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     3901 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     3961 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     4021 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     4081 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     4141 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     4201 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     4261 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     4321 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     4381 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     4441 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     4501 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     4561 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     4621 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     4681 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     4741 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     4801 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     4861 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     4921 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     4981 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     5041 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     5101 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     5161 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     5221 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     5281 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     5341 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     5401 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     5461 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     5521 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     5581 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     5641 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     5701 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     5761 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     5821 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     5881 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     5941 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     6001 agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg
     6061 ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga agtctgctaa
     6121 catgcggtga cgtcgaggag aatcctggcc caatgcctga accctctaag tctgctccag
     6181 cccctaaaaa gggttctaag aaggctatca ctaaggcgca gaagaaggat ggtaagaagc
     6241 gtaagcgcag ccgcaaggag agctattcta tctatgtgta caaggttctg aagcaggtcc
     6301 accccgacac cggcatctca tccaaggcca tggggatcat gaactccttc gtcaacgaca
     6361 tcttcgagcg catcgcgggc gaggcttctc gcctggctca ctacaataag cgctcgacca
     6421 tcacctccag ggagattcag acggctgtgc gcctgctgct gcctggggag ctggctaagc
     6481 atgctgtgtc cgagggcact aaggcagtta ccaagtacac tagctctaag gatccaccgg
     6541 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     6601 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     6661 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     6721 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     6781 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     6841 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     6901 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     6961 gccccgtaat gcagaagaaa accatgggct gggaggcctc ctccgagcgg atgtaccccg
     7021 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     7081 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     7141 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     7201 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggaat
     7261 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     7321 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     7381 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     7441 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     7501 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     7561 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     7621 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     7681 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     7741 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     7801 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7861 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7921 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7981 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     8041 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     8101 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     8161 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     8221 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     8281 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     8341 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     8401 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     8461 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     8521 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8581 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8641 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8701 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8761 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8821 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8881 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8941 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     9001 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     9061 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     9121 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     9181 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     9241 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     9301 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     9361 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     9421 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     9481 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9721 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     9781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
    10021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
    10081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
    10141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
    10201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat
    10261 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
    10321 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
    10381 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
    10441 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcatcggg
    10501 gtcagggtcg ccggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
    10561 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
    10621 aaaataaggc tagtccgttt ttagcgcgtg cgccaat
//
