LOCUS       sequence-493009-       11974 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  A CRISPR-based technology to study chromosome-specific aneuploidy by
            targeting human centromeres.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11974)
  AUTHORS   Bosco N, Goldberg A, Zhao X, Mays JC, Cheng P, Johnson AF, Bianchi 
            JJ, Toscani C, Di Tommaso E, Katsnelson L, Annuar D, Mei S, 
            Faitelson RE, Pesselev IY, Mohamed KS, Mermerian A, 
            Camacho-Hernandez EM, Gionco CA, Manikas J, Tseng YS, Sun Z, Fani S,
            Keegan S, Lippman SM, Fenyo D, Giunta S, Santaguida S, Davoli T
  TITLE     KaryoCreate: A CRISPR-based technology to study chromosome-specific 
            aneuploidy by targeting human centromeres.
  JOURNAL   Cell. 2023 Apr 27;186(9):1985-2001.e19. doi: 
            10.1016/j.cell.2023.03.029. Epub 2023 Apr 18.
  PUBMED    37075754
REFERENCE   2  (bases 1 to 11974)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2023.03.029"; journalName: "Cell"; date: 
            "2023-04-27- 27"; volume: "186"; issue: "9"; pages: "1985-2001.e19"
COMMENT     Sequence Label: pHAGE-dCas9
FEATURES             Location/Qualifiers
     source          1..11974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(25..45)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(52..117)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(141..165)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(273..297)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    307..325
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    328..346
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(348..551)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(377..397)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        552..855
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    912..1029
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1322..1366)
                     /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    1551..1784
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2281..2406
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2453..3086
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3278..3297)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3347..3363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3347..3363)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3360..3382)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3371..3387
                     /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(3395..3401,3402..3419,3420..3425))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3440..3461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3701..3718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3872..4460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3952..3971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4631..5422,5423..5491))
                     /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     5254..5273
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5492..5596)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5664..5682
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     LTR             5801..6034
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    6109..6697
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             6215..6226
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6624..6644
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6764..7162)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        complement(7183..7682)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7231..7250)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     7624..7647
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(7715..7741)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    7782..7806
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(7826..7873)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(7874..9457,9458..9460,9461..11947,
                     11948..11950,11951..11974))
                     /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             complement(7874..11974)
                     /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"
ORIGIN
        1 ggctgctggg actccgtgga taccgacctt ccgcttcttc tttggggcca tcttatcgtc
       61 atcgtctttg taatcaatat catgatcctt gtagtctccg tcgtggtcct tatagtccat
      121 ggtgaagggg gcggccgcgg agcctgcttt tttgtacaaa cttgttgatg ggctcgagga
      181 tatcaccggt agggatcctc tagagtccgg aggctggatc ggtcccggtg tcttctatgg
      241 aggtcaaaac agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctcgt
      301 cgacgatctc tatcactgat agggagatct ctatcactga tagggagagc tctgcttata
      361 tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga
      421 cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg
      481 tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa
      541 aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag
      601 tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
      661 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
      721 aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac
      781 gtcattattg acgtcaatgg gcgggggtcg tggggcggtc agccaggcgg gccatttacc
      841 gtaagttatg taacgacatg catggctagc ctagtccaaa ctggatctct gctgtccctg
      901 taataaaccc gaaaattttg aatttttgta atttgttttt gtaattcttt agtttgtatg
      961 tctgttgcta ttatgtctac tattctttcc cctgcactgt accccccaat cccccctttt
     1021 cttttaaaat tgtggatgaa tactgccatt tgtgaattcg gcgataccgt cgagatccgt
     1081 tcactaatcg aatggatctg tctctgtctc tctctccacc ttcttcttct attccttcgg
     1141 gcctgtcggg tcccctcggg gttgggaggt gggtctgaaa cgataatggt gaatatccct
     1201 gcctaactct attcactata gaaagtacag caaaaactat tcttaaacct accaagcctc
     1261 ctactatcat tatgaataat tttatatacc acagccaatt tgttatgtta aaccaattcc
     1321 acaaacttgc ccatttatct aattccaata attcttgttc attcttttct tgctggtttt
     1381 gcgattcttc aattaaggag tgtattaagc ttgtgtaatt gttaatttct ctgtcccact
     1441 ccatccaggt cgtgtgattc caaatctgtt ccagagattt attactccaa ctagcattcc
     1501 aaggcacagc agtggtgcaa atgagttttc cagagcaacc ccaaatcccc aggagctgtt
     1561 gatcctttag gtatctttcc acagccagga ttcttgcctg gagctgcttg atgccccaga
     1621 ctgtgagttg caacagatgc tgttgcgcct caatagccct cagcaaattg ttctgctgct
     1681 gcactatacc agacaataat tgtctggcct gtaccgtcag cgtcattgac gctgcgccca
     1741 tagtgcttcc tgctgctccc aagaacccaa ggaacaaagc tcctattccc actgctcttt
     1801 tttctctctg caccactctt ctctttgcct tggtgggtgc tactcctaat ggttcaattt
     1861 ttactacttt atatttatat aattcacttc tccaattgtc cctcatatct cctcctccag
     1921 gtctgaagat cagcggccgg ccgcttgctg tgcggtggtc ttacttttgt tttgctcttc
     1981 ctctatcttg tctaaagctt ccttggtgtc ttttatctct atcctttgat gcacacaata
     2041 gagggttgct actgtattat ataatgatct aagttcttct gatcctgtct gaagggatgg
     2101 ttgtagctgt cccagtattt gtctacagcc ttctgatgtt tctaacaggc caggattaac
     2161 tgcgaatcgt tctagctccc tgcttgccca tactatatgt tttaatttat attttttctt
     2221 tccccctggc cttaaccgaa ttttttccca tcgcgatcta attctccccc gcttaatact
     2281 gacgctctcg cacccatctc tctccttcta gcctccgcta gtcaaaattt ttggcgtact
     2341 caccagtcgc cgcccctcgc ctcttgccgt gcgcgcttca gcaagccgag tcctgcgtcg
     2401 agagagctcc tctggtttcc ctttcgcttt caagtccctg ttcgggcgcc actgctagag
     2461 attttccaca ctgactaaaa gggtctgagg gatctctagt taccagagtc acacaacaga
     2521 cgggcacaca ctacttgaag cactcaaggc aagctttatt gaggcttaag cagtgggttc
     2581 cctagttagc cagagagctc ccaggctcag atctggtcta accagagaga cccagtacag
     2641 gcaaaaagca gctgcttata tgcaggatct gagggctcgc cactccccag tcccgcccag
     2701 gccacgcctc cctggaaagt ccccagcgga aagtcccttg tagcaagctc gatatcagca
     2761 gttcttgaag tactccggat gcagctctcg ggccacgtga tgaaatgcta ggcggctgtc
     2821 aaacctccac tctaacactt ctctctccgg gtcatccatc ccatgcaggc tcacagggtg
     2881 taacaagctg gtgttctctc ctttattggc ctcttctacc ttatctggct caactggtac
     2941 tagcttgtag caccatccaa aggtcagtgg atatctgacc cctggccctg gtgtgtagtt
     3001 ctgctaatca gggaagtagc cttgtgtgtg gtagatccac agatcaagga tatcttgtct
     3061 tctttgggag tgaattagcc cttccaacta ctaagtttgt agtacatatt taacaaatac
     3121 aatttcttta aaatgaaaat aattcagagg aatcacaggt ttagagtaaa tgaaaccaca
     3181 ggtaattggc agtggtaata gggtatgggg tgggaagttt gggatgattt tggttagctt
     3241 gagttatcca gttgatccag acatgataag atacattgat gagtttggac aaaccacaac
     3301 tagaatgcag tgaaaaaaat gctttatttg tgaaattcgt aatcatgtca tagctgtttc
     3361 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3421 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3481 ccgctttcca gtcgggaaac ctgtcgtgcc acggggagct ttttgcaaaa gcctaggcct
     3541 ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct
     3601 gcataaataa aaaaaattag tcagccatga gcttgctgca ttaatgaatc ggccaacgcg
     3661 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3721 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3781 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3841 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3901 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3961 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4021 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4081 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4141 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4201 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4261 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4321 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4381 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4441 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4501 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4561 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4621 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4681 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4741 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4801 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4861 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4921 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4981 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5041 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5101 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5161 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5221 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     5281 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5341 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5401 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5461 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5521 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5581 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5641 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     5701 gatctagccc tttccttaat taacccgggc ctctcactct ctgatattca tttctttgca
     5761 agttataaat actgaataat aagatgacat gaactactac tgctagagat tttccacact
     5821 gactaaaagg gtctgaggga tctctagtta ccagagtcac acaacagacg ggcacacact
     5881 acttgaagca ctcaaggcaa gctttattga ggcttaagca gtgggttccc tagttagcca
     5941 gagagctccc aggctcagat ctggtctaac cagagagacc cagtacaagc aaaaagcaga
     6001 tcttgtcttc gttgggagtg aattagccct tccagtcccc ccttttcttt taaaaagtgg
     6061 ctaagatcta cagctgcctt gtaagtcatt ggtcttaaag gatctcaggc ggggaggcgg
     6121 cccaaaggga gatccgactc gtctgagggc gaaggcgaag acgcggaaga ggccgcagag
     6181 ccggcagcag gccgcgggaa ggaaggtccg ctggattgag ggccgaaggg acgtagcaga
     6241 aggacgtccc gcgcagaatc caggtggcaa cacaggcgag cagccaagga aaggacgatg
     6301 atttccccga caacaccacg gaattgtcag tgcccaacag ccgagcccct gtccagcagc
     6361 gggcaaggca ggcggcgatg agttccgccg tggcaatagg gagggggaaa gcgaaagtcc
     6421 cggaaaggag ctgacaggtg gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca
     6481 cagtgcacac cacgccacgt tgcctgacaa cgggccacaa ctcctcataa agagacagca
     6541 accaggattt atacaaggag gagaaaatga aagccatacg ggaagcaata gcatgataca
     6601 aaggcattaa agcagcgtat ccacatagcg taaaaggagc aacatagtta agaataccag
     6661 tcaatctttc acaaattttg taatccagag gttgattagg atctatcgat gcatggggtc
     6721 gtgcgctcct ttcggtcggg cgctgcgggt cgtggggcgg gcgttagccc tcccacacat
     6781 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
     6841 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
     6901 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
     6961 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
     7021 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
     7081 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     7141 cttgagacaa aggcttggcc atggtaagct tgggctgcag gtcgaaaggc ccggagatga
     7201 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     7261 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     7321 accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt ggccgctgcc
     7381 ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga gactagtgag
     7441 acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg gggggaactt
     7501 cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg gagccggttg
     7561 gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt
     7621 gcccagcggg gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac
     7681 ccggtagaat ttctagttta attaatcatt actaagcgta gtctgggacg tcgtatgggt
     7741 attcgaaccg cgggccctct aggctaccta gctatgcgca aaccactttg tacaagaaag
     7801 ctgggtcggc gcgcccaccc ttttactttt tcttttttgc ctggccggcc tttttcgtgg
     7861 ccgccggcct tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg tacaggccgg
     7921 tgatgctctg gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg tacctcttcc
     7981 ggtcgatggt ggtgtcaaag tacttgaagg cggcaggggc tcccagattg gtcagggtaa
     8041 acaggtggat gatattctcg gcctgctctc tgatgggctt atcccggtgc ttgttgtagg
     8101 cggacagcac tttgtccaga ttagcgtcgg ccaggatcac tctcttggag aactcgctga
     8161 tctgctcgat gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc tgtttctgct
     8221 cattatcctc gggggagccc ttcagcttct catagtggct ggccaggtac aggaagttca
     8281 catatttgga gggcagggcc agttcgtttc ccttctgcag ttcgccggca gaggccagca
     8341 ttctcttccg gccgttttcc agctcgaaca gggagtactt aggcagcttg atgatcaggt
     8401 cctttttcac ttctttgtag cccttggctt ccagaaagtc gatgggattc ttctcgaagc
     8461 tgcttctttc catgatggtg atccccagca gctctttcac actcttcagt ttcttggact
     8521 tgcccttttc cactttggcc accaccagca cagaataggc cacggtgggg ctgtcgaagc
     8581 cgccgtactt cttagggtcc cagtccttct ttctggcgat cagcttatcg ctgttcctct
     8641 tgggcaggat agactctttg ctgaagccgc ctgtctgcac ctcggtcttt ttcacgatat
     8701 tcacttgggg catgctcagc actttccgca cggtggcaaa atcccggccc ttatcccaca
     8761 cgatctcccc ggtttcgccg tttgtctcga tcagaggccg cttccggatc tcgccgttgg
     8821 ccagggtaat ctcggtcttg aaaaagttca tgatgttgct gtagaagaag tacttggcgg
     8881 tagccttgcc gatttcctgc tcgctcttgg cgatcatctt ccgcacgtcg tacaccttgt
     8941 agtcgccgta cacgaactcg ctttccagct tagggtactt tttgatcagg gcggttccca
     9001 cgacggcgtt caggtaggcg tcgtgggcgt ggtggtagtt gttgatctcg cgcactttgt
     9061 aaaactggaa atccttccgg aaatcggaca ccagcttgga cttcagggtg atcactttca
     9121 cttcccggat cagcttgtca ttctcgtcgt acttagtgtt catccgggag tccaggatct
     9181 gtgccacgtg ctttgtgatc tgccgggttt ccaccagctg tctcttgatg aagccggcct
     9241 tatccagttc gctcaggccg cctctctcgg ccttggtcag attgtcgaac tttctctggg
     9301 taatcagctt ggcgttcagc agctgccgcc agtagttctt catcttcttc acgacctctt
     9361 cggagggcac gttgtcgctc ttgccccggt tcttgtcgct tctggtcagc accttgttgt
     9421 cgatggagtc gtccttcaga aagctctgag gcacgatagc gtccacatcg tagtcggaca
     9481 gccggttgat gtccagttcc tggtccacgt acatatcccg cccattctgc aggtagtaca
     9541 ggtacagctt ctcgttctgc agctgggtgt tttccacggg gtgttctttc aggatctggc
     9601 tgcccagctc tttgatgccc tcttcgatcc gcttcattct ctcgcggctg ttcttctgtc
     9661 ccttctgggt ggtctggttc tctctggcca tttcgatcac gatgttctcg ggcttgtgcc
     9721 ggcccatcac tttcacgagc tcgtccacca ccttcactgt ctgcaggatg cccttcttaa
     9781 tggcggggct gccggccaga ttggcaatgt gctcgtgcag gctatcgccc tggccggaca
     9841 cctgggcttt ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc agctgcatga
     9901 agtttctgtt ggcgaagccg tcggacttca ggaaatccag gattgtcttg ccggactgct
     9961 tgtcccggat gccgttgatc agcttccggc tcagcctgcc ccagccggtg tatctccgcc
    10021 gcttcagctg cttcatcact ttgtcgtcga acaggtgggc ataggttttc agccgttcct
    10081 cgatcatctc tctgtcctca aacagtgtca gggtcagcac gatatcttcc agaatgtcct
    10141 cgttttcctc attgtccagg aagtccttgt ccttgataat tttcagcaga tcgtggtatg
    10201 tgcccaggga ggcgttgaac cgatcttcca cgccggagat ttccacggag tcgaagcact
    10261 cgattttctt gaagtagtcc tctttcagct gcttcacggt cactttccgg ttggtcttga
    10321 acagcaggtc cacgatggcc tttttctgct cgccgctcag gaaggcgggc tttctcattc
    10381 cctcggtcac gtatttcact ttggtcagct cgttatacac ggtgaagtac tcgtacagca
    10441 ggctgtgctt gggcagcacc ttctcgttgg gcaggttctt atcgaagttg gtcatccgct
    10501 cgatgaagct ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc cagggggtga
    10561 tggtttcctc gctctttctg gtcatccagg cgaatctgct gtttcccctg gccagagggc
    10621 ccacgtagta ggggatgcgg aaggtcagga tcttctcgat cttttcccgg ttgtccttca
    10681 ggaatgggta aaaatcttcc tgccgccgca gaatggcgtg cagctctccc aggtggatct
    10741 ggtgggggat gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc tctctgttca
    10801 gcttcacgag cagttcctcg gtgccgtcca tcttttccag gatgggcttg atgaacttgt
    10861 agaactcttc ctggctggct ccgccgtcaa tgtagccggc gtagccgttc ttgctctggt
    10921 cgaagaaaat ctctttgtac ttctcaggca gctgctgccg cacgagagct ttcagcaggg
    10981 tcaggtcctg gtggtgctcg tcgtatctct tgatcataga ggcgctcagg ggggccttgg
    11041 tgatctcggt gttcactctc aggatgtcgc tcagcaggat ggcgtcggac aggttcttgg
    11101 cggccagaaa caggtcggcg tactggtcgc cgatctgggc cagcaggttg tccaggtcgt
    11161 cgtcgtaggt gtccttgctc agctgcagtt tggcatcctc ggccaggtcg aagttgctct
    11221 tgaagttggg ggtcaggccc aggctcaggg caatcaggtt tccgaacagg ccattcttct
    11281 tctcgccggg cagctgggcg atcagatttt ccagccgtct gctcttgctc agtctggcag
    11341 acaggatggc cttggcgtcc acgccgctgg cgttgatggg gttttcctcg aacagctggt
    11401 tgtaggtctg caccagctgg atgaacagct tgtccacgtc gctgttgtcg gggttcaggt
    11461 cgccctcgat caggaagtgg ccccggaact tgatcatgtg ggccagggcc agatagatca
    11521 gccgcaggtc ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg tagatggtgg
    11581 ggtacttctc gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg tgccgctcgt
    11641 gcttcttatc ctcttccacc aggaaggact cttccagtct gtggaagaag ctgtcgtcca
    11701 ccttggccat ctcgttgctg aagatctctt gcagatagca gatccggttc ttccgtctgg
    11761 tgtatcttct tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg ccgctgtcga
    11821 acagcagggc tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg cccagcacct
    11881 tgaatttctt gctgggcacc ttgtactcgt cggtgatcac ggcccagccc acagagttgg
    11941 tgccgatggc caggccgatg ctgtacttct tgtc
//
