LOCUS       sequence-336115-       10657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing two gRNAs targeting mouse 
            Ascl1..
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: Ascl1 x2)
FEATURES             Location/Qualifiers
     source          1..10657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     513..532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        674..922
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        944..1019
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1115..1363
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1385..1460
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1708..2890
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1939..2881
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2838..2858
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2896..2905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2905..2970
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2977..2997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3022..7122
                     /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             3022..7122
                     /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             7123..7170
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7186..7239
                     /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             7240..7617
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      7630..7639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7636..8343
                     /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(7781..7799)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(8371..8388)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8377..8584
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8593..8733
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8593..8722
                     /label=AAV2 ITR
     rep_origin      8808..9263
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8895..8914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9105..9126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9280..9299)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9399..9421
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9459..9477)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9545..9649
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9650..9718,9719..10510)
                     /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(9868..9887)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
       61 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      121 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
      181 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      241 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      301 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      361 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      421 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      481 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      541 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      601 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      661 tttgctcaca tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      721 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      781 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      841 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      901 cttgtggaaa ggacgaaaca ccgagcctgc ttctttgcga ccggttttag agctagaaat
      961 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1021 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg
     1081 cgccaattct gcagacaaat ggctctagac atgtgagggc ctatttccca tgattccttc
     1141 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     1201 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     1261 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     1321 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgaggggg cggtcacaag
     1381 tcaggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     1441 aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt taaaataagg
     1501 ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctctaca ggtaccagta
     1561 attcatacaa aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc
     1621 actaacgtag aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat
     1681 cactgaggtg gagaagagca tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca
     1741 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     1801 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     1861 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     1921 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
     1981 ggttatggcc cttgcgtgcc ttgaattact tccacgcccc tggctgcagt acgtgattct
     2041 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
     2101 cccttcgcct cgtgcttgag ttgaggcctg gcttgggcgc tggggccgcc gcgtgcgaat
     2161 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
     2221 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
     2281 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
     2341 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     2401 gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc
     2461 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     2521 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     2581 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     2641 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga gtacgtcgtc
     2701 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     2761 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     2821 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     2881 ggtgtcgtga ccggtgccac catggactat aaggaccacg acggagacta caaggatcat
     2941 gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt
     3001 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac
     3061 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     3121 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     3181 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     3241 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     3301 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     3361 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     3421 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     3481 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     3541 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     3601 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     3661 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     3721 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     3781 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     3841 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     3901 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     3961 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     4021 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     4081 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     4141 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     4201 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     4261 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     4321 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     4381 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     4441 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     4501 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     4561 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     4621 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     4681 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     4741 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     4801 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     4861 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     4921 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     4981 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     5041 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     5101 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     5161 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     5221 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     5281 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     5341 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     5401 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     5461 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     5521 gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac
     5581 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     5641 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     5701 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     5761 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     5821 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     5881 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     5941 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     6001 accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag
     6061 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     6121 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     6181 ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     6241 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     6301 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     6361 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     6421 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     6481 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     6541 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     6601 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     6661 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     6721 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     6781 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     6841 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     6901 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     6961 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     7021 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     7081 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     7141 aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg cagaggaagt
     7201 ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tgcctgaacc ctctaagtct
     7261 gctccagccc ctaaaaaggg ttctaagaag gctatcacta aggcgcagaa gaaggatggt
     7321 aagaagcgta agcgcagccg caaggagagc tattctatct atgtgtacaa ggttctgaag
     7381 caggtccacc ccgacaccgg catctcatcc aaggccatgg ggatcatgaa ctccttcgtc
     7441 aacgacatct tcgagcgcat cgcgggcgag gcttctcgcc tggctcacta caataagcgc
     7501 tcgaccatca cctccaggga gattcagacg gctgtgcgcc tgctgctgcc tggggagctg
     7561 gctaagcatg ctgtgtccga gggcactaag gcagttacca agtacactag ctctaaggat
     7621 ccaccggtcg ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag
     7681 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     7741 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     7801 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     7861 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     7921 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     7981 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     8041 tccgacggcc ccgtaatgca gaagaaaacc atgggctggg aggcctcctc cgagcggatg
     8101 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     8161 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     8221 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     8281 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     8341 aaggaattct aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     8401 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     8461 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     8521 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     8581 gggagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     8641 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     8701 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     8761 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     8821 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     8881 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     8941 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     9001 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     9061 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     9121 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     9181 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     9241 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     9301 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     9361 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     9421 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     9481 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     9541 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     9601 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     9661 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     9721 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     9781 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     9841 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     9901 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     9961 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
    10021 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
    10081 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
    10141 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
    10201 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
    10261 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
    10321 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
    10381 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
    10441 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
    10501 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
    10561 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
    10621 ttaacgtgag ttttcgttcc actgagcgtc agacccc
//
