LOCUS       sequence-446489-       10334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To establish stable dCas9-KRAB knock-in line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10334)
  AUTHORS   Zhang J, Ataei L, Mittal K, Wu L, Caldwell L, Huynh L, Sarajideen S,
            Tse K, Simon MM, Mazid MA, Cook DP, Trcka D, Kwan T, Hoffman MM, 
            Wrana JL, Esteban MA, Ramalho-Santos M
  TITLE     LINE1 and PRC2 control nucleolar organization and repression of the 
            8C state in human ESCs.
  JOURNAL   Dev Cell. 2024 Oct 14:S1534-5807(24)00574-4. doi: 
            10.1016/j.devcel.2024.09.024.
  PUBMED    39413784
REFERENCE   2  (bases 1 to 10334)
  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: "Dev Cell. 
            2024 Oct 14:S1534-5807(24)00574-4. doi: 
            10.1016/j.devcel.2024.09.024."
COMMENT     Sequence Label: pT2-hUbC-dCas9-KRAB-T2a-Puro
FEATURES             Location/Qualifiers
     source          1..10334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1163..1182)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1164..1182)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1192..1209)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1192..1208)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1201..1223)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1349..1804)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1486..1507)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1698..1717
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1831..1935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1936..2004,2005..2796)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2154..2173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2967..3555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3456..3475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3709..3726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3843..3864
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3879..3884,3885..3902,3903..3909)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3917..3933
                     /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     3922..3944
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3941..3957
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3941..3957
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3976..3996
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3978..3996
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    4471..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4508..4527)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4562..4581
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(4657..5256)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4740..4760)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5237..5256
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(5257..5310)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(5317..5337)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5404..5598)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             complement(5632..5652)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(5665..7179,7180..7182,7183..7248,7249..7254,
                     7255..9738,9739..9741,9742..9768))
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(join(5665..7248,7249..7251,7252..9738,9739..9741,
                     9742..9768))
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(5665..9768)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(9778..9798)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(9805..9870)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      9870..9879
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(join(9908..10334,1..785))
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     complement(10052..10071)
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
ORIGIN
        1 ccataactgc cgcccccgca acagacgaca aaccgagttc tccagtcagt gacaaacttc
       61 acgtcagggt ccccagatgg tgccccagcc catctcaccc gaataagagc tttcccgcat
      121 tagcgaaggc ctcaagacct tgggttcttg ccgcccacca tgccccccac cttgtttcaa
      181 cgacctcaca gcccgcctca caagcgtctt ccattcaaga ctcgggaaca gccgccattt
      241 tgctgcgctc cccccaaccc ccagttcagg gcaaccttgc tcgcggaccc agactacagc
      301 ccttggcggt ctctccacac gcttccgtcc caccgagcgg cccggcggcc acgaaagccc
      361 cggccagccc agcagcccgc tactcaccaa gtgacgatca cagcgatcca caaacaagaa
      421 ccgcgaccca aatcccggct gcgacggaac tagctgtgcc acacccggcg cgtccttata
      481 taatcatcgg cgttcaccgc cccacggaga tccctccgca gaatcgccga gaagggacta
      541 cttttcctcg cctgttccgc tctctggaaa gaaaaccagt gccctagagt cacccaagtc
      601 ccgtcctaaa atgtccttct gctgatactg gggttctaag gccgagtctt atgagcagcg
      661 ggccgctgtc ctgagcgtcc gggcggaagg atcaggacgc tcgctgcgcc cttcgtctga
      721 cgtggcagcg ctcgccgtga ggaggggggc gcccgcggga ggcgccaaaa cccggcgcgg
      781 aggccagatc tggagccgac acgggttaat taatgcggcc gcgcgcacta gtgaattcca
      841 tggatatcaa gcttctaaag ccatgacatc attttctgga attttccaag ctgtttaaag
      901 gcacagtcaa cttagtgtat gtaaacttct gacccactgg aattgtgata cagtgaatta
      961 taagtgaaat aatctgtctg taaacaattg ttggaaaaat gacttgtgtc atgcacaaag
     1021 tagatgtcct aactgacttg ccaaaactat tgtttgttaa caagaaattt gtggagtagt
     1081 tgaaaaacga gttttaatga ctccaactta agtgtatgta aacttccgac ttcaactgta
     1141 tagggatccg agctccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
     1201 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     1261 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     1321 acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc
     1381 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1441 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1501 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1561 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1621 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1681 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1741 gtagcggtca cgctgcgcgt aaccaccact cccgccgcgc ttaatgcgcc gctacagggc
     1801 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2161 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaagtta
     2341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2461 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2641 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3361 tgagaaagcg ccacggttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3601 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3661 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3721 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3781 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3841 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     3901 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     3961 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg taccgggccc
     4021 cccctcgagg tcgactctag ctagaggatc cctatacagt tgaagtcgga agtttacata
     4081 caccttagcc aaatacattt aaactcactt tttcacaatt cctgacattt aatcctagta
     4141 aaaattccct gtcttaggtc agttaggatc accactttat tttaagaatg tgaaatatca
     4201 gaataatagt agagagaatg attcatttca gcttttattt ctttcatcac attcccagtg
     4261 ggtcagaagt ttacatacac tcaattagta tttggtagca ttgcctttaa attgtttaac
     4321 ttgggtcaaa catttcgagt agccttccac aagctagatc cttatcgatc cctcttaagt
     4381 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4441 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4501 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4561 tgtggtttgt ccaaactcat cgagctcgag atcttgatca cccgggcgcg ccaccggtcg
     4621 tgctgcagaa gctagtaacg gccgccagtg tgctggtcag gcaccgggct tgcgggtcat
     4681 gcaccaggtg cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga agccgagccg
     4741 ctcgtagaag gggaggttgc ggggcgcgga ggtctccagg aaggcgggca ccccggcgcg
     4801 ctcggccgcc tccactccgg ggagcacgac ggcgctgccc agacccttgc cctggtggtc
     4861 gggcgacacg ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt gcggcgccag
     4921 gaggccttcc atctgttgct gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg
     4981 gccgatctcg gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc agaccgccac
     5041 cgcggcgccg tcgtccgcga cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa
     5101 gagttcttgc agctcggtga cccgctcgat gtggcggtcc ggatcgacgg tgtggcgcgt
     5161 ggcggggtag tcggcgaacg cggcggcgag ggtgcgtacg gccctgggga cgtcgtcgcg
     5221 ggtggcgagg cgcaccgtgg gcttgtactc ggtcataggg ccgggattct cctccacgtc
     5281 accgcatgtt agaagacttc ctctgccctc gctagcaact ttgcgtttct ttttcggaac
     5341 tgatgatttg atttcaaatg cagtctctga atcaggatgg gtctcttggt gaatttctct
     5401 ctccaccagc cagggctctt ctcccttctc caaccggagg atcacatctg gcttagtaag
     5461 ctgataaccc aaggaaacca ggttcttata gttctccagc atcacatttc tgtacaggat
     5521 ctgctgagca gtgtccagca gcttccactc ctccctggtg aagtccacaa acacatcctt
     5581 gaaggtcacc agtgtccggg accaggcagt cagtgactta gcatcgctag ccaccttcct
     5641 cttcttcttg gggtcagccc tgctgtctcc accgagctga gagaggtcga ttcttgtttc
     5701 atagagcccc gtaattgact gatgaatcag tgtggcgtcc aggacctcct ttgtagaggt
     5761 gtaccgcttt ctgtctatgg tggtgtcgaa gtacttgaag gctgcaggcg cgcccaagtt
     5821 ggtcagagta aacaagtgga taatgttttc tgcctgctcc ctgatgggct tatccctgtg
     5881 cttattgtaa gcagaaagca ccttatcgag gttagcgtcg gcgaggatca ctcttttgga
     5941 gaattcgctt atttgctcga tgatctcatc aaggtagtgt ttgtgttgtt ccacgaacag
     6001 ctgcttctgc tcattatctt cgggagaccc tttgagcttt tcatagtggc tggccagata
     6061 caagaaatta acgtatttag agggcagtgc cagctcgtta cctttctgca gctcgcccgc
     6121 actagcgagc attcgtttcc ggccgttttc aagctcaaag agagagtact tgggaagctt
     6181 aatgatgagg tcttttttga cctctttata tcctttcgcc tcgagaaagt cgatggggtt
     6241 tttttcgaag cttgatcgct ccatgattgt gatgcccagc agttccttga cgcttttgag
     6301 ttttttagac ttccctttct ccactttggc cacaaccagt acactgtaag cgactgtagg
     6361 agaatcgaat ccgccgtatt tcttggggtc ccaatctttt ttgcgtgcga tcagcttgtc
     6421 gctgttcctt ttcgggagga tactttcctt ggagaagcct ccggtctgta cttcggtctt
     6481 tttaacgatg ttcacctgcg gcatggacag gaccttccgg actgtcgcga aatccctacc
     6541 cttgtcccac acgatttctc ctgtttctcc gtttgtttcg ataagtggtc gcttccgaat
     6601 ctctccattg gccagtgtaa tctcggtctt gaaaaaattc ataatattgc tgtaaaagaa
     6661 gtacttagcg gtggccttgc ctatttcctg ctcagacttt gcgatcattt tcctaacatc
     6721 gtacacttta tagtctccgt aaacaaattc agattcaagc ttgggatatt ttttgataag
     6781 tgcagtgcct accactgcat tcaggtaggc atcatgcgca tggtggtaat tgttgatctc
     6841 tctcacctta taaaactgaa agtcctttct gaaatctgag accagcttag acttcagagt
     6901 aataactttc acctctcgaa tcagtttgtc attttcatcg tacttggtgt tcatgcgtga
     6961 atcgagaatt tgggccacgt gcttggtgat ctggcgtgtc tcaacaagct gccttttgat
     7021 gaagccggct ttatccaact cagacaggcc acctcgttca gccttagtca gattatcgaa
     7081 cttccgttgt gtgatcagtt tggcgttcag cagctgccgc caataatttt tcattttctt
     7141 gacaacttct tctgagggga cgttatcact cttccctcta tttttatcgg atcttgtcaa
     7201 cactttatta tcaatagaat catctttgag aaaagactgg ggcacgatgg catccacgtc
     7261 gtagtcggag agccgattga tgtccagttc ctgatccacg tacatgtccc tgccgttctg
     7321 caggtagtac aggtagagct tctcattctg aagctgggtg ttttcaactg ggtgttcctt
     7381 aaggatttgg gaccccagtt cttttatacc ctcttcaatc ctcttcatcc tttccctact
     7441 gttcttctgt cccttctggg tagtttggtt ctctcgggcc atctcgataa cgatattctc
     7501 gggcttatgc cttcccatta ctttgacgag ttcatccacg accttaacgg tctgcagtat
     7561 tccctttttg atagctgggc tacctgcaag attagcgatg tgctcgtgaa gactgtcccc
     7621 ctggccagaa acttgtgctt tctggatgtc ctccttaaag gtgagagagt catcatggat
     7681 caactgcatg aagttccggt tggcaaatcc atcggactta agaaaatcca ggattgtctt
     7741 tccactctgc ttgtctcgga tcccattgat cagttttctt gacagccgcc cccatcctgt
     7801 atatcggcgc ctcttgagct gtttcatgac tttgtcgtcg aagagatgag cgtaagtttt
     7861 caagcgttct tcaatcatct ccctatcttc aaacaacgta agggtgagga caatgtcctc
     7921 aagaatgtcc tcgttctcct cattgtccag gaagtccttg tctttaatga ttttcaggag
     7981 atcgtgatac gttcccaggg atgcgttgaa gcgatcctcc actccgctga tttcaacaga
     8041 gtcgaaacat tcaatctttt tgaaatagtc ttctttgagc tgtttcacgg taactttccg
     8101 gttcgtcttg aagaggaggt ccacgatagc tttcttctgc tctccagaca ggaatgctgg
     8161 ctttctcatc ccttctgtga cgtatttgac cttggtgagc tcgttataaa ctgtgaagta
     8221 ctcgtacagc agagagtgtt taggaagcac cttttcgtta ggcagatttt tatcaaagtt
     8281 agtcatcctt tcgatgaagg actgggcaga ggccccctta tccacgactt cctcgaagtt
     8341 ccagggagtg atggtctctt ctgatttgcg agtcatccac gcgaatctgg aatttccccg
     8401 ggcgaggggg cctacatagt agggtatccg aaatgtgagg attttctcaa tcttttccct
     8461 gttatctttc aaaaaggggt agaaatcctc ttgccgcctg aggatagcgt gcagttcgcc
     8521 caggtgaatc tggtggggga tgcttccatt gtcgaaagtg cgctgtttgc gcaacagatc
     8581 ttctctgtta agctttacca gcagctcctc ggtgccgtcc attttttcca agatgggctt
     8641 aataaatttg taaaattcct cctggcttgc tccgccgtca atgtatccgg cgtagccatt
     8701 tttagactga tcgaagaaaa tttccttgta cttctcaggc agttgctgtc tgacaagggc
     8761 cttcagcaaa gtcaagtctt ggtggtgctc atcatagcgc ttgatcatac tagcgctcag
     8821 cggagctttg gtgatctccg tgttcactcg cagaatatca ctcagcagaa tggcgtctga
     8881 caggttcttt gccgccaaaa aaaggtctgc gtactggtcg ccgatctggg ccagcagatt
     8941 gtcgagatca tcatcgtagg tgtctttgct cagttgaagc ttggcatctt cggccaggtc
     9001 gaagttagat ttaaagttgg gggtcagccc gagtgacagg gcgataagat taccaaacag
     9061 gccgttcttc ttctccccag ggagctgtgc gatgaggttt tcgagccgcc gggatttgga
     9121 cagcctagcg ctcaggattg ctttggcgtc aactccggat gcgttgatcg ggttctcttc
     9181 gaaaagctga ttgtaagtct gaaccagttg gataaagagt ttgtcgacat cgctgttgtc
     9241 tgggttcagg tccccctcga tgaggaagtg tccccgaaat ttgatcatat gcgccagcgc
     9301 gagatagatc aaccgcaagt cagccttatc agtactgtct acaagcttct tcctcagatg
     9361 atatatggtt gggtactttt catggtacgc cacctcgtcc acgatattgc caaagattgg
     9421 gtggcgctcg tgctttttat cctcctccac caaaaaggac tcctccagcc tatggaagaa
     9481 agagtcatcc accttagcca tctcattact aaagatctcc tgcaggtagc agatccgatt
     9541 ctttctgcgg gtatatctgc gccgtgctgt tcttttgagc cgcgtggctt cggcggtttc
     9601 cccggagtcg aacaggaggg cgccaatgag gttcttcttt atgctgtggc gatcggtatt
     9661 gcccagaact ttgaattttt tgctcggcac cttgtactcg tccgtaatga cggcccagcc
     9721 gacgctgttt gtgccgatgg cgagcccaat ggagtacttc ttgtccattc cgcggcccac
     9781 cttcctcttc ttcttggggg ccatcttgtc atcgtcatcc ttgtaatcga tgtcatgatc
     9841 tttataatca ccgtcatggt ctttgtagtc catggtggct ctagagtcga cctgcagccc
     9901 aagcttcgtc taacaaaaaa gccaaaaacg gccagaattt agcggacaat ttactagtct
     9961 aacactgaaa attacatatt gacccaaatg attacatttc aaaaggtgcc taaaaaactt
    10021 cacaaaacac actcgccaac cccgagcgca tagttcaaaa ccggagcttc agctacttaa
    10081 gaagataggt acataaaacc gaccaaagaa actgacgcct cacttatccc tcccctcacc
    10141 agaggtccgg cgcctgtcga ttcaggagag cctaccctag gcccgaaccc tgcgtcctgc
    10201 gacggagaaa agcctaccgc acacctaccg gcaggtggcc ccaccctgca ttataagcca
    10261 acagaacggg tgacgtcacg acacgacgag ggcgcgcgct cccaaaggta cgggtgcact
    10321 gcccaacggc accg
//
