LOCUS       sequence-333975-       12371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for transposon stgRNA library construction (library 4) 
            (SpCas9 expression is induced upon Cre-loxP recombination.).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12371)
  AUTHORS   Park J, Lim JM, Jung I, Heo SJ, Park J, Chang Y, Kim HK, Jung D, Yu 
            JH, Min S, Yoon S, Cho SR, Park T, Kim HH
  TITLE     Recording of elapsed time and temporal information about biological 
            events using Cas9.
  JOURNAL   Cell. 2021 Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014.
  PUBMED    33539780
REFERENCE   2  (bases 1 to 12371)
  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: "Cell. 2021 
            Jan 28. pii: S0092-8674(21)00014-3. doi: 
            10.1016/j.cell.2021.01.014."
COMMENT     Sequence Label: Cre-responsive Cas9 expressing transposon libr...
FEATURES             Location/Qualifiers
     source          1..12371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..30
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     22..39
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     23..39
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    312..536
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(564..597)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             complement(625..1224)
                     /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(708..728)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1205..1224
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     protein_bind    complement(1270..1303)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1310..2487)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1319..2256
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(1342..1362)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        complement(2607..2855)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2665..2684)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2835..2855)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    2867..2900
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    3087..3221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3341..3475
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3595..3729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3789..3822
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3832..3841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3838..7941
                     /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             3838..7941
                     /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             7957..7977
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7978..8004
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             7978..8004
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             8020..8079
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             join(8086..8088,8089..8091,8092..8805)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     8739..8760
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    8857..8938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9606..9626)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9606..9624)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9645..9661)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9645..9661)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9658..9680)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9669..9685
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9693..9699,9700..9717,9718..9723))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9738..9759
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9876..9893)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10047..10635)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10127..10146)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10806..11597,11598..11666))
                     /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     11429..11448
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11667..11771)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      11798..12253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11885..11904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12095..12116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gggttttccc agtcacgacg ttgtaaaacg acggccagtg gcgcgcctac agttgaagtc
       61 ggaagtttac atacacttaa ggacaggatt ggtgacagaa aagccccatc cttaggcagc
      121 tagtcaggta gtaaaaggtc tcaactaggc agtggcagag caggattcaa attcagggct
      181 gttgtgatgc ctccgcagac tctgagcgcc acctggtggt aatttgtctg tgcctcttct
      241 gacgtggaag aacagcaact aacacactaa cacggcattt actatgggcc agccattgtt
      301 ctaagctagc cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
      361 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
      421 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
      481 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggggta
      541 cctcgtatgc cgtcttctgc ttgataactt cgtataggat actttatacg aagttataac
      601 ttacttgtac agctcgtaac gcgttcaggc accgggcttg cgggtcatgc accaggtgcg
      661 cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct cgtagaaggg
      721 gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct cggccgcctc
      781 cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg gcgagactcc
      841 gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga ggccttccat
      901 ctgttgctgc gcggccagcc gggaaccgct caactcggcc atgcgcgggc cgatctcggc
      961 gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccacgg cggcgccgtc
     1021 gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga gttcttgcag
     1081 ctcggtgacc cgctcgatgt ggcggtccgg atcgacggtg tggcgcgtgg cggggtagtc
     1141 ggcgaacgcg gcggcgaggg tgcgtacggc cctggggacg tcgtcgcggg tggcgaggcg
     1201 caccgtgggc ttgtactcgg tcatggtggc gatatcatgt aagttgtcac gaagttgcag
     1261 tgcagatgga taacttcgta tagcatacat tatacgaagt tatcgtacgt cacgacacct
     1321 gaaatggaag aaaaaaactt tgaaccactg tctgaggctt gagaatgaac caagatccaa
     1381 actcaaaaag ggcaaattcc aaggagaatt acatcaagtg ccaagctggc ctaacttcag
     1441 tctccaccca ctcagtgtgg ggaaactcca tcgcataaaa cccctccccc caacctgaag
     1501 acgacgtact ccaaaagctc gagaactaat cgaggtgcct ggacggcgcc cggtactccg
     1561 tggagtcaca tgaagcgacg gctgaggacg gaaaggccct tttcctttgt gtgggtgact
     1621 cacccgcccg ctctcccgag cgccgcgtcc tccattttga gctccctgca gcagggccgg
     1681 gaagcggcca tctttccgct cacgcaactg gtgccgaccg ggccagcctt gccgcccagg
     1741 gcggggcgat acacggcggc gcgaggccag gcaccagagc aggccggcca gcttgagact
     1801 acccccgtcc gattctcggt ggccgcgctc gcaggccccg cctcgccgaa catgtgcgct
     1861 gggacgcacg ggccccgtcg ccgcccgcgg ccccaaaaac cgaaatacca gtgtgcagat
     1921 cttggcccgc atttacaaga ctatcttgcc agaaaaaaag cgtcgcagca ggtcatcaaa
     1981 aattttaaat ggctagagac ttatcgaaag cagcgagaca ggcgcgaagg tgccaccaga
     2041 ttcgcacgcg gcggccccag cgcccaggcc aggcctcaac tcaagcacga ggcgaagggg
     2101 ctccttaagc gcaaggcctc gaactctccc acccacttcc aacccgaagc tcgggatcaa
     2161 gaatcacgta ctgcagccag tggaagtaat tcaaggcacg caagggccat aacccgtaaa
     2221 gaggccaggc ccgcgggaac cacacacggc acttacctgt gttctggcgg caaacccgtt
     2281 gcgaaaaaga acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg
     2341 aaaaaggcgg agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta
     2401 ggcaccggat caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg
     2461 ctctgcccac tgacgggcac cggagcctta aaaggcgcgt ctagaataaa aaaagcaccg
     2521 actcggtgcc actttttcaa gttgataacg gactagcctt aggttaactt gctatttcta
     2581 gctctaaccc agagacgtcc gtctccggtg tttcgtcctt tccacaagat atataaagcc
     2641 aagaaatcga aatactttca agttacggta agcatatgat agtccatttt aaaacataat
     2701 tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat
     2761 atctttgtgt ttacagtcaa attaattcta attatctctc taacagcctt gtatcgtata
     2821 tgcaaatatg aaggaatcat gggaaatagg ccctcactag ttttttataa cttcgtataa
     2881 agtatcctat acgaagttat cctagaccgg tttttaaaag aaaaggggaa taaatgaaag
     2941 cttgcagatc tgcgactcta gaggatctgc gactctagag gatcataatc agccatacca
     3001 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     3061 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     3121 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3181 gtttgtccaa actcatcaat gtatcttatc atgtctggat ctgcgactct agaggatcat
     3241 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3301 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     3361 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3421 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgcga
     3481 ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3541 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3601 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3661 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3721 gtctggatcc ccatcaagct gatccggaac ccttcaaaaa caaattacaa aaattcaaaa
     3781 ttttgatcat aacttcgtat aatgtatgct atacgaagtt atgctcccgg ggccaccatg
     3841 gacaagaagt acagcatcgg cctggacatc ggtaccaaca gcgtgggctg ggccgtgatc
     3901 accgacgagt acaaggtgcc cagcaagaag ttcaaggtgc tgggcaacac cgaccgccac
     3961 agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggcgagac cgccgaggcc
     4021 acccgcctga agcgcaccgc ccgccgccgc tacacccgcc gcaagaaccg catctgctac
     4081 ctgcaggaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccaccgcctg
     4141 gaggagagct tcctggtgga ggaggacaag aagcacgagc gccaccccat cttcggcaac
     4201 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gcgcaagaag
     4261 ctggtggaca gcaccgacaa ggccgacctg cgcctgatct acctggccct ggcccacatg
     4321 atcaagttcc gcggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     4381 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga gaaccccatc
     4441 aacgccagcg gcgtggacgc caaggccatc ctgagcgccc gcctgagcaa gagccgccgc
     4501 ctggagaacc tgatcgccca gctgcccggc gagaagaaga acggcctgtt cggcaacctg
     4561 atcgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggac
     4621 gccaagctgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     4681 atcggcgacc agtacgccga cctgttcctg gccgccaaga acctgagcga cgccatcctg
     4741 ctgagcgaca tcctgcgcgt gaacaccgag atcaccaagg cccccctgag cgccagcatg
     4801 atcaagcgct acgacgagca ccaccaggac ctgaccctgc tgaaggccct ggtgcgccag
     4861 cagctgcccg agaagtacaa ggagatcttc ttcgaccaga gcaagaacgg ctacgccggc
     4921 tacatcgacg gcggcgccag ccaggaggag ttctacaagt tcatcaagcc catcctggag
     4981 aagatggacg gcaccgagga gctgctggtg aagctgaacc gcgaggacct gctgcgcaag
     5041 cagcgcacct tcgacaacgg cagcatcccc caccagatcc acctgggcga gctgcacgcc
     5101 atcctgcgcc gccaggagga cttctacccc ttcctgaagg acaaccgcga gaagatcgag
     5161 aagatcctga ccttccgcat cccctactac gtgggccccc tggcccgcgg caacagccgc
     5221 ttcgcctgga tgacccgcaa gagcgaggag accatcaccc cctggaactt cgaggaggtg
     5281 gtggacaagg gcgccagcgc ccagagcttc atcgagcgca tgaccaactt cgacaagaac
     5341 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtac
     5401 aacgagctga ccaaggtgaa gtacgtgacc gagggcatgc gcaagcccgc cttcctgagc
     5461 ggcgagcaga agaaggccat cgtggacctg ctgttcaaga ccaaccgcaa ggtgaccgtg
     5521 aagcagctga aggaggacta cttcaagaag atcgagtgct tcgacagcgt ggagatcagc
     5581 ggcgtggagg accgcttcaa cgccagcctg ggcacctacc acgacctgct gaagatcatc
     5641 aaggacaagg acttcctgga caacgaggag aacgaggaca tcctggagga catcgtgctg
     5701 accctgaccc tgttcgagga ccgcgagatg atcgaggagc gcctgaagac ctacgcccac
     5761 ctgttcgacg acaaggtgat gaagcagctg aagcgccgcc gctacaccgg ctggggccgc
     5821 ctgagccgca agcttatcaa cggcatccgc gacaagcaga gcggcaagac catcctggac
     5881 ttcctgaaga gcgacggctt cgccaaccgc aacttcatgc agctgatcca cgacgacagc
     5941 ctgaccttca aggaggacat ccagaaggcc caggtgagcg gccagggcga cagcctgcac
     6001 gagcacatcg ccaacctggc cggcagcccc gccatcaaga agggcatcct gcagaccgtg
     6061 aaggtggtgg acgagctggt gaaggtgatg ggccgccaca agcccgagaa catcgtgatc
     6121 gagatggccc gcgagaacca gaccacccag aagggccaga agaacagccg cgagcgcatg
     6181 aagcgcatcg aggagggcat caaggagctg ggcagccaga tcctgaagga gcaccccgtg
     6241 gagaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa cggccgcgac
     6301 atgtacgtgg accaggagct ggacatcaac cgcctgagcg actacgacgt ggaccacatc
     6361 gtgccccaga gcttcctgaa ggacgacagc atcgacaaca aggtgctgac ccgcagcgac
     6421 aagaaccgcg gcaagagcga caacgtgccc agcgaggagg tggtgaagaa gatgaagaac
     6481 tactggcgcc agctgctgaa cgccaagctg atcacccagc gcaagttcga caacctgacc
     6541 aaggccgagc gcggcggcct gagcgagctg gacaaggccg gcttcatcaa gcgccagctg
     6601 gtggagaccc gccagatcac caagcacgtg gcccagatcc tggacagccg catgaacacc
     6661 aagtacgacg agaacgacaa gctgatccgc gaggtgaagg tgatcaccct gaagagcaag
     6721 ctggtgagcg acttccgcaa ggacttccag ttctacaagg tgcgcgagat caacaactac
     6781 caccacgccc acgacgccta cctgaacgcc gtggtgggca ccgccctgat caagaagtac
     6841 cccaagctgg agagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcgcaagatg
     6901 atcgccaaga gcgagcagga gatcggcaag gccaccgcca agtacttctt ctacagcaac
     6961 atcatgaact tcttcaagac cgagatcacc ctggccaacg gcgagatccg caagcgcccc
     7021 ctgatcgaga ccaacggcga gaccggcgag atcgtgtggg acaagggccg cgacttcgcc
     7081 accgtgcgca aggtgctgag catgccccag gtgaacatcg tgaagaagac cgaggtgcag
     7141 accggcggct tcagcaagga gagcatcctg cccaagcgca acagcgacaa gctgatcgcc
     7201 cgcaagaagg actgggaccc caagaagtac ggcggcttcg acagccccac cgtggcctac
     7261 agcgtgctgg tggtggccaa ggtggagaag ggcaagagca agaagctgaa gagcgtgaag
     7321 gagctgctgg gcatcaccat catggagcgc agcagcttcg agaagaaccc catcgacttc
     7381 ctggaggcca agggctacaa ggaggtgaag aaggacctga tcatcaagct gcccaagtac
     7441 agcctgttcg agctggagaa cggccgcaag cgcatgctgg ccagcgccgg cgagctgcag
     7501 aagggcaacg agctggccct gcccagcaag tacgtgaact tcctgtacct ggccagccac
     7561 tacgagaagc tgaagggcag ccccgaggac aacgagcaga agcagctgtt cgtggagcag
     7621 cacaagcact acctggacga gatcatcgag cagatcagcg agttcagcaa gcgcgtgatc
     7681 ctggccgacg ccaacctgga caaggtgctg agcgcctaca acaagcaccg cgacaagccc
     7741 atccgcgagc aggccgagaa catcatccac ctgttcaccc tgaccaacct gggcgccccc
     7801 gccgccttca agtacttcga caccaccatc gaccgcaagc gctacaccag caccaaggag
     7861 gtgctggacg ccaccctgat ccaccagagc atcaccggtc tgtacgagac ccgcatcgac
     7921 ctgagccagc tgggcggcga cggcggctcc ggacctccaa agaaaaagag aaaagtatac
     7981 ccctacgacg tgcccgacta cgccgaattc ggaagcgggc agtgcaccaa ctacgccctg
     8041 ctgaagctgg ccggcgacgt ggagagcaac cccggccccg gatccatggt gagcaagggc
     8101 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     8161 cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg
     8221 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     8281 ggctacggcg tggcctgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     8341 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tctctttcaa ggacgacggt
     8401 acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     8461 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     8521 ttcaacagcc actacgtcta tatcacggcc gacaagcaga agaactgcat caaggctaac
     8581 ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     8641 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagccatcag
     8701 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     8761 accgccgccg ggattacaca tggcatggac gagctgtaca agtgacccgg cgcgttaagt
     8821 cgagctcgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     8881 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     8941 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     9001 aagcaagtaa aacctctaca aatgtggtac ttaagaggga tcgataagga tctagcttgt
     9061 ggaaggctac tcgaaatgtt tgacccaagt taaacaattt aaaggcaatg ctaccaaata
     9121 ctaattgagt gtatgtaaac ttctgaccca ctgggaatgt gatgaaagaa ataaaagctg
     9181 aaatgaatca ttctctctac tattattctg atatttcaca ttcttaaaat aaagtggtcc
     9241 taggagctag tcaggtagta aaaggtctca actaggcagt ggcagagcag gattcaaatt
     9301 cagggctgtt gtgatgcctc cgcagactct gagcgccacc tggtggtaat ttgtctgtgc
     9361 ctcttctgac gtggaagaac agcaactaac acactaacac ggcatttact atgggccagc
     9421 cattgttcta agatcctaac tgacctaaga cagggaattt ttactaggat taaatgtcag
     9481 gaattgtgaa aaagtgagtt taaatgtatt tggctaaggt gtatgtaaac ttccgacttc
     9541 aactgtatag ggatcctcta gctagagtcg acctcgaggg ggggcccggt acccagcttt
     9601 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     9661 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     9721 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     9781 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     9841 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     9901 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     9961 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
    10021 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
    10081 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
    10141 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
    10201 ctgtccgcct ttctcccttc gggaaccgtg gcgctttctc atagctcacg ctgtaggtat
    10261 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
    10321 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
    10381 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
    10441 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
    10501 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
    10561 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
    10621 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
    10681 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
    10741 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
    10801 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
    10861 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
    10921 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
    10981 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
    11041 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
    11101 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
    11161 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
    11221 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaacttggc cgcagtgtta
    11281 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
    11341 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
    11401 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
    11461 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
    11521 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
    11581 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
    11641 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
    11701 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
    11761 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta
    11821 agcgcggcgg gagtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
    11881 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
    11941 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
    12001 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
    12061 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
    12121 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
    12181 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
    12241 acgcttacaa tttccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
    12301 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
    12361 gggtaacgcc a
//
