LOCUS       sequence-328112-        9826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9 expressing plasmid containing the gRNA targeting mouse 
            Ascl1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9826)
  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 9826)
  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-GFP (gRNA: Ascl1-1)
FEATURES             Location/Qualifiers
     source          1..9826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(209..228)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     556..577
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(647..664)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    653..860
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   869..1009
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   869..998
                     /label=AAV2 ITR
     rep_origin      1084..1539
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1171..1190)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1381..1402
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1556..1575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1675..1697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1735..1753)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1821..1925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1926..1994,1995..2786)
                     /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(2144..2163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2957..3545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3446..3465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3607..3855
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3607..3627
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3778..3797
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3877..3952
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4200..5382
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4431..5373
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5330..5350
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      5388..5397
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5397..5462
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5469..5489
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5514..9614
                     /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             5514..9614
                     /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             9615..9662
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9678..9731
                     /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             join(9732..9734,9735..9826,1..619)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(9732..9734,9735..9826,1..619)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
ORIGIN
        1 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
       61 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
      121 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      181 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
      241 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
      301 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
      361 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
      421 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
      481 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
      541 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
      601 catggacgag ctgtacaagg aattctaact agagctcgct gatcagcctc gactgtgcct
      661 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
      721 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
      781 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
      841 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc
      901 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
      961 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     1021 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     1081 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1141 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1201 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1261 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     1321 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1381 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     1441 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     1501 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     1561 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     1621 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     1681 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     1741 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     1801 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1861 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     1921 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     1981 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2041 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2101 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2161 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2221 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2281 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2341 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2401 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2461 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2521 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2581 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     2641 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     2701 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     2761 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     2821 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     2881 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2941 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3001 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3061 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3121 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3181 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3241 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3301 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3361 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3421 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3481 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3541 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3601 acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     3661 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     3721 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     3781 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     3841 aaaggacgaa acaccgagcc tgcttctttg cgaccggttt tagagctaga aatagcaagt
     3901 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
     3961 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
     4021 tctgcagaca aatggctcta gaggtaccag taattcatac aaaaggactc gcccctgcct
     4081 tggggaatcc cagggaccgt cgttaaactc ccactaacgt agaacccaga gatcgctgcg
     4141 ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg tggagaagag catgcgtgag
     4201 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
     4261 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     4321 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     4381 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     4441 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     4501 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     4561 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     4621 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     4681 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     4741 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     4801 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     4861 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     4921 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     4981 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     5041 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     5101 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     5161 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     5221 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     5281 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     5341 acagtggttc aaagtttttt tcttccattt caggtgtcgt gaccggtgcc accatggact
     5401 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata
     5461 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     5521 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     5581 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     5641 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     5701 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     5761 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     5821 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     5881 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     5941 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     6001 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     6061 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     6121 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     6181 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     6241 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     6301 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     6361 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     6421 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     6481 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     6541 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     6601 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     6661 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     6721 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     6781 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     6841 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     6901 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     6961 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     7021 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     7081 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     7141 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     7201 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     7261 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     7321 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     7381 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     7441 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     7501 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     7561 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     7621 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     7681 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     7741 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     7801 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     7861 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     7921 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     7981 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     8041 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     8101 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     8161 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     8221 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     8281 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     8341 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     8401 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     8461 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     8521 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     8581 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     8641 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     8701 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     8761 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     8821 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     8881 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     8941 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     9001 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     9061 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     9121 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     9181 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     9241 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     9301 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     9361 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     9421 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     9481 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     9541 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     9601 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     9661 aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     9721 atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     9781 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcga
//
