LOCUS       sequence-455546-       12061 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of tp1 (Notch) dependent Cas9-GFP and U6-driven 4 sgRNAs 
            in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12061)
  AUTHORS   Siniscalco AM, Perera RP, Greenslade JE, Veeravenkatasubramanian H, 
            Masters A, Doll HM, Raj B
  TITLE     Barcoding Notch signaling in the developing brain.
  JOURNAL   Development. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102.
  PUBMED    39575683
REFERENCE   2  (bases 1 to 12061)
  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. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102."
COMMENT     Sequence Label: p104Tol2-tp1:Cas9-t2A-GFP, 4xU6:sgRNA
FEATURES             Location/Qualifiers
     source          1..12061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             2394..2402
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2497..2517
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2533..2586
                     /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(2593..2595,2596..2598,2599..3312)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2593..2595,2596..2598,2599..3309)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2638..2659)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2899..2918)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3246..3267
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(3349..3367)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(3396..3420)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3422..3556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(3619..3639)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(3647..3663)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    5836..5970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(6409..6596,6597..6608)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6642..6658)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6642..6658)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(6695..6713)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6734..6750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6747..6769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6758..6774
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6782..6788,6789..6806,6807..6812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6827..6848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6965..6982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7136..7724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7216..7235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7895..8686,8687..8755))
                     /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     8518..8537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8756..8860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9023..9044)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(9393..9404,9405..9567)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     9940..9956
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    9976..9996
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    10291..10315
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     10317..10333
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10343..10362
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        10343..10361
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      10397..10406
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             10415..10435
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(10442..12061,1..2481)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes MGAS15252 Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL
                     ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 tcaggagagc agaagaaagc aatcgtggat ctgctgttta aaaccaacag gaaagtgact
       61 gtcaagcagc tgaaggagga ctacttcaag aaaattgaat gcttcgattc cgtggagatc
      121 agcggagtcg aagacagatt taacgcaagc ctgggcgctt accacgatct gctgaagatc
      181 attaaggata aagacttcct ggacaacgag gaaaatgagg atatcctgga agacattgtg
      241 ctgacactga ccctgtttga ggacagagga atgatcgagg aaagactgaa aacctatgct
      301 catctgttcg atgacaaggt gatgaaacag ctgaagagaa ggcgctacac tggctggggt
      361 agactgagca ggaagctgat caacggcatt agggataaac agtcaggaaa gacaatcctg
      421 gactttctga aaagtgatgg cttcgccaac cgcaatttta tgcagctgat tcacgatgac
      481 agtctgacct tcaaagagga catccagaag gctcaggtgt ctggacaggg ccactccctg
      541 catgagcaga ttgcaaacct ggctggaagc ccagccatca agaaaggcat tctgcagaca
      601 gtgaaaatcg tcgatgagct ggtgaaagtc atgggccata agcccgaaaa catcgtgatt
      661 gagatggctc gcgaaaatca gacaacccag aagggtcaga agaacagtag agagaggatg
      721 aaaagaatcg aggaaggcat taaggagctg ggtagccaga tcctgaaaga gcacccagtg
      781 gaaaacacac agctgcagaa tgagaagctg tatctgtact atctgcagaa tggaagagat
      841 atgtacgtgg accaggagct ggatattaac aggctgtctg attacgacgt ggatcatatc
      901 gtcccccaga gtttcatcaa agatgacagc attgacaaca aggtgctgac caggtccgac
      961 aaaaacagag gaaaatcaga taatgtccct agtgaggaag tggtcaagaa aatgaagaac
     1021 tactggagac agctgctgaa tgccaaactg atcactcaga ggaagtttga taacctgaca
     1081 aaagcagagc gcggtggact gtcagaactg gacaaagctg gattcatcaa gaggcagctg
     1141 gtggaaacac gccagatcac taaacacgtc gcacagattc tggatagtcg catgaacaca
     1201 aagtacgatg agaatgacaa actgatcaga gaagtgaagg tcattaccct gaagagtaaa
     1261 ctggtcagcg actttaggaa agatttccag ttttataagg tccgcgagat taacaattat
     1321 caccatgccc atgacgcata cctgaacgcc gtggtcggta ccgcactgat caagaaatac
     1381 ccaaaactgg agagcgaatt cgtgtacgga gactataagg tgtacgatgt cagaaaaatg
     1441 atcgccaagt ccgagcagga aattggaaaa gctactgcca agtatttctt ttactcaaac
     1501 atcatgaatt tctttaagac agagatcacc ctggccaatg gagaaatccg caaaaggccc
     1561 ctgattgaga caaacggaga gacaggcgaa atcgtgtggg acaaaggcag agattttgca
     1621 accgtgagga aggtcctgag catgcctcaa gtgaatatcg tcaagaaaac tgaggtgcag
     1681 acaggcggtt tctcaaaaga aagtattctg ccaaaacgca actctgataa gctgatcgct
     1741 agaaagaaag actgggaccc taagaagtat ggaggctttg actctcccac tgtggcatac
     1801 tccgtcctgg tggtcgctaa ggtggagaag ggcaaaagca agaaactgaa atctgtcaag
     1861 gagctgctgg gtatcacaat tatggagaga agctctttcg agaagaaccc aatcgatttt
     1921 ctggaggcca aaggttataa ggaagtgaag aaagacctga tcattaaact gcccaagtac
     1981 agtctgtttg agctggaaaa cggcaggaaa cgcatgctgg caagcgctgg agagctgcag
     2041 aaaggcaatg aactggccct gccttctaag tacgtgaact tcctgtatct ggcaagccac
     2101 tacgagaagc tgaaaggatc tccagaggat aacgaacaga aacagctgtt tgtggagcag
     2161 cacaagcatt atctggacga gatcattgaa cagattagcg agttctctaa aagagtgatc
     2221 ctggccgacg caaatctgga taaggtcctg tctgcttaca acaaacacag agataagccc
     2281 atcagggagc aggccgaaaa tatcattcat ctgttcactc tgacaaacct gggcgcacct
     2341 gcagctttca agtacttcga cactacaatc gatagaaaga ggtacacctc cactaaggag
     2401 gtgctggacg ctacactgat ccatcagagt attaccggcc tgtacgaaac aaggattgac
     2461 ctgtctcagc tgggtggcga ctcaccggtg ggatccccta agaagaagag aaaggtgagg
     2521 tctggtggcg gagagggcag aggcagtctg ctgacatgcg gtgacgtgga agagaatccc
     2581 ggcccttcta gaatggtgag caagggcgaa gagctgttca ccggggtggt gcccatcctg
     2641 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2701 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     2761 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     2821 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     2881 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2941 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3001 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3061 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3121 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3181 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3241 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3301 ctgtacaagt aagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg
     3361 gttaattgcg cgcttggcgt aatcatggtc atagcaccca gctttcttgt acaaagtggg
     3421 ggatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3481 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3541 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3601 ggtgtgggag gttttttcca actttattat acatagttga taattcactg gccgtcgttt
     3661 tacggtacca ggtttaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     3721 ctagccttat ttaaacttgc tatgctgttt ccagcatagc tcttaaaccg tagtagtagc
     3781 cgggcagcga actaggagcc tggagaactg ctatataaac ccgggacttc tgggtatgtt
     3841 tctgccatgt ggtaagtgga taaacctact tattcaactc atttacctca aacatgacag
     3901 taaacctctt cggggcaaac ctgcaaacgc ttaaaacctt caaagtttga aaccagcagt
     3961 tttgtagagg tttaccgtag tatttttgaa ctgtaaagca tatgcagagg aagctggttt
     4021 taggcttgtg tgattggatg agttcttctg tgacgtaact gatcttttgt cctcgtgtcc
     4081 ttcgccttcc cccacaggca tgcgcggacc ttcccccgcc gcagaccaga ggacaacatg
     4141 ctcagacgcc agcaaacggc ccatcgaaac attcacagac tctcggattg gtttaaaaaa
     4201 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt taaacttgct
     4261 atgctgtttc cagcatagct cttaaactgt agatggactt gaactccgaa ctgggagtct
     4321 ggaggacggc tatatatacc agtgacttct gggtatgtgt ttcatgtgtg gtatatggct
     4381 tacttactta ttcaactcat ttacacttac atgtgtcagt aaatcactgt gtagtagtaa
     4441 acctgtaaac ccttaaattc tttaaaattt gaaaaccagc agtttatgta ggtttaccgt
     4501 agagttttgg agctgtaagc gtatgcgaag gaaacaggcc gtttctttag agtgattgga
     4561 tgagttcctg aacttcctgt gtgacatcga ggagcttttg ttcacgtgtc cttctcctcc
     4621 ccccacaggc atgcgcagat cccccctgcc ttggataaga gcacaaaaca ctccgacaaa
     4681 aggaagctgc tctcactaga gcagagccca tgaaaaccca gacagacacg tggagggtaa
     4741 tgagtttaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     4801 atttaaactt gctatgctgt ttccagcata gctcttaaac ccgtccttca gcttcagggc
     4861 gaaccaagag ctggagggag agctatatat accagggact tctgggtatg tttttgggag
     4921 gtggtgagtg actaaaccac ttattcagct cccttagatc aagtctgacc ctatatcatg
     4981 gtgacataaa cctgcaaact gataaaacct gaaggatctc aaatccagag tttgtgtgag
     5041 ggattaccgt ggtaattttc aaactgtaaa gcatatgcaa aattatctgg tcttggcttg
     5101 agtgattggg tgtctcggtg tgatgcaggg acgttttcag tgacgtgtcc ttctccctcc
     5161 cccacaggca tgcgcagaac atttcccccc tccttgaaga ccagaacaaa agacgccgag
     5221 agcaggaaac tcgtcttact gaatgaccga ggctggagaa acgctgagag acttgactaa
     5281 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt atttaaactt
     5341 gctatgctgt ttccagcata gctcttaaac ccccagaagt cctccagtcc gaaccaagag
     5401 ctggagggag agctatatat accagggact tctgggtatg tttttgggag gtggtgagtg
     5461 actaaaccac ttattcagct cccttagatc aagtctgacc ctatatcatg gtgacataaa
     5521 cctgcaaact gataaaacct gaaggatctc aaatccagag tttgtgtgag ggattaccgt
     5581 ggtaattttc aaactgtaaa gcatatgcaa aattatctgg tcttggcttg agtgattggg
     5641 tgtctcggtg tgatgcaggg acgttttcag tgacgtgtcc ttctccctcc cccacaggca
     5701 tgcgcagaac atttcccccc tccttgaaga ccagaacaaa agacgccgag agcaggaaac
     5761 tcgtcttact gaatgaccga ggctggagaa acgctgagag acttactgat gcatttcatt
     5821 taatgtatcg atgatgatcc agacatgata agatacattg atgagtttgg acaaaccaca
     5881 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     5941 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     6001 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     6061 atggctgatt atgatcctct agatcagatc tgcgaagata cggccacggg tgctcttgat
     6121 cctgtggctg attttggact gtgctgctcg cagctgctga tgaatcacat acttcctcca
     6181 ttttcttcca ctgattgact gttataattt ccctaatttc caggtcaagg tgctgtgcat
     6241 tgtggtaata gatgtgacat gacgtcactt ccaaaggacc aatgaacatg tctgaccaat
     6301 ttcatataat gtgaaaacga ttttcatagg cagaataaat aacatttaaa ttaaactggg
     6361 catcagcgca attcaattgg tttggtaata gcaagggaaa atagaatgaa gtgatctcca
     6421 aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta cttttttttc
     6481 taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact caagtaaagt
     6541 aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc aagtacttta
     6601 cacctctggt tcttgacccc ctaccttcag caagcccagc agatccacta gttctagagc
     6661 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     6721 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6781 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6841 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6901 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6961 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     7021 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     7081 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     7141 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     7201 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     7261 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7321 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7381 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7441 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7501 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7561 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7621 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7681 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7741 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaaggcatt ttggtcatga
     7801 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7861 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7921 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7981 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     8041 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     8101 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     8161 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     8221 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     8281 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8341 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8401 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8461 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8521 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8581 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8641 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8701 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8761 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8821 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8881 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     8941 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     9001 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     9061 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccacgct ggctaagaac
     9121 tcatcagcct ccccggtcca tctacccacg taccaatgca ccaattggcc acaatgacgg
     9181 ctactacatg gtgccattcc ttcctcttta taggaatgga gactacctcc tgtccaacaa
     9241 tgctcttgga tacgagtacg cctacctgtt ggacccaggt cattgcacaa caccagaaat
     9301 gccctctgat ctgcaaaaga cgtgaatatc tgttcagaca cccatatcca ctctgttcca
     9361 cacaggtcag aggtttgtcc aggagttctt gacagaggtg taaaaagtac tcaaaaattt
     9421 tactcaagtg aaagtacaag tacttaggga aaattttact caattaaaag taaaagtatc
     9481 tggctagaat cttacttgag taaaagtaaa aaagtactcc attaaaattg tacttgagta
     9541 ttaaggaagt aaaagtaaaa gcaagaaaga aaactagaga ttcttgttta agcttttaat
     9601 ctcaaaaaac attaaatgaa atgcatacaa ggttttatcc tgctttagaa ctgtttgtat
     9661 ttaattatca aactataaga cagacaatct aatgccagta cacgctactc aaagttgtaa
     9721 aacctcagat ttaacttcag tagaagctga ttctcaaaat tgttagtgtc aagcctagct
     9781 cttttggggc tgaaaagcaa tcctgcagtg ctgaaaagcc tctcacaggc agccgatgcg
     9841 ggaagaggtg tattagtctt gatagagagg ctgcaaatag caggaaacgt gagcagagac
     9901 tccctggtgt ctgaaacaca ggccagatgg gccctcgagc aggaaacagc tatgaccatg
     9961 attacgccaa gctatcaact ttgtatagaa aagttgtgca ggtcgactct agaggatccc
    10021 gactcatggg aaaatgggcg gaagggcacc gtgggaaaat agtagatccc gactcgtggg
    10081 aaaatgggcg gaagggcacc gtgggaaaat agtagatccc gactcgtggg aaaatgggcg
    10141 gaagggcacc gtgggaaaat agtagatcca ctgtatgcat cttgggcata aaaggcagat
    10201 cacttcagct tctgcttaca cttgcttttg acacaactgt gtttacttgc aatccctcga
    10261 atcgctagca tcgattcgaa ttcaaggatc acaagtttgt acaaaaaagc aggcttgtaa
    10321 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc
    10381 ccccctcgag gtcgacgcca ccatggcttc tccacctaag aagaagagaa aggtgggaag
    10441 catggataag aagtatagca tcggcctgga tattggaact aactccgtgg gttgggcagt
    10501 gattacagac gactacaagg tccctagcaa gaaatttaag gtgctgggta acaccgacag
    10561 gcacagcatc aagaaaaatc tgattggagc cctgctgttc ggttctggag agactgccga
    10621 agcaacacgc ctgaaaagaa cagcaagaag gcgctatacc agaaggaaga atagaatctg
    10681 ttacctgcag gagattttct ctaacgaaat ggctaaggtg gacgattcat tctttcatag
    10741 gctggaggaa agtttcctgg tcgaggaaga taagaaacac gagcgccatc ctatctttgg
    10801 aaacattgtg gacgaggtcg cctatcacga aaaataccca accatctatc atctgcgcaa
    10861 gaaactggct gactctactg ataaagccga cctgagactg atctatctgg ctctggccca
    10921 catgattaag ttcaggggtc attttctgat cgagggcgat ctgaaccccg acaattccga
    10981 tgtggacaag ctgttcatcc agctggtcca gatttacaat cagctgtttg aggaaaaccc
    11041 tattaatgct tccagagtgg acgcaaaagc tatcctgtca gccaggctgt ccaagtcacg
    11101 cagactggag aacctgattg cacagctgcc cggagaaaag aggaacggtc tgtttggaaa
    11161 tctgatcgct ctgagtctgg gcctgactcc taacttcaaa agcaattttg atctggctga
    11221 ggacgccaaa ctgcagctgt caaaggacac atatgacgat gacctggata acctgctggc
    11281 acagatcgga gatcagtacg ctgacctgtt cctggctgcc aaaaatctgt ccgacgcaat
    11341 cctgctgtca gatattctga gagtgaacag cgagattaca aaagcacctc tgagtgccag
    11401 catgatcaag agatatgacg agcaccatca ggatctgacc ctgctgaagg ctctggtcag
    11461 gcagcagctg ccagagaagt acaaggaaat tttctttgat cagtccaaga acggctacgc
    11521 cggttatatc gacggaggcg catcacagga ggaattctac aagtttatca aacctattct
    11581 ggagaagatg gacggaactg aggaactgct ggtgaaactg aatagagagg acctgctgag
    11641 gaagcagcgc acatttgata acggttccat cccacaccag attcatctgg gagagctgca
    11701 cgctatcctg aggcgccagg aagacttcta cccctttctg aaagataacc gcgagaagat
    11761 cgaaaaaatt ctgaccttca gaatccctta ctatgtgggt ccactggctc gcggaaacag
    11821 cagatttgcc tggatgactc gcaaatccga ggaaaccatt actccttgga acttcgagga
    11881 agtggtcgat aagggcgcct ctgcacagtc cttcatcgag agaatgacta attttgacaa
    11941 aaacctgccc aatgagaaag tgctgcctaa gcactccctg ctgtacgagt atttcactgt
    12001 ctataacgaa ctgacaaagg tgaaatacgt caccgagggc atgagaaagc cagccttcct
    12061 g
//
