LOCUS       sequence-336101-       10216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing the gRNA targeting mouse 
            Neurog2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10216)
  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 10216)
  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 (gRNA: Neurog2-2)
FEATURES             Location/Qualifiers
     source          1..10216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        75..323
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     75..95
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     246..265
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        345..420
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        668..1850
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          899..1841
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1798..1818
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1856..1865
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1865..1930
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1937..1957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1982..6082
                     /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             1982..6082
                     /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             6083..6130
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6146..6199
                     /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             6200..6577
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6590..6599
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6596..7303
                     /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(6741..6759)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(7331..7348)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7337..7544
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7553..7693
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7553..7682
                     /label=AAV2 ITR
     rep_origin      7768..8223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7855..7874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8065..8086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8240..8259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8359..8381
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8419..8437)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8505..8609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8610..8678,8679..9470)
                     /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(8828..8847)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9641..10216,1..13)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10130..10149
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
       61 ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca
      121 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
      181 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
      241 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
      301 tcttgtggaa aggacgaaac accgctcccg aggtgccaag acgggtttta gagctagaaa
      361 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
      421 ttttttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt tttagcgcgt
      481 gcgccaattc tgcagacaaa tggctctaga ggtaccagta attcatacaa aaggactcgc
      541 ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga
      601 tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca
      661 tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
      721 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg
      781 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
      841 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt
      901 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc
      961 ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag cttcgggttg
     1021 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     1081 ttgaggcctg gcttgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     1141 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     1201 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     1261 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     1321 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     1381 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     1441 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     1501 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     1561 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     1621 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     1681 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     1741 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     1801 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga ccggtgccac
     1861 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
     1921 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
     1981 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
     2041 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     2101 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
     2161 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     2221 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     2281 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     2341 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     2401 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     2461 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     2521 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     2581 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     2641 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     2701 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     2761 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     2821 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     2881 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     2941 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     3001 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     3061 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     3121 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     3181 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     3241 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     3301 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     3361 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     3421 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     3481 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     3541 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     3601 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     3661 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     3721 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     3781 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     3841 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     3901 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
     3961 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
     4021 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
     4081 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     4141 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
     4201 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     4261 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
     4321 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     4381 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
     4441 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     4501 cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga
     4561 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     4621 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     4681 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     4741 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     4801 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     4861 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     4921 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     4981 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     5041 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     5101 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
     5161 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     5221 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     5281 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     5341 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     5401 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
     5461 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     5521 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     5581 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
     5641 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
     5701 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     5761 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     5821 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     5881 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     5941 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
     6001 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
     6061 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     6121 aaagaaaaag gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     6181 cgaggagaat cctggcccaa tgcctgaacc ctctaagtct gctccagccc ctaaaaaggg
     6241 ttctaagaag gctatcacta aggcgcagaa gaaggatggt aagaagcgta agcgcagccg
     6301 caaggagagc tattctatct atgtgtacaa ggttctgaag caggtccacc ccgacaccgg
     6361 catctcatcc aaggccatgg ggatcatgaa ctccttcgtc aacgacatct tcgagcgcat
     6421 cgcgggcgag gcttctcgcc tggctcacta caataagcgc tcgaccatca cctccaggga
     6481 gattcagacg gctgtgcgcc tgctgctgcc tggggagctg gctaagcatg ctgtgtccga
     6541 gggcactaag gcagttacca agtacactag ctctaaggat ccaccggtcg ccaccatggt
     6601 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     6661 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     6721 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     6781 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     6841 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     6901 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     6961 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     7021 gaagaaaacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     7081 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     7141 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     7201 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     7261 cgagggccgc cactccaccg gcggcatgga cgagctgtac aaggaattct aactagagct
     7321 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     7381 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     7441 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     7501 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc
     7561 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     7621 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     7681 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     7741 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     7801 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     7861 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     7921 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     7981 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     8041 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     8101 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     8161 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     8221 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     8281 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     8341 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     8401 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     8461 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     8521 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     8581 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     8641 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     8701 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     8761 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     8821 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     8881 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     8941 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     9001 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     9061 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9121 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     9181 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     9241 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     9301 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     9361 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     9421 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     9481 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     9541 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     9601 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     9661 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     9721 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     9781 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     9841 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     9901 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     9961 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
    10021 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
    10081 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
    10141 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
    10201 gctcgtcagg ggggcg
//
