LOCUS       sequence-170099-       13357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dSpyCas9-3XmCherry and telomere-targeting Spy sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13357)
  AUTHORS   Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, 
            Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR
  TITLE     Naturally Occurring Off-Switches for CRISPR-Cas9.
  JOURNAL   Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 
            10.1016/j.cell.2016.11.017. Epub 2016 Dec 8.
  PUBMED    27984730
REFERENCE   2  (bases 1 to 13357)
  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: 
            "10.1016/j.cell.2016.11.017"; journalName: "Cell"; date: 
            "2016-12-15- 15"; volume: "167"; issue: "7"; pages: "1829-1838.e9"
COMMENT     Sequence Label: pEJS477-pHAGE-TO-Spy dCas9
            3XmCherry-SgRNA/Tel...
FEATURES             Location/Qualifiers
     source          1..13357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2299..2602
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2603..2806
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2757..2777
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2808..2826
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2829..2847
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2857..2881
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2974..2983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2986..3006
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3025..3048,3049..3051,3052..5535,5536..5541,5542..5607,
                     5608..5610,5611..7125)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(3025..3048,3049..3051,3052..5538,5539..5541,
                     5542..7125)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3025..7125
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7144..7164
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7177..7884
                     /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"
     CDS             7903..8610
                     /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"
     CDS             8629..9336
                     /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"
     misc_feature    9358..9946
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9411..9431)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9829..9840)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10021..10254
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        10350..10598
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     10350..10370
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     10521..10540
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        10620..10695
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(10762..10780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10848..10952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10953..11021,11022..11813)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(11171..11190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11984..12572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12473..12492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12726..12743
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12983..13004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13019..13024,13025..13042,13043..13049)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13057..13073
                     /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)."
     primer_bind     13062..13084
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13081..13097
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13081..13097
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13147..13166
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
     2221 gctagctgat gcgggcccta gttattaata gtaatcaatt acggggtcat tagttcatag
     2281 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2341 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2401 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2461 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     2521 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     2581 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     2641 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     2701 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2761 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
     2821 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
     2881 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
     2941 cctaccggtt aaggtgaacg cgtgcaggct ggcgccacca tggcccctaa aaagaaacgc
     3001 aaggtcgaat tcggaagcgg aagcgacaag aagtactcca ttgggctcgc tatcggtacc
     3061 aacagcgtcg gctgggccgt cattacggac gagtacaagg tgccgagcaa aaaattcaaa
     3121 gttctgggca ataccgatcg ccacagcata aagaagaacc tcattggagc cctcctgttc
     3181 gactccgggg agacggccga agccacgcgg ctcaaaagaa cagcacggcg cagatatacc
     3241 cgcagaaaga atcggatctg ctacctgcag gagatcttta gtaatgagat ggctaaggtg
     3301 gatgactctt tcttccatag gctggaggag tcctttttgg tggaggagga taaaaagcac
     3361 gagcgccacc caatctttgg caatatcgtg gacgaggtgg cgtaccatga aaagtaccca
     3421 accatatatc atctgaggaa gaagctggta gacagtactg ataaggctga cttgcggttg
     3481 atctatctcg cgctggcgca catgatcaaa tttcggggac acttcctcat cgagggggac
     3541 ctgaacccag acaacagcga tgtcgacaaa ctctttatcc aactggttca gacttacaat
     3601 cagcttttcg aggagaaccc gatcaacgca tccggcgttg acgccaaagc aatcctgagc
     3661 gctaggctgt ccaaatcccg gcggctcgaa aacctcatcg cacagctccc tggggagaag
     3721 aagaacggcc tgtttggtaa tcttatcgcc ctgtcactcg ggctgacccc caactttaaa
     3781 tctaacttcg acctggccga agatgccaag ctgcaactga gcaaagacac ctacgatgat
     3841 gatctcgaca atctgctggc ccagatcggc gaccagtacg cagacctttt tttggcggca
     3901 aagaacctgt cagacgccat tctgctgagt gatattctgc gagtgaacac ggagatcacc
     3961 aaagctccgc tgagcgctag tatgatcaag cgctatgatg agcaccacca agacttgact
     4021 ttgctgaagg cccttgtcag acagcaactg cctgagaagt acaaggaaat tttcttcgat
     4081 cagtctaaaa atggctacgc cggatacatt gacggcggag caagccagga ggaattttac
     4141 aaatttatta agcccatctt ggaaaaaatg gacggcaccg aggagctgct ggtaaagctg
     4201 aacagagaag atctgttgcg caaacagcgc actttcgaca atggaagcat cccccaccag
     4261 attcacctgg gcgaactgca cgctatcctc aggcggcaag aggatttcta cccctttttg
     4321 aaagataaca gggaaaagat tgagaaaatc ctcacatttc ggatacccta ctatgtaggc
     4381 cccctcgctc ggggaaattc cagattcgcg tggatgactc gcaaatcaga agagaccatc
     4441 actccctgga acttcgagga agtcgtggat aagggggcct ctgcccagtc cttcatcgaa
     4501 aggatgacta actttgataa aaatctgcct aacgaaaagg tgcttcctaa acactctctg
     4561 ctgtacgagt acttcacagt ttataacgag ctcaccaagg tcaaatacgt cacagaaggg
     4621 atgagaaagc cagcattcct gtctggagag cagaagaaag ctatcgtgga cctcctcttc
     4681 aagacgaacc ggaaagttac cgtgaaacag ctcaaagaag actatttcaa aaagattgaa
     4741 tgtttcgact ctgttgaaat cagcggagtg gaggatcgct tcaacgcatc cctgggaacg
     4801 tatcacgatc tcctgaaaat cattaaagac aaggacttcc tggacaatga ggagaacgag
     4861 gacattcttg aggacattgt cctcaccctt acgttgtttg aagataggga gatgattgaa
     4921 gaacgcttga aaacttacgc tcatctcttc gacgacaaag tcatgaaaca gctcaagaga
     4981 cgccgatata caggatgggg gcggctgtca agaaaactga tcaatggcat ccgagacaag
     5041 cagagtggaa agacaatcct ggattttctt aagtccgatg gatttgccaa ccggaacttc
     5101 atgcagttga tccatgatga ctctctcacc tttaaggagg acatccagaa agcacaagtt
     5161 tctggccagg gggacagtct tcacgagcac atcgctaatc ttgcaggtag cccagctatc
     5221 aaaaagggaa tactgcagac cgttaaggtc gtggatgaac tcgtcaaagt aatgggaagg
     5281 cataagcccg agaatatcgt tatcgagatg gcccgagaga accaaactac ccagaaggga
     5341 cagaagaaca gtagggaaag gatgaagagg attgaagagg gtataaaaga actggggtcc
     5401 caaatcctta aggaacaccc agttgaaaac acccagcttc agaatgagaa gctctacctg
     5461 tactacctgc agaacggcag ggacatgtac gtggatcagg aactggacat caaccggttg
     5521 tccgactacg acgtggatgc tatcgtgccc caaagctttc tcaaagatga ttctattgat
     5581 aataaagtgt tgacaagatc cgataaaaat agagggaaga gtgataacgt cccctcagaa
     5641 gaagttgtca agaaaatgaa aaattattgg cggcagctgc tgaacgccaa actgatcaca
     5701 caacggaagt tcgataatct gactaaggct gaacgaggtg gcctgtctga gttggataaa
     5761 gccggcttca tcaaaaggca gcttgttgag acacgccaga tcaccaagca cgtggcccaa
     5821 attctcgatt cacgcatgaa caccaagtac gatgaaaatg acaaactgat tcgagaggtg
     5881 aaagttatta ctctgaagtc taagctggtc tcagatttca gaaaggactt tcagttttat
     5941 aaggtgagag agatcaacaa ttaccaccat gcgcatgatg cctacctgaa tgcagtggta
     6001 ggcactgcac ttatcaaaaa atatcccaag ctggaatctg aatttgttta cggagactat
     6061 aaagtgtacg atgttaggaa aatgatcgca aagtctgagc aggaaatagg caaggccacc
     6121 gctaagtact tcttttacag caatattatg aattttttca agaccgagat tacactggcc
     6181 aatggagaga ttcggaagcg accacttatc gaaacaaacg gagaaacagg agaaatcgtg
     6241 tgggacaagg gtagggattt cgcgacagtc cgcaaggtcc tgtccatgcc gcaggtgaac
     6301 atcgttaaaa agaccgaagt acagaccgga ggcttctcca aggaaagtat cctcccgaaa
     6361 aggaacagcg acaagctgat cgcacgcaaa aaagattggg accccaagaa atacggcgga
     6421 ttcgattctc ctacagtcgc ttacagtgta ctggttgtgg ccaaagtgga gaaagggaag
     6481 tctaaaaaac tcaaaagcgt caaggaactg ctgggcatca caatcatgga gcgatccagc
     6541 ttcgagaaaa accccatcga ctttctcgaa gcgaaaggat ataaagaggt caaaaaagac
     6601 ctcatcatta agctgcccaa gtactctctc tttgagcttg aaaacggccg gaaacgaatg
     6661 ctcgctagtg cgggcgagct gcagaaaggt aacgagctgg cactgccctc taaatacgtt
     6721 aatttcttgt atctggccag ccactatgaa aagctcaaag ggtctcccga agataatgag
     6781 cagaagcagc tgttcgtgga acaacacaaa cactaccttg atgagatcat cgagcaaata
     6841 agcgagttct ccaaaagagt gatcctcgcc gacgctaacc tcgataaggt gctttctgct
     6901 tacaataagc acagggataa gcccatcagg gagcaggcag aaaacattat ccacttgttt
     6961 actctgacca acttgggcgc gcctgcagcc ttcaagtact tcgacaccac catagacaga
     7021 aagcggtaca cctctacaaa ggaggtcctg gacgccacac tgattcatca gtcaattacg
     7081 gggctctatg aaacaagaat cgacctctct cagctcggtg gagacggctc tactagtggc
     7141 tctcccaaga agaagaggaa ggtaggatcc ggaagtatgg tgagcaaggg cgaggaggat
     7201 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     7261 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     7321 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     7381 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     7441 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     7501 gtggtgaccg tgacccagga ctcctcactg caggacggcg agttcatcta caaggtgaag
     7561 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     7621 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     7681 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     7741 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     7801 cacaacgagg actacaccat cgtggaacag tacgaacgcg ctgagggccg ccactccacc
     7861 ggcggcatgg acgagctgta caagtctggc ggccgctctg gcatggtgag caagggcgag
     7921 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     7981 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     8041 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     8101 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     8161 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     8221 ggcggcgtgg tgaccgtgac ccaggactcc tcactgcagg acggcgagtt catctacaag
     8281 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     8341 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     8401 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     8461 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     8521 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgctga gggccgccac
     8581 tccaccggcg gcatggacga gctgtacaag ggtagcatcg atggtagcat ggtgagcaag
     8641 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     8701 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     8761 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     8821 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     8881 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     8941 gaggacggcg gcgtggtgac cgtgacccag gactcctcac tgcaggacgg cgagttcatc
     9001 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     9061 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     9121 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     9181 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     9241 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgctgagggc
     9301 cgccactcca ccggcggcat ggacgagctg tacaagctcg agtagggtaa tctagataat
     9361 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     9421 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     9481 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     9541 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     9601 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     9661 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     9721 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     9781 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     9841 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     9901 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctga gatcctttaa
     9961 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
    10021 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
    10081 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
    10141 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
    10201 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
    10261 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
    10321 agaggcccgg ggcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt
    10381 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
    10441 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
    10501 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
    10561 tcttggcttt atatatcttg tggaaaggac gaaacaccgt tagggttagg gttagggttg
    10621 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
    10681 gcaccgagtc ggtgcttttt ttgaattctc gacccccggg ttaattaagg aaagggctag
    10741 atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
    10801 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
    10861 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
    10921 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
    10981 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
    11041 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
    11101 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
    11161 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
    11221 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
    11281 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
    11341 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
    11401 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
    11461 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    11521 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    11581 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    11641 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    11701 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    11761 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    11821 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    11881 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    11941 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    12001 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    12061 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    12121 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    12181 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    12241 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    12301 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    12361 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    12421 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    12481 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    12541 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    12601 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    12661 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    12721 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
    12781 ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt tttttattta
    12841 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
    12901 tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc gactggaaag
    12961 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
    13021 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
    13081 caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt tcactgcatt
    13141 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa
    13201 ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt
    13261 acctgtggtt tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa
    13321 attgtatttg ttaaatatgt actacaaact tagtagt
//
