LOCUS       sequence-328126-       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: Neurog2-1)
FEATURES             Location/Qualifiers
     source          1..10216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        118..1300
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          349..1291
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1248..1268
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1306..1315
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1315..1380
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1387..1407
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1432..5532
                     /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             1432..5532
                     /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             5533..5580
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5596..5649
                     /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             5650..6027
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6040..6049
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6046..6753
                     /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(6191..6209)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6781..6798)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6787..6994
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7003..7143
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7003..7132
                     /label=AAV2 ITR
     rep_origin      7218..7673
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7305..7324)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7515..7536
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7690..7709)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7809..7831
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7869..7887)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7955..8059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8060..8128,8129..8920)
                     /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(8278..8297)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9091..9679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9580..9599
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9741..9989
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9741..9761
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9912..9931
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10011..10086
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga tcgctgcgtt
       61 cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca tgcgtgaggc
      121 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      181 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      241 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
      301 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg
      361 tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact
      421 tccacgcccc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
      481 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
      541 gcttgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
      601 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
      661 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
      721 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
      781 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
      841 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
      901 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
      961 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1021 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1081 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1141 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1201 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1261 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ccggtgccac catggactat
     1321 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag
     1381 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
     1441 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1501 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1561 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1621 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1681 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1741 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1801 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1861 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1921 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1981 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     2041 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     2101 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     2161 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     2221 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2281 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2341 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2401 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2461 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2521 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2581 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2641 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2701 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2761 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2821 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2881 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2941 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     3001 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     3061 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     3121 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     3181 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     3241 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     3301 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3361 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3421 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3481 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3541 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3601 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3661 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3721 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3781 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3841 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3901 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3961 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     4021 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     4081 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     4141 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     4201 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     4261 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     4321 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4381 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4441 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4501 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4561 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4621 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4681 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4741 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4801 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     4861 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4921 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4981 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     5041 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     5101 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     5161 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     5221 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     5281 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     5341 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     5401 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     5461 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5521 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     5581 gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     5641 cctggcccaa tgcctgaacc ctctaagtct gctccagccc ctaaaaaggg ttctaagaag
     5701 gctatcacta aggcgcagaa gaaggatggt aagaagcgta agcgcagccg caaggagagc
     5761 tattctatct atgtgtacaa ggttctgaag caggtccacc ccgacaccgg catctcatcc
     5821 aaggccatgg ggatcatgaa ctccttcgtc aacgacatct tcgagcgcat cgcgggcgag
     5881 gcttctcgcc tggctcacta caataagcgc tcgaccatca cctccaggga gattcagacg
     5941 gctgtgcgcc tgctgctgcc tggggagctg gctaagcatg ctgtgtccga gggcactaag
     6001 gcagttacca agtacactag ctctaaggat ccaccggtcg ccaccatggt gagcaagggc
     6061 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     6121 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     6181 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     6241 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     6301 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     6361 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     6421 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaaaacc
     6481 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     6541 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     6601 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     6661 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     6721 cactccaccg gcggcatgga cgagctgtac aaggaattct aactagagct cgctgatcag
     6781 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     6841 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     6901 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     6961 aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg
     7021 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     7081 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     7141 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     7201 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     7261 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     7321 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     7381 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     7441 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     7501 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     7561 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     7621 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     7681 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     7741 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     7801 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     7861 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     7921 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     7981 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     8041 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     8101 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     8161 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     8221 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     8281 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     8341 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     8401 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     8461 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     8521 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     8581 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     8641 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     8701 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     8761 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     8821 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     8881 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     8941 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     9001 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     9061 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     9121 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     9181 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     9241 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     9301 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     9361 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     9421 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     9481 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     9541 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     9601 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     9661 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     9721 ctggcctttt gctcacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac
     9781 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     9841 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     9901 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     9961 tatatatctt gtggaaagga cgaaacaccg catcggggtc agggtcgccg gttttagagc
    10021 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
    10081 cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta
    10141 gcgcgtgcgc caattctgca gacaaatggc tctagaggta ccagtaattc atacaaaagg
    10201 actcgcccct gccttg
//
