LOCUS       sequence-328109-       10213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for cloning gRNA sequences for DNA targeting. 
            Contains an T2A linked H2B mCherry nuclear reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10213)
  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 10213)
  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
FEATURES             Location/Qualifiers
     source          1..10213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        67..1249
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          298..1240
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1197..1217
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1255..1264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1264..1329
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1336..1356
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1381..5481
                     /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             1381..5481
                     /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             5482..5529
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5545..5598
                     /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             5599..5976
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      5989..5998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5995..6702
                     /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(6140..6158)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6730..6747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6736..6943
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6952..7092
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6952..7081
                     /label=AAV2 ITR
     rep_origin      7167..7622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7254..7273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7464..7485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7639..7658)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7758..7780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7818..7836)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7904..8008
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8009..8077,8078..8869)
                     /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(8227..8246)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9040..9628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9529..9548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9690..9938
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9690..9710
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9861..9880
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9957..10032
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cgctgcgttc ccgccccctc acccgcccgc tctcgtcatc actgaggtgg agaagagcat
       61 gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
      121 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      181 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
      241 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
      301 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
      361 tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg
      421 aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt
      481 tgaggcctgg cttgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg
      541 tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct
      601 ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt
      661 ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
      721 gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc
      781 tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg
      841 gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc
      901 aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag
      961 ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag
     1021 gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt
     1081 ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca
     1141 cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa
     1201 gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgac cggtgccacc
     1261 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
     1321 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
     1381 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     1441 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     1501 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     1561 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     1621 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     1681 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1741 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1801 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1861 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1921 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     1981 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     2041 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     2101 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     2161 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     2221 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     2281 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     2341 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     2401 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     2461 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     2521 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     2581 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     2641 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     2701 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2761 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2821 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2881 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     2941 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     3001 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     3061 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     3121 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     3181 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     3241 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     3301 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     3361 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     3421 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     3481 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     3541 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     3601 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     3661 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3721 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3781 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3841 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3901 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     3961 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     4021 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     4081 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     4141 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     4201 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     4261 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     4321 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     4381 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     4441 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     4501 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     4561 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     4621 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     4681 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4741 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     4801 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4861 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4921 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     4981 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     5041 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     5101 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     5161 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     5221 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     5281 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     5341 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     5401 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     5461 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     5521 aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc
     5581 gaggagaatc ctggcccaat gcctgaaccc tctaagtctg ctccagcccc taaaaagggt
     5641 tctaagaagg ctatcactaa ggcgcagaag aaggatggta agaagcgtaa gcgcagccgc
     5701 aaggagagct attctatcta tgtgtacaag gttctgaagc aggtccaccc cgacaccggc
     5761 atctcatcca aggccatggg gatcatgaac tccttcgtca acgacatctt cgagcgcatc
     5821 gcgggcgagg cttctcgcct ggctcactac aataagcgct cgaccatcac ctccagggag
     5881 attcagacgg ctgtgcgcct gctgctgcct ggggagctgg ctaagcatgc tgtgtccgag
     5941 ggcactaagg cagttaccaa gtacactagc tctaaggatc caccggtcgc caccatggtg
     6001 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     6061 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     6121 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     6181 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     6241 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     6301 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     6361 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     6421 aagaaaacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     6481 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     6541 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     6601 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     6661 gagggccgcc actccaccgg cggcatggac gagctgtaca aggaattcta actagagctc
     6721 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     6781 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     6841 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     6901 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg caggaacccc
     6961 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     7021 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     7081 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     7141 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     7201 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     7261 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     7321 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     7381 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     7441 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     7501 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     7561 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     7621 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     7681 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     7741 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     7801 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     7861 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     7921 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     7981 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     8041 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     8101 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     8161 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     8221 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     8281 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     8341 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     8401 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     8461 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     8521 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     8581 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     8641 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     8701 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     8761 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     8821 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     8881 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     8941 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     9001 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     9061 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     9121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     9181 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     9241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     9301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     9361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     9421 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     9481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     9541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     9601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     9661 ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt ccttcatatt
     9721 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     9781 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     9841 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     9901 tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtcttcgaga agacctgttt
     9961 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
    10021 ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
    10081 tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtaccag taattcatac
    10141 aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt
    10201 agaacccaga gat
//
