LOCUS       sequence-42182-e       17238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of ChR2(H134R)-EYFP, for optogenetic 
            excitation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 17238)
  AUTHORS   Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, Hsu YW, 
            Garcia AJ 3rd, Gu X, Zanella S, Kidney J, Gu H, Mao Y, Hooks BM, 
            Boyden ES, Buzsaki G, Ramirez JM, Jones AR, Svoboda K, Han X, Turner
            EE, Zeng H
  TITLE     A toolbox of Cre-dependent optogenetic transgenic mice for 
            light-induced activation and silencing.
  JOURNAL   Nat Neurosci. 2012 Mar 25;15(5):793-802. doi: 10.1038/nn.3078.
  PUBMED    22446880
REFERENCE   2  (bases 1 to 17238)
  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"; doi: "10.1038/nn"; 
            journalName: "Nat Neurosci"; date: "2012-03-25- 25"; volume: "15"; 
            issue: "5"; pages: "793-802"
COMMENT     Sequence Label: Ai32
FEATURES             Location/Qualifiers
     source          1..17238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    3..1087
                     /label=Rosa26 left arm
                     /note="left homology arm from intron 1 of the mouse Rosa26 
                     gene (Soriano, 1999)"
     enhancer        1181..1484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1486..1763
                     /label=chicken beta-actin promoter
     intron          1765..2782
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2706..2728
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2790..2809
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    2858..2891
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    2915..2948
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3116..3250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3370..3504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3624..3758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3787..3820
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3928..3937
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3934..4860
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             3934..4860
                     /codon_start=1
                     /product="N-terminal fragment of channelrhodopsin-2 from 
                     the alga Chlamydomonas reinhardtii (Nagel et al., 2003)"
                     /label=ChR2
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      4870..4879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4876..4878,4879..4881,4882..5592)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4921..4942)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5182..5201)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5529..5550
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5686..6274
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5739..5759)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6157..6168)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6298..6505
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6523..6556
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        6578..7077
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     protein_bind    7097..7130
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     regulatory      7131..7140
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7137..7940
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7200..7219)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7810..7829
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7950..8411
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     misc_feature    8475..12744
                     /label=Rosa26 right arm
                     /note="right homology arm from intron 1 of the mouse Rosa26
                     gene (Soriano, 1999)"
     promoter        12793..13286
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             13313..13882
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphteria toxin Fragment A"
                     /label=DTA
                     /translation="DDVVILLIFCMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDD
                     DWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELG
                     LSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEIN
                     FETRGKRGQDAMYEYMAQACAGNRVRR"
     intron          13959..14024
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_signal    14098..14322
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(14390..14410)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(14390..14408)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(14429..14445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14429..14445)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14442..14464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14453..14469
                     /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(14477..14483,14484..14501,14502..14507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14522..14543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14660..14677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14831..15419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14911..14930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15590..16381,16382..16450))
                     /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     16213..16232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16451..16555)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(16581..17036)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(16718..16739)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     16930..16949
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     17163..17185
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     17177..17194
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     17178..17194
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     17204..17223
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        17204..17222
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ccgcggcagg ccctccgagc gtggtggagc cgttctgtga gacagccggg tacgagtcgt
       61 gacgctggaa ggggcaagcg ggtggtgggc aggaatgcgg tccgccctgc agcaaccgga
      121 gggggaggga gaagggagcg gaaaagtctc caccggacgc ggccatggct cggggggggg
      181 ggggcagcgg aggascgctt ccggccgacg tctcgtcgct gattggctty ttttcctccc
      241 gccgtgtgtg aaaacacaaa tggcgtgttt tggttggcgt aaggcgcctg tcagttaacg
      301 gcagccggag tgcgcagccg ccggcagcct cgctctgccc actgggtggg gcgggaggta
      361 ggtggggtga ggcgagctgn acgtgcgggc gcggtcggcc tctggcgggg cgggggaggg
      421 gagggagggt cagcgaaagt agctcgcgcg cgagcggccg cccaccctcc ccttcctctg
      481 ggggagtcgt tttacccgcc gccggccggg cctcgtcgtc tgattggctc tcggggccca
      541 gaaaactggc ccttgccatt ggctcgtgtt cgtgcaagtt gagtccatcc gccggccagc
      601 gggggcggcg aggaggcgct cccaggttcc ggccctcccc tcggccccgc gccgcagagt
      661 ctggccgcgc gcccctgcgc aacgtggcag gaagcgcgcg ctgggggcgg ggacgggcag
      721 tagggctgag cggctgcggg gcgggtgcaa gcacgtttcc gacttgagtt gcctcaagag
      781 gggcgtgctg agccagacct ccatcgcgca ctccggggag tggagggaag gagcgagggc
      841 tcagttgggc tgttttggag gcaggaagca cttgctctcc caaagtcgct ctgagttgtt
      901 atcagtaagg gagctgcagt ggagtaggcg gggagaaggc cgcacccttc tccggagggg
      961 ggaggggagt gttgcaatac ctttctggga gttctctgct gcctcctggc ttctgaggac
     1021 cgccctgggc ctgggagaat cccttgcccc ctcttcccct cgtgatctgc aactccagtc
     1081 tttctagtta attaaccgtt taaacaattc tgcaggaatc tagttattaa tagtaatcaa
     1141 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1201 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1261 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1321 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1381 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1441 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     1501 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     1561 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
     1621 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     1681 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     1741 aaaagcgaag cgcgcggcgg gcggggagtc gctgcgacgc tgccttcgcc ccgtgccccg
     1801 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     1861 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
     1921 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
     1981 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
     2041 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
     2101 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
     2161 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
     2221 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
     2281 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
     2341 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
     2401 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
     2461 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
     2521 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
     2581 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     2641 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     2701 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     2761 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     2821 ttttggcaaa gaattgattt gataccgcgg gccctaagaa gttcctattc tctagaaagt
     2881 ataggaactt cgtcgacatt taaatcattt aaatataact tcgtataatg tatgctatac
     2941 gaagttattc gcgatgaata aatgaaagct tgcagatctg cgactctaga ggatctgcga
     3001 ctctagagga tcataatcag ccntaccaca ttttgtagag gttttactng ctttaaaaaa
     3061 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3121 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3181 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3241 tgtctggatc tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta
     3301 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3361 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3421 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3481 aatgtatctt atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat
     3541 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3601 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3661 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3721 tgtccaaact catcaatgta tcttatcatg tctggatccc catcaagctg atccggaacc
     3781 cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc gacctgcagc
     3841 ccaagctaga tcgaattcgg ccggcccctt cacgtgttgc acttaacgcg gcctaggtta
     3901 cgcgccattt aaatcctggc cggccttgcc accatggatt atggaggcgc cctgagtgcc
     3961 gttgggcgcg agctgctatt tgtaacgaac ccagtagtcg tcaatggctc tgtacttgtg
     4021 cctgaggacc agtgttactg cgcgggctgg attgagtcgc gtggcacaaa cggtgcccaa
     4081 acggcgtcga acgtgctgca atggcttgct gctggcttct ccatcctact gcttatgttt
     4141 tacgcctacc aaacatggaa gtcaacctgc ggctgggagg agatctatgt gtgcgctatc
     4201 gagatggtca aggtgattct cgagttcttc ttcgagttta agaacccgtc catgctgtat
     4261 ctagccacag gccaccgcgt ccagtggttg cgttacgccg agtggcttct cacctgcccg
     4321 gtcattctca ttcgcctgtc aaacctgacg ggcttgtcca acgactacag caggcgcacc
     4381 atgggtctgc ttgtgtctga tattggcaca attgtgtggg gcgccacttc cgccatggcc
     4441 accggatacg tcaaggtcat cttcttctgc ctgggtctgt gttatggtgc taacacgttc
     4501 tttcacgctg ccaaggccta catcgagggt taccacaccg tgccgaaggg ccggtgtcgc
     4561 caggtggtga ctggcatggc ttggctcttc ttcgtatcat ggggtatgtt ccccatcctg
     4621 ttcatcctcg gccccgaggg cttcggcgtc ctgagcgtgt acggctccac cgtcggccac
     4681 accatcattg acctgatgtc gaagaactgc tggggtctgc tcggccacta cctgcgcgtg
     4741 ctgatccacg agcatatcct catccacggc gacattcgca agaccaccaa attgaacatt
     4801 ggtggcactg agattgaggt cgagacgctg gtggaggacg aggccgaggc tggcgcagta
     4861 cccgcggccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     4921 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4981 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     5041 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac
     5101 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     5161 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     5221 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     5281 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     5341 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     5401 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     5461 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
     5521 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     5581 gagctgtaca agggttccgg ataaagcggc cgccggcaag tgaattcagg ccggccattt
     5641 aaatggcgcg ttttatctta ccgaaggttc ctagggctta tcgataatca acctctggat
     5701 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     5761 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     5821 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     5881 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     5941 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     6001 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     6061 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     6121 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     6181 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     6241 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgcctagg gcctcgactg
     6301 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     6361 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     6421 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     6481 aagacaatag caggcatgct ggggatatct ttcaccggtg tggtgccagg gcgtgccctt
     6541 gggctccccg ggcgcggcgg ccgcatcgaa ttctaccggg taggggaggc gcttttccca
     6601 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     6661 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     6721 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     6781 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     6841 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     6901 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     6961 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     7021 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     7081 tgcaatcgcc gctagcgaag ttcctattct ctagaaagta taggaacttc gccaccatgg
     7141 gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     7201 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     7261 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     7321 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     7381 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     7441 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     7501 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     7561 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     7621 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     7681 ccgacggcga tgatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     7741 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     7801 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     7861 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     7921 ttcttgacga gttcttctga ggggatccgc tgtaagtctg cagaaattga tgatctatta
     7981 aacaataaag atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg
     8041 agaacagagt acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg
     8101 ggattagata aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt
     8161 tcatagttgg atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc
     8221 ccactcatga tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc
     8281 cactttgtgg ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg
     8341 agatcagcag cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa
     8401 ttccatcaga aagcttgcag aagatctccc caactggggt aacctttgag ttctctcagt
     8461 tgggggggcg cgccctagaa gatgggcggg agtcttctgg gcaggcttaa aggctaacct
     8521 ggtgtgtggg cgttgtcctg caggggaatt gaacaggtgt aaaattggag ggacaagact
     8581 tcccacagat tttcggtttt gtcgggaagt tttttaatag gggcaaatag gaaaatggag
     8641 gataggagtc atctggggtt tatgcagcaa aactacaggt atattgcttg tatccgcctc
     8701 ggagatttcc atgaggagat aaagacatgt cacccgagtt tatactctcc tgcttagatc
     8761 ctactacagt atgaaataca gtgtygcgag gtagactatg taagcagatt taatcatttt
     8821 aaagagccca gtacttcata tccatttctc ccgctccttc tgcagcctta tcaaaaggta
     8881 tttagaacac tcattttagc cccattttca tttattatac tggcttatcc aacccctaga
     8941 cagagcattg gcattttccc tttcctgatc ttagaagtct gatgactcat gaaaccagac
     9001 agattagtta catacaccac aaatcgaggc tgtagctggg gcctcaacac tgcagttctt
     9061 ttataactcc ttagtacact ttttgttgat cctttgcctt gatccttaat tttcagtgtc
     9121 tatcacctct cccgtcaggt ggtgttccac atttgggcct attctcagtc cagggagttt
     9181 tacaacaata gatgtattga gaatccaacc taaagcttaa ctttccactc ccatgaatgc
     9241 ctctctcctt tttctccatt ataactgagc tatwaccatt aatggtttca ggtggatgtc
     9301 tcctccccca atatacctga tgtatctaca tattgccagg ctgatatttt aagacatwaa
     9361 aggtatattt cattattgag ccacatggta ttgattactg ctactaaaat tttgtcattg
     9421 tacacatctg taaaaggtgg ttccttttgg aatgcaaagt tcaggtgttt gttgtctttc
     9481 ctgacctaag gtcttgtgag cttgtatttt ttctatttaa gcagtgcttt ctcttggact
     9541 ggcttgactc atggcattct acacgttatt gctggtctaa atgtgatttt gccaagcttc
     9601 ttcaggacct ataattttgc ttgacttgta gccaaacaca agtaaaatga ttaagcaaca
     9661 aatgtatttg tgaagcttgg tttttaggtt gttgtgttgt gtgtgcttgt gctctataat
     9721 aatactatcc aggggctgga gaggtggctc ggagttcaag agcacagact gctcttccag
     9781 aagtcctgag ttcaattccc agcaaccaca tggtggctca caaccatctg taatgggatc
     9841 tgatgccctc ttctggtgtg tctgaagacc acaagtgtat tcacattaaa taaataatcc
     9901 tccttcttct tctttttttt tttttaaaga gaatwctgtc tccagtagaa ttactgaagt
     9961 aatgaaatac tttgtgtttg ttccaatatg gwagccaata atcaaatact cttwagcact
    10021 ggaaatgtac caaggaacta ttttatttaa gtgwactgtg gacagaggag ccataactgc
    10081 agacttgtgg gatacagaag accaatgcag acttaatgtc ttttctctta cactaagcaa
    10141 taaagaaata aaaattgaac ttctagtatc ctatttgtta aactgctagc tttactaact
    10201 tttgtgcttc atctatacaa agctgaaagc taagtctgca gccattacta aacatgaaag
    10261 caagtaatga taattttgga tttcaaaaat gtagggccag agtttagcca gccagtggtg
    10321 gtgcttgcct ttatgcctta atcccagcac tctggaggca gagacaggca gatctctgag
    10381 tttgagccca gcctggtcta cacatcaagt tctatctagg atagccagga atacacacag
    10441 aaaccctgtt ggggaggggg gctctgagat ttcataaaat tataattgaa gcattcccta
    10501 atgagccact atggatgtgg ctaaatccgt ctacctttct gatgagattt gggtattatt
    10561 ttttctgtct ctgctgttgg ttgggtcttt tgacactgtg ggctttctta aagcctcctt
    10621 ccctgccatg tggtctcttg tttgctacta acttcccatg gcttaaatgg catggctttt
    10681 tgccttctaa gggcagctgc tgagwtttgc agcctgattt ccagggtggg gttgggaaat
    10741 ctttcaaaca ctaaaattgt cctttaattt ttttttaaaa aatgggttat ataataaacc
    10801 tcataaaata gttatgagga gtgaggtgga ctaatattaa tgagtccctc ccctataaaa
    10861 gagctattaa ggctttttgt cttatactaa cttttttttt aaatgtggta tctttagaac
    10921 caagggtctt agagttttag tatacagaaa ctgttgcatc gcttaatcag attttctagt
    10981 ttcaaatcca gagaatccaa attcttcaca gccaaagtca aattaagaat ttctgacttt
    11041 aatgttattt gctactgtga atataaaatg atagcttttc ctgaggcagg gtctcactat
    11101 gtatctctgc ctgatctgca acaagatatg tagactaaag ttctgcctgc ttttgtctcc
    11161 tgaatactaa ggttaaaatg tagtaatact tttggaactt gcaggtcaga ttcttttata
    11221 ggggacacac taagggagct tgggtgatag ttggtaaatg tgtttaagtg atgaaaactt
    11281 gaattattat caccgcaacc tactttttaa aaaaaaaagc caggcctgtt agagcatgct
    11341 aagggatccc taggacttgc tgagcacaca agagtagtac ttggcaggct cctggtgaga
    11401 gcatatttca aaaaacaagg cagacaacca agaaactaca gtaaggttac ctgtctttaa
    11461 ccatctgcat atacacaggg atattaaaat attccaaata atatttcatt caagttttcc
    11521 cccatcaaat tgggacatgg atttctccgg tgaataggca gagttggaaa ctaaacaaat
    11581 gttggttttg tgatttgtga aattgttttc aagtgatagt taaagcccat gagatacaga
    11641 acaaagctgc tatttcgagg tctcttggtt atactcagaa gcacttcttt gggtttccct
    11701 gcactatcct gatcatgtgc taggcctwcc ttaggctgat tgttgttcaa ataacttaag
    11761 tttcctgtca ggtgatgtca tatgatttca tatatcaagg caaaacatgt tatatatgtt
    11821 aaacatttgk acttaatgtg aaagttaggt ctttgtgggt tttgatttta atttcaaaac
    11881 ctgagctaaa taagtcattt tacatgtctt acatttggtg aattgtatat tgtggtttgc
    11941 aggcaagact ctctgaccta gtaaccctcc tatagagcac tttgctgggt cacaagtcta
    12001 ggagtcaagc atttcacctt gaagttgaga cgttttgtta gtgtatacta gttatatgtt
    12061 ggaggacatg tttatccaga agatattcag gactattttt gactgggcta aggaattgat
    12121 tctgattagc actgttagtg agcattgagt ggcctttagg cttgaattgg agtcacttgt
    12181 atatctcaaa taatgctggc cttttttwaa aagcccttgt tctttatcac cctgttttct
    12241 acataatttt tgttcaaaga aatacttgtt tggatctcct tttgacaaca atagcatgtt
    12301 ttcaagccat attttttttc cttttttttt ttttttttgg tttttcgaga cagggtttct
    12361 ctgtatagcc ctggctgtcc tggaactcac tttgtagacc aggctggcct cgaactcaga
    12421 aatccgcctg cctctgcctc ctgagtgccg ggattaaagg cgtgcaccac cacgcctggc
    12481 taagttggat attttgtata taactataac caatactaac tccactgggt ggatttttaa
    12541 ttcagtcagt agtcttaagt ggtctttatt ggcccttatt aaaatctact gttcactcta
    12601 acagaggctg ttggactagt ggsactaagc aacttcctac ggatatacta gcagataagg
    12661 gtcagggata gaaactagtc tagcgttttg tatacctacc agcttatact accttgttct
    12721 gatagaaata tttaggacat ctagcttatc gatccgtcga cggtatcgat aagcttgata
    12781 tcgaattcta ccgggtaggg gaggcgcttt tccaaggcag tctgagcatg cgcttagcag
    12841 ccccgctggc acttggcgct acacaagtgg cctytggcct cgcacacatt ccacatccac
    12901 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctwctcctcc
    12961 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tsaggacgtg acaaatggaa
    13021 gtagcacgtc tcactagtct cgtcagatgg acagcaccgc tgagcaatgg aagcgggtag
    13081 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
    13141 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
    13201 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
    13261 cctcttcctc atctccgggc ctttcgacct gcaggtcctc gccatggatc ctgatgatgt
    13321 tgttattctt ctaatctttt gtatggaaaa cttttcttcg taccacggga ctaaacctgg
    13381 ttatgtagat tccattcaaa aaggtataca aaagccaaaa tctggtacac aaggaaatta
    13441 tgacgatgat tggaaagggt tttatagtac cgacaataaa tacgacgctg cgggatactc
    13501 tgtagataat gaaaacccgc tctctggaaa agctggaggc gtggtcaaag tgacgtatcc
    13561 aggactgacg aaggttctcg cactaaaagt ggataatgcc gaaactatta agaaagagtt
    13621 aggtttaagt ctcactgaac cgttgatgga gcaagtcgga acggaagagt ttatcaaaag
    13681 gttcggtgat ggtgcttcgc gtgtagtgct cagccttccc ttcgctgagg ggagttctag
    13741 cgttgaatat attaataact gggaacaggc gaaagcgtta agcgtagaac ttgagattaa
    13801 ttttgaaacc cgtggaaaac gtggccaaga tgcgatgtat gagtatatgg ctcaagcctg
    13861 tgcaggaaat cgtgtcaggc gatctctttg tgaaggaacc ttacttctgt ggtgtgacat
    13921 aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa tttttaagtg
    13981 tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca acctatggaa
    14041 ctgatgaatg ggagcagtgg tggaatgcag atcctagagc tcgctgatca gcctcgactg
    14101 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
    14161 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
    14221 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
    14281 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
    14341 ccagctgggg ctcgacctcg agggggggcc cggtacccag cttttgttcc ctttagtgag
    14401 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
    14461 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
    14521 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
    14581 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
    14641 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
    14701 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
    14761 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
    14821 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
    14881 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
    14941 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
    15001 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
    15061 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
    15121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
    15181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
    15241 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
    15301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
    15361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
    15421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
    15481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
    15541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
    15601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
    15661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
    15721 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
    15781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
    15841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
    15901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
    15961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
    16021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
    16081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
    16141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
    16201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
    16261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
    16321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
    16381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
    16441 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
    16501 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
    16561 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
    16621 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
    16681 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
    16741 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
    16801 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
    16861 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
    16921 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
    16981 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
    17041 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
    17101 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
    17161 ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata
    17221 gggcgaattg gagctcca
//
