LOCUS       sequence-336130-       10656 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing two gRNAs targeting mouse 
            Olig2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10656)
  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 10656)
  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: Olig2 x2)
FEATURES             Location/Qualifiers
     source          1..10656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        248..1430
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          479..1421
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1378..1398
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1436..1445
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1445..1510
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1517..1537
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1562..5662
                     /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             1562..5662
                     /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             5663..5710
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5726..5779
                     /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             5780..6157
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6170..6179
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6176..6883
                     /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(6321..6339)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6911..6928)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6917..7124
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7133..7273
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7133..7262
                     /label=AAV2 ITR
     rep_origin      7348..7803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7435..7454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7645..7666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7820..7839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7939..7961
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7999..8017)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8085..8189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8190..8258,8259..9050)
                     /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(8408..8427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9221..9809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9710..9729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9871..10119
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        10140..10215
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        10311..10559
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        10581..10656
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
       61 gcgccaattc tgcagacaaa tggctctaca ggtaccagta attcatacaa aaggactcgc
      121 ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga
      181 tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca
      241 tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
      301 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
      361 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
      421 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
      481 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc
      541 ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag cttcgggttg
      601 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
      661 ttgaggcctg gcttgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
      721 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
      781 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
      841 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
      901 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
      961 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     1021 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     1081 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     1141 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     1201 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     1261 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     1321 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     1381 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga ccggtgccac
     1441 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
     1501 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
     1561 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
     1621 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     1681 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
     1741 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     1801 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     1861 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     1921 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     1981 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     2041 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     2101 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     2161 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     2221 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     2281 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     2341 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     2401 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     2461 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     2521 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     2581 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     2641 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     2701 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     2761 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     2821 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     2881 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     2941 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     3001 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     3061 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     3121 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     3181 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     3241 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     3301 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     3361 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     3421 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     3481 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     3541 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     3601 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     3661 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     3721 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     3781 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     3841 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     3901 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     3961 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     4021 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     4081 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     4141 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     4201 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     4261 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     4321 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     4381 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     4441 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     4501 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     4561 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     4621 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     4681 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     4741 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     4801 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     4861 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     4921 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     4981 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     5041 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     5101 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     5161 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     5221 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     5281 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     5341 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     5401 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     5461 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     5521 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     5581 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     5641 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     5701 aaagaaaaag gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     5761 cgaggagaat cctggcccaa tgcctgaacc ctctaagtct gctccagccc ctaaaaaggg
     5821 ttctaagaag gctatcacta aggcgcagaa gaaggatggt aagaagcgta agcgcagccg
     5881 caaggagagc tattctatct atgtgtacaa ggttctgaag caggtccacc ccgacaccgg
     5941 catctcatcc aaggccatgg ggatcatgaa ctccttcgtc aacgacatct tcgagcgcat
     6001 cgcgggcgag gcttctcgcc tggctcacta caataagcgc tcgaccatca cctccaggga
     6061 gattcagacg gctgtgcgcc tgctgctgcc tggggagctg gctaagcatg ctgtgtccga
     6121 gggcactaag gcagttacca agtacactag ctctaaggat ccaccggtcg ccaccatggt
     6181 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     6241 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     6301 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     6361 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     6421 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     6481 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     6541 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     6601 gaagaaaacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     6661 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     6721 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     6781 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     6841 cgagggccgc cactccaccg gcggcatgga cgagctgtac aaggaattct aactagagct
     6901 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6961 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     7021 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     7081 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc
     7141 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     7201 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     7261 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     7321 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     7381 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     7441 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     7501 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     7561 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     7621 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     7681 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     7741 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     7801 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     7861 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     7921 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     7981 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     8041 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     8101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     8161 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     8221 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     8281 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     8341 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     8401 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     8461 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     8521 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     8581 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     8641 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     8701 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     8761 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     8821 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     8881 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     8941 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     9001 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     9061 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     9121 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     9181 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     9241 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     9301 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     9361 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     9421 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     9481 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     9541 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     9601 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     9661 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     9721 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     9781 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     9841 tggccttttg ctggcctttt gctcacatgt gagggcctat ttcccatgat tccttcatat
     9901 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     9961 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
    10021 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
    10081 ttcttggctt tatatatctt gtggaaagga cgaaacaccg cagcagcggc ttcacaggag
    10141 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
    10201 gcaccgagtc ggtgcttttt tgttttagag ctagaaatag caagttaaaa taaggctagt
    10261 ccgtttttag cgcgtgcgcc aattctgcag acaaatggct ctagacatgt gagggcctat
    10321 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
    10381 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
    10441 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
    10501 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
    10561 ctcctcgtcc acgtcctcgg gttttagagc tagaaatagc aagttaaaat aaggctagtc
    10621 cgttatcaac ttgaaaaagt ggcaccgagt cggtgc
//
