LOCUS       sequence-328127-       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: Neurog2-3)
FEATURES             Location/Qualifiers
     source          1..10216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        81..1263
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          312..1254
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1211..1231
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1269..1278
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1278..1343
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1350..1370
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1395..5495
                     /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             1395..5495
                     /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             5496..5543
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5559..5612
                     /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             5613..5990
                     /codon_start=1
                     /gene="HIST1H2BB"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPSKSAPAPKKGSKKAITKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSSK"
     regulatory      6003..6012
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6009..6716
                     /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(6154..6172)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6744..6761)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6750..6957
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6966..7106
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6966..7095
                     /label=AAV2 ITR
     rep_origin      7181..7636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7268..7287)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7478..7499
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7653..7672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7772..7794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7832..7850)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7918..8022
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8023..8091,8092..8883)
                     /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(8241..8260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9054..9642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9543..9562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        9704..9952
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     9704..9724
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     9875..9894
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        9974..10049
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tagaacccag agatcgctgc gttcccgccc cctcacccgc ccgctctcgt catcactgag
       61 gtggagaaga gcatgcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      121 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      181 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      241 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      301 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      361 gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc
      421 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
      481 cctcgtgctt gagttgaggc ctggcttggg cgctggggcc gccgcgtgcg aatctggtgg
      541 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
      601 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
      661 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
      721 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
      781 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
      841 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
      901 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
      961 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     1021 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     1081 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     1141 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     1201 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     1261 tgaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat catgatattg
     1321 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
     1381 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     1441 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     1501 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     1561 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     1621 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     1681 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     1741 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     1801 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     1861 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     1921 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     1981 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     2041 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     2101 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     2161 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     2221 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     2281 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     2341 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     2401 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     2461 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     2521 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     2581 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     2641 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     2701 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     2761 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     2821 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     2881 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     2941 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     3001 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     3061 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     3121 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     3181 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     3241 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     3301 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     3361 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     3421 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     3481 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     3541 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     3601 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     3661 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     3721 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     3781 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     3841 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     3901 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     3961 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     4021 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     4081 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     4141 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     4201 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     4261 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     4321 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     4381 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     4441 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     4501 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     4561 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     4621 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     4681 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     4741 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     4801 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     4861 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     4921 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     4981 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     5041 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     5101 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     5161 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     5221 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     5281 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     5341 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     5401 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     5461 agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg
     5521 ccggccaggc aaaaaagaaa aaggaattcg gcagtggaga gggcagagga agtctgctaa
     5581 catgcggtga cgtcgaggag aatcctggcc caatgcctga accctctaag tctgctccag
     5641 cccctaaaaa gggttctaag aaggctatca ctaaggcgca gaagaaggat ggtaagaagc
     5701 gtaagcgcag ccgcaaggag agctattcta tctatgtgta caaggttctg aagcaggtcc
     5761 accccgacac cggcatctca tccaaggcca tggggatcat gaactccttc gtcaacgaca
     5821 tcttcgagcg catcgcgggc gaggcttctc gcctggctca ctacaataag cgctcgacca
     5881 tcacctccag ggagattcag acggctgtgc gcctgctgct gcctggggag ctggctaagc
     5941 atgctgtgtc cgagggcact aaggcagtta ccaagtacac tagctctaag gatccaccgg
     6001 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     6061 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     6121 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     6181 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     6241 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     6301 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     6361 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     6421 gccccgtaat gcagaagaaa accatgggct gggaggcctc ctccgagcgg atgtaccccg
     6481 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     6541 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     6601 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     6661 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggaat
     6721 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     6781 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     6841 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     6901 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     6961 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     7021 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     7081 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     7141 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     7201 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     7261 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7321 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7381 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7441 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7501 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     7561 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7621 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7681 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     7741 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     7801 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     7861 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     7921 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     7981 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     8041 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8101 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8161 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8221 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     8281 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8341 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8401 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     8461 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     8521 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     8581 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     8641 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     8701 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     8761 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     8821 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     8881 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     8941 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     9001 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     9061 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     9121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     9181 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     9241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     9301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     9361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     9481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     9541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     9601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     9661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat
     9721 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     9781 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     9841 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     9901 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtcgtgag
     9961 cttggcatcc tcggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
    10021 aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt
    10081 aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag
    10141 gtaccagtaa ttcatacaaa aggactcgcc cctgccttgg ggaatcccag ggaccgtcgt
    10201 taaactccca ctaacg
//
