LOCUS       sequence-497463-       13507 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Expression of Cas9 with Thy1.1 cell surface marker under a 
            doxycycline responsive promoter. Requires additional expression of 
            transactivator..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13507)
  AUTHORS   Muhar MF, Farnung J, Cernakova M, Hofmann R, Henneberg LT, 
            Pfleiderer MM, Denoth-Lippuner A, Kalcic F, Nievergelt AS, Peters 
            Al-Bayati M, Sidiropoulos ND, Beier V, Mann M, Jessberger S, Jinek 
            M, Schulman BA, Bode JW, Corn JE
  TITLE     C-terminal amides mark proteins for degradation via SCF-FBXO31.
  JOURNAL   Nature. 2025 Feb;638(8050):519-527. doi: 10.1038/s41586-024-08475-w.
  PUBMED    39880951
REFERENCE   2  (bases 1 to 13507)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 15, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nature"; date: "2025-02"; volume: "638"; issue: "8050"; pages: 
            "519-527"
COMMENT     Sequence Label: pJC2297 - pHR-TRE3G-hSpCas9-NLS-FLAG-2A-Thy1.1
FEATURES             Location/Qualifiers
     source          1..13507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(830..850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        835..1164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1015..1150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          2298..2363
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             2493..2513
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2938..3072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2975..2994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3029..3048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             3240..3873
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3920..4045
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4542..4775
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4960..5004
                     /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    5289..5406
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5468..5817
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    5476..5494
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5512..5530
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5548..5566
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5584..5602
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5620..5638
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5656..5674
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5692..5710
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5756..5780
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      5824..5833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5830..9933
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5830..9933
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9934..9981
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9982..10005
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10021..10077
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             10084..10572
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     misc_feature    10610..11198
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10663..10683)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11081..11092)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11201..11217)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11202..11218)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11308..11541
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(11566..11584)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(11567..11584)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(11874..11893)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12107..12126)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12226..12248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(12286..12304)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12372..12476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12477..12545,12546..13337)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(12695..12714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgtg gaatgtgtgt
      841 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      901 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      961 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     1021 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     1081 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     1141 tttggaggcc taggcttttg caaaaagctt ggacacaaga caggcttgcg agatatgttt
     1201 gagaatacca ctttatcccg cgtcagggag aggcagtgcg taaaaagacg cggactcatg
     1261 tgaaatactg gtttttagtg cgccagatct ctataatctc gcgcaaccta ttttcccctc
     1321 gaacactttt taagccgtag ataaacaggc tgggacactt cacatgagcg aaaaatacat
     1381 cgtcacctgg gacatgttgc agatccatgc acgtaaactc gcaagccgac tgatgccttc
     1441 tgaacaatgg aaaggcatta ttgccgtaag ccgtggcggt ctgtaccggg tgcgttactg
     1501 gcgcgtgaac tgggtattcg tcatgtcgat accgtttgta tttccagcta cgatcacgac
     1561 aaccagcgcg agcttaaagt gctgaaacgc gcagaaggcg atggcgaagg cttcatcgtt
     1621 attgatgacc tggtggatac cggtggtact gcggttgcga ttcgtgaaat gtatccaaaa
     1681 gcgcactttg tcaccatctt cgcaaaaccg gctggtcgtc cgctggttga tgactatgtt
     1741 gttgatatcc cgcaagatac ctggattgaa cagccgtggg atatgggcgt cgtattcgtc
     1801 ccgccaatct ccggtcgcta atcttttcaa cgcctggcac tgccgggcgt tgttcttttt
     1861 aacttcaggc gggttacaat agtttccagt aagtattctg gaggctgcat ccatgacaca
     1921 ggcaaacctg agcgaaaccc tgttcaaacc ccgctttaaa catcctgaaa cctcgacgct
     1981 agtccgccgc tttaatcacg gcgcacaacc gcctgtgcag tcggcccttg atggtaaaac
     2041 catccctcac tggtatcgca tgattaaccg tctgatgtgg atctggcgcg gcattgaccc
     2101 acgcgaaatc ctcgacgtcc aggcacgtat tgtgatgagc gatgccgaac gtaccgacga
     2161 tgatttatac gatacggtga ttggctaccg tggcggcaac tggatttatg agtgggcccc
     2221 ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag
     2281 agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga
     2341 ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt
     2401 ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg
     2461 aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag gtagaagacc
     2521 ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa
     2581 ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa
     2641 ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat cataacatac
     2701 tgttttttct tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat
     2761 tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg
     2821 ccttgactag agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2881 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2941 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3001 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3061 catgtctgga tcaactggat aactcaagct aaccaaaatc atcccaaact tcccacccca
     3121 taccctatta ccactgccaa ttacctgtgg tttcatttac tctaaacctg tgattcctct
     3181 gaattatttt cattttaaag aaattgtatt tgttaaatat gtactacaaa cttagtagtt
     3241 ggaagggcta attcactccc aaagaagaca agatatcctt gatctgtgga tctaccacac
     3301 acaaggctac ttccctgatt agcagaacta cacaccaggg ccaggggtca gatatccact
     3361 gacctttgga tggtgctaca agctagtacc agttgagcca gataaggtag aagaggccaa
     3421 taaaggagag aacaccagct tgttacaccc tgtgagcctg catgggatgg atgacccgga
     3481 gagagaagtg ttagagtgga ggtttgacag ccgcctagca tttcatcacg tggcccgaga
     3541 gctgcatccg gagtacttca agaactgctg atatcgagct tgctacaagg gactttccgc
     3601 tggggacttt ccagggaggc gtggcctggg cgggactggg gagtggcgag ccctcagatc
     3661 ctgcatataa gcagctgctt tttgcctgta ctgggtctct ctggttagac cagatctgag
     3721 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     3781 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     3841 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
     3901 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
     3961 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
     4021 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
     4081 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
     4141 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     4201 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     4261 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     4321 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     4381 agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
     4441 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     4501 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     4561 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     4621 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     4681 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     4741 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     4801 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     4861 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     4921 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     4981 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     5041 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     5101 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     5161 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     5221 agagacagat ccattcgatt agtgaacgga tctcgacggt atcgccaaat ggcagtattc
     5281 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     5341 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     5401 aattttcggg tttattacag ggacagcaga gatccagttt atcgatgagg ccctttcgtc
     5461 ttcactcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     5521 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     5581 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     5641 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     5701 gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg cgcctataaa agcagagctc
     5761 gtttagtgaa ccgtcagatc gcctggagca attccacaac acttttgtct tatacttacg
     5821 cgtgccacca tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
     5881 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
     5941 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
     6001 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
     6061 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
     6121 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
     6181 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
     6241 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
     6301 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
     6361 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
     6421 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     6481 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     6541 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     6601 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     6661 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     6721 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     6781 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     6841 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     6901 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     6961 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     7021 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     7081 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     7141 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     7201 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     7261 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     7321 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     7381 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     7441 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     7501 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     7561 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     7621 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     7681 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     7741 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     7801 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     7861 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     7921 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     7981 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     8041 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     8101 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     8161 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     8221 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     8281 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     8341 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     8401 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     8461 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     8521 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     8581 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     8641 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     8701 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     8761 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     8821 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     8881 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     8941 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     9001 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     9061 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     9121 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     9181 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     9241 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     9301 aagagtgtga aagagctact ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     9361 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     9421 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     9481 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     9541 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     9601 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     9661 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     9721 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     9781 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     9841 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     9901 acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct
     9961 ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc cggcagcggc
    10021 gccaccaact tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc cggccccatg
    10081 ggtatgaacc cagccatcag cgtcgctctc ctgctctcag tcttgcaggt gtcccgaggg
    10141 cagaaggtga ccagcctgac agcctgcctg gtgaaccaaa accttcgcct ggactgccgc
    10201 catgagaata acaccaagga taactccatc cagcatgagt tcagcctgac ccgagagaag
    10261 aggaagcacg tgctctcagg cacccttggg atacccgagc acacgtaccg ctcccgcgtc
    10321 accctctcca accagcccta tatcaaggtc cttaccctag ccaacttcac caccaaggat
    10381 gagggcgact acttttgtga gcttcgcgtc tcgggcgcga atcccatgag ctccaataaa
    10441 agtatcagtg tgtatagaga caagctggtc aagtgtggcg gcataagcct gctggttcag
    10501 aacacatcct ggatgctgct gctgctgctt tccctctccc tcctccaagc cctggacttc
    10561 atttctctgt gacctgcagg catgcaagct tgatatcaag cttatcgata atcaacctct
    10621 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
    10681 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
    10741 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
    10801 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
    10861 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
    10921 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
    10981 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
    11041 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
    11101 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
    11161 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgagg
    11221 gaattaattc gagctcggta cctttaagac caatgactta caaggcagct gtagatctta
    11281 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
    11341 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
    11401 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
    11461 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
    11521 cagtgtggaa aatctctagc agcatctaga attaattccg tgtattctat agtgtcacct
    11581 aaatcgtatg tgtatgatac ataaggttat gtattaattg tagccgcgtt ctaacgacaa
    11641 tatgtacaag cctaattgtg tagcatctgg cttactgaag cagaccctat catctctctc
    11701 gtaaactgcc gtcagagtcg gtttggttgg acgaaccttc tgagtttctg gtaacgccgt
    11761 cccgcacccg gaaatggtca gcgaaccaat cagcagggtc atcgctagcc agatcctcta
    11821 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
    11881 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
    11941 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
    12001 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
    12061 aatgcaggag tcgcataagg gagagcgtcg aatggtgcac tctcagtaca atctgctctg
    12121 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
    12181 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
    12241 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
    12301 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
    12361 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
    12421 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
    12481 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
    12541 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
    12601 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
    12661 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
    12721 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
    12781 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
    12841 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
    12901 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
    12961 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
    13021 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
    13081 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
    13141 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
    13201 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
    13261 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
    13321 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
    13381 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
    13441 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
    13501 gatcttc
//
