LOCUS       sequence-390299-       14183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CENPT-Delta-C-dCas9-3xGFP protein in mammalian cells. When
            coupled with a guide RNA against a high repeat target locus, this 
            can be applied to seed ectopic kinetochores at the target locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14183)
  AUTHORS   Tovini L, Johnson SC, Guscott MA, Andersen AM, Spierings DCJ, 
            Wardenaar R, Foijer F, McClelland SE
  TITLE     Targeted assembly of ectopic kinetochores to induce 
            chromosome-specific segmental aneuploidies.
  JOURNAL   EMBO J. 2023 May 15;42(10):e111587. doi: 10.15252/embj.2022111587. 
            Epub 2023 Apr 17.
  PUBMED    37063065
REFERENCE   2  (bases 1 to 14183)
  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: "EMBO J."; 
            date: "2023-05-15"; volume: "42(10):e111587. doi"; pages: " 
            10.15252/embj.2022111587. Epub 2023 Apr 17"
COMMENT     Sequence Label: pCENPT-Delta-C-dCas9-3xGFP
FEATURES             Location/Qualifiers
     source          1..14183
                     /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(4222..4245,4246..4248,4249..6732,6733..6738,6739..6804,
                     6805..6807,6808..8322)
                     /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(4222..4245,4246..4248,4249..6735,6736..6738,
                     6739..8322)
                     /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             4222..8322
                     /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             8341..8361
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8374..9087
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             9106..9819
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             9838..10551
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     misc_feature    10573..11161
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10626..10646)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11044..11055)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11236..11469
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11588..11606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        11674..11778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11779..11847,11848..12639)
                     /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(11997..12016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12810..13398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13299..13318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13552..13569
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13809..13830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13845..13850,13851..13868,13869..13875)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13883..13899
                     /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     13888..13910
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13907..13923
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13907..13923
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13973..13992
                     /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 tcggtggagg cggatcaggg ggaggaggta gtgagatggc agaccacaac
     3061 cctgacagcg attctacacc tcgtacactg ctgcgtcgtg tgctggatac tgctgatcct
     3121 cgtactcctc gtcgtcctcg tagcgctcgt gctggtgctc gccgtgccct gctggaaaca
     3181 gcctctcctc gtaaactgag tggccagacc cgtactattg cccgtgggcg ttcacatgga
     3241 gcccgctcgg tcggtcgtag tgctcacatt caagcctcgg gacacctgga agaacaaacc
     3301 cctcgtaccc tgctgaaaaa catcctgctg actgcccctg aaagctctat tctgatgccg
     3361 gaaagcgtcg ttaaaccggt tcctgctcct caagccgttc aaccttctcg tcaagagtcc
     3421 tcctgtggat ctctggagct gcaactgcct gaactggagc ctcctacaac actggcaccg
     3481 gggctgctgg cgcctggtcg tcgtaaacaa cgtctgcgtc tgagcgtgtt tcaacaaggt
     3541 gttgatcagg gcctgagtct gtctcaagaa cctcaaggca acgctgatgc ttctagtctg
     3601 acccgttctc tgaacctgac ctttgctact ccactgcaac ctcaaagcgt ccaacgtcct
     3661 ggactggctc gccgccctcc tgctcgccgt gccgttgatg tgggtgcttt tctgcgtgac
     3721 ctgcgtgata caagcctggc acctccgaat attgtgctgg aggatacaca gccgttttca
     3781 cagcctatgg ttggtagccc taacgtttat cactcactgc cgtgtacccc tcacactggt
     3841 gccgaagatg ctgaacaagc tgctggtcgt aaaacccaaa gctctggacc gggactgcaa
     3901 aaaaactctc cggggaaacc tgcccaattc ctggcgggtg aagctgaaga agtcaacgcc
     3961 tttgctctgg gatttctgtc taccagctcg ggtgttagcg gagaagatga ggtggaacct
     4021 ctgcatgatg gtgtggaaga agccgagaag aaaatggaag aggaaggggt gtctgtctct
     4081 gaaatggaag cgactggtgc tcaaggacca agtcgtgtcg aagaggcaga aggccatacc
     4141 gaggttaccg aagccgaagg aagccaaggg gaaggcggtg gtggttcagg tggaggtgga
     4201 agcgaattcg gaagcggaag cgacaagaag tactccattg ggctcgctat cggtaccaac
     4261 agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt
     4321 ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggagccct cctgttcgac
     4381 tccggggaga cggccgaagc cacgcggctc aaaagaacag cacggcgcag atatacccgc
     4441 agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc taaggtggat
     4501 gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa aaagcacgag
     4561 cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa gtacccaacc
     4621 atatatcatc tgaggaagaa gctggtagac agtactgata aggctgactt gcggttgatc
     4681 tatctcgcgc tggcgcacat gatcaaattt cggggacact tcctcatcga gggggacctg
     4741 aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac ttacaatcag
     4801 cttttcgagg agaacccgat caacgcatcc ggcgttgacg ccaaagcaat cctgagcgct
     4861 aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg ggagaagaag
     4921 aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa ctttaaatct
     4981 aacttcgacc tggccgaaga tgccaagctg caactgagca aagacaccta cgatgatgat
     5041 ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt ggcggcaaag
     5101 aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga gatcaccaaa
     5161 gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga cttgactttg
     5221 ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt cttcgatcag
     5281 tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga attttacaaa
     5341 tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt aaagctgaac
     5401 agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc ccaccagatt
     5461 cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc ctttttgaaa
     5521 gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta tgtaggcccc
     5581 ctcgctcggg gaaattccag attcgcgtgg atgactcgca aatcagaaga gaccatcact
     5641 ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt catcgaaagg
     5701 atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg
     5761 tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac agaagggatg
     5821 agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct cctcttcaag
     5881 acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa gattgaatgt
     5941 ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct gggaacgtat
     6001 cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga gaacgaggac
     6061 attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat gattgaagaa
     6121 cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct caagagacgc
     6181 cgatatacag gatgggggcg gctgtcaaga aaactgatca atggcatccg agacaagcag
     6241 agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg gaacttcatg
     6301 cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc acaagtttct
     6361 ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc agctatcaaa
     6421 aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat
     6481 aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca gaagggacag
     6541 aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact ggggtcccaa
     6601 atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct ctacctgtac
     6661 tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa ccggttgtcc
     6721 gactacgacg tggatgctat cgtgccccaa agctttctca aagatgattc tattgataat
     6781 aaagtgttga caagatccga taaaaataga gggaagagtg ataacgtccc ctcagaagaa
     6841 gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact gatcacacaa
     6901 cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc
     6961 ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt ggcccaaatt
     7021 ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg agaggtgaaa
     7081 gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca gttttataag
     7141 gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc agtggtaggc
     7201 actgcactta tcaaaaaata tcccaagctg gaatctgaat ttgtttacgg agactataaa
     7261 gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa ggccaccgct
     7321 aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac actggccaat
     7381 ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga aatcgtgtgg
     7441 gacaagggta gggatttcgc gacagtccgc aaggtcctgt ccatgccgca ggtgaacatc
     7501 gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct cccgaaaagg
     7561 aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata cggcggattc
     7621 gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa agggaagtct
     7681 aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg atccagcttc
     7741 gagaaaaacc ccatcgactt tctcgaagcg aaaggatata aagaggtcaa aaaagacctc
     7801 atcattaagc tgcccaagta ctctctcttt gagcttgaaa acggccggaa acgaatgctc
     7861 gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa atacgttaat
     7921 ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag
     7981 aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga gcaaataagc
     8041 gagttctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct ttctgcttac
     8101 aataagcaca gggataagcc catcagggag caggcagaaa acattatcca cttgtttact
     8161 ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat agacagaaag
     8221 cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc aattacgggg
     8281 ctctatgaaa caagaatcga cctctctcag ctcggtggag acggctctac tagtggctct
     8341 cccaagaaga agaggaaggt aggatccgga agtatgcgta aaggcgaaga gctgttcact
     8401 ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg
     8461 cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact
     8521 actggtaaac tgccggtacc ttggccgact ctggtaacga cgctgactta tggtgttcag
     8581 tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg
     8641 gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt
     8701 gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac
     8761 tttaaagaag acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat
     8821 gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac
     8881 aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt
     8941 gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa
     9001 gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc
     9061 acgcatggta tggatgaact gtacaaatct ggcggccgct ctggcatgcg taaaggcgaa
     9121 gagctgttca ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat
     9181 aagttttccg tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag
     9241 ttcatctgta ctactggtaa actgccggta ccttggccga ctctggtaac gacgctgact
     9301 tatggtgttc agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag
     9361 tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg
     9421 tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg
     9481 aaaggcattg actttaaaga agacggcaat atcctgggcc ataagctgga atacaatttt
     9541 aacagccaca atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt
     9601 aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac
     9661 actccaatcg gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc
     9721 gttctgtcta aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc
     9781 gcagcgggca tcacgcatgg tatggatgaa ctgtacaaag gtagcatcga tggtagcatg
     9841 cgtaaaggcg aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat
     9901 gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa
     9961 ctgacgctga agttcatctg tactactggt aaactgccgg taccttggcc gactctggta
    10021 acgacgctga cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat
    10081 gacttcttca agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag
    10141 gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac
    10201 cgcattgagc tgaaaggcat tgactttaaa gaagacggca atatcctggg ccataagctg
    10261 gaatacaatt ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt
    10321 aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac
    10381 taccagcaaa acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg
    10441 agcacgcaaa gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg
    10501 gagttcgtaa ccgcagcggg catcacgcat ggtatggatg aactgtacaa actcgagtag
    10561 ggtaatctag ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
    10621 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
    10681 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
    10741 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
    10801 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
    10861 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
    10921 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
    10981 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
    11041 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
    11101 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
    11161 cctgagatcc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
    11221 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
    11281 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
    11341 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
    11401 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
    11461 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
    11521 gaatatcaga gagtgagagg cccgggttaa ttaaggaaag ggctagatca ttcttgaaga
    11581 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
    11641 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
    11701 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
    11761 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
    11821 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
    11881 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
    11941 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
    12001 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
    12061 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
    12121 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
    12181 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
    12241 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
    12301 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
    12361 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
    12421 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
    12481 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
    12541 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
    12601 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
    12661 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
    12721 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
    12781 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
    12841 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
    12901 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
    12961 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
    13021 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
    13081 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
    13141 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
    13201 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
    13261 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
    13321 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
    13381 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
    13441 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
    13501 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
    13561 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
    13621 attcattaat gcagcaagct catggctgac taattttttt tatttatgca gaggccgagg
    13681 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
    13741 tttgcaaaaa gctccccgtg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
    13801 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
    13861 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
    13921 gacatgatta cgaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
    13981 tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa
    14041 aatcatccca aacttcccac cccataccct attaccactg ccaattacct gtggtttcat
    14101 ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa
    14161 atatgtacta caaacttagt agt
//
