LOCUS       sequence-386828-       11317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting the Rosa26 locus in a thid generation Cas9 backbone 
            with TagBFP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11317)
  AUTHORS   Girish V, Lakhani AA, Thompson SL, Scaduto CM, Brown LM, Hagenson 
            RA, Sausville EL, Mendelson BE, Kandikuppa PK, Lukow DA, Yuan ML, 
            Stevens EC, Lee SN, Schukken KM, Akalu SM, Vasudevan A, Zou C, 
            Salovska B, Li W, Smith JC, Taylor AM, Martienssen RA, Liu Y, Sun R,
            Sheltzer JM
  TITLE     Oncogene-like addiction to aneuploidy in human cancers.
  JOURNAL   Science. 2023 Jul 6:eadg4521. doi: 10.1126/science.adg4521.
  PUBMED    37410869
REFERENCE   2  (bases 1 to 11317)
  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: "Science. 
            2023 Jul 6:eadg4521. doi: 10.1126/science.adg4521."
COMMENT     Sequence Label: Rosa26_gRNA
FEATURES             Location/Qualifiers
     source          1..11317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        128..339
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      359..368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             368..391
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             398..418
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             443..4543
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             443..4543
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4544..4591
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4607..4663
                     /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             4664..5365
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    5372..5960
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5425..5445)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5843..5854)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6032..6265
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6343..6464
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6380..6399)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6434..6453
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6504..6639
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6659..6678)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6660..6678)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6688..6705)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6688..6704)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6697..6719)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6846..7301
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6933..6952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7143..7164
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7327..7431
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7432..7500,7501..8292)
                     /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(7650..7669)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8463..9051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8952..8971
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9205..9222
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9339..9360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9375..9380,9381..9398,9399..9405)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9413..9429
                     /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     9418..9440
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9437..9453
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9437..9453
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9472..9492
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9474..9492
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        9520..9746
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             9747..9927
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    9974..10099
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    10592..10825
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        complement(11004..11252)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(11062..11081)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(11232..11252)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    join(11259..11317,1..59)
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
       61 gggtttatta cagggacagc agagatccac tttggcgccg gctcgagtgg ctccggtgcc
      121 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      181 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      241 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      301 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc
      361 caccatggat tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg
      421 tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa
      481 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
      541 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
      601 cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag
      661 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
      721 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
      781 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
      841 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
      901 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
      961 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     1021 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     1081 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     1141 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     1201 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     1261 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     1321 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     1381 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     1441 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     1501 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     1561 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     1621 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     1681 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     1741 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     1801 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     1861 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     1921 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     1981 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     2041 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     2101 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     2161 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     2221 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     2281 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     2341 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     2401 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca
     2461 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat
     2521 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc
     2581 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa
     2641 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca
     2701 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca
     2761 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca
     2821 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta
     2881 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc
     2941 cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa
     3001 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga
     3061 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca
     3121 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc
     3181 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat
     3241 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa
     3301 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa
     3361 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg
     3421 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa
     3481 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     3541 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     3601 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     3661 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     3721 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     3781 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     3841 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     3901 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     3961 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     4021 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     4081 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     4141 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     4201 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     4261 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     4321 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     4381 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     4441 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     4501 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaagcgtc ctgctgctac
     4561 taagaaagct ggtcaagcta agaaaaagaa agctagcggc agcggcgcca ccaacttcag
     4621 cctgctgaag caggccggcg acgtggagga gaaccccggc cccatgagcg agctgattaa
     4681 ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg
     4741 cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt
     4801 cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag
     4861 caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga
     4921 gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca
     4981 ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt
     5041 cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgaaac
     5101 gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg
     5161 cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac ccgctaagaa
     5221 cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa
     5281 caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag
     5341 caaactgggg cacaagctta attgagtcga caatcaacct ctggattaca aaatttgtga
     5401 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     5461 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     5521 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     5581 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     5641 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     5701 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     5761 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5821 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5881 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5941 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     6001 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     6061 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     6121 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     6181 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     6241 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     6301 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     6361 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6421 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     6481 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     6541 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     6601 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     6661 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     6721 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6781 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     6841 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6901 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6961 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     7021 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     7081 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7141 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7201 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7261 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     7321 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7381 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     7441 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     7501 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     7561 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7621 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7681 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7741 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7801 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7861 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7921 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7981 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     8041 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     8101 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     8161 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     8221 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     8281 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     8341 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     8401 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     8461 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     8521 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     8581 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     8641 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8701 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8761 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8821 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8881 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8941 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     9001 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     9061 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     9121 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     9181 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     9241 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     9301 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     9361 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     9421 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     9481 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
     9541 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
     9601 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
     9661 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
     9721 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
     9781 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     9841 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     9901 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     9961 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
    10021 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
    10081 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
    10141 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
    10201 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
    10261 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
    10321 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
    10381 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
    10441 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
    10501 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
    10561 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
    10621 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
    10681 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
    10741 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
    10801 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcgccattt
    10861 gtctcaagat ctagaattca aaaaaagcac cgactcggtg ccactttgct gtttccagca
    10921 aagttgataa cggactagcc ttatttaaac ttgctatgct gtttccagca tagctcttaa
    10981 accgggttag acaacttgct gtcggtgttt cgtcctttcc acaagatata taaagccaag
    11041 aaatcgaaat actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt
    11101 aaaactgcaa actacccaag aaattattac tttctacgtc acgtattttg tactaatatc
    11161 tttgtgttta cagtcaaatt aattctaatt atctctctaa cagccttgta tcgtatatgc
    11221 aaatatgaag gaatcatggg aaataggccc tcccacaatt ttaaaagaaa aggggggatt
    11281 ggggggtaca gtgcagggga aagaatagta gacataa
//
