LOCUS       sequence-174573-       15327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid contains destabilized Cas9 and has Venus after P2A 
            sequence. This vector also contains filler sequence which required 
            to be remove for cloning of desired sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15327)
  AUTHORS   Senturk S, Shirole NH, Nowak DG, Corbo V, Pal D, Vaughan A, Tuveson 
            DA, Trotman LC, Kinney JB, Sordella R
  TITLE     Rapid and tunable method to temporally control gene editing based on
            conditional Cas9 stabilization.
  JOURNAL   Nat Commun. 2017 Feb 22;8:14370. doi: 10.1038/ncomms14370.
  PUBMED    28224990
REFERENCE   2  (bases 1 to 15327)
  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: "Nat 
            Commun."; date: "2017-02-22"; volume: "8:14370. doi"; pages: " 
            10.1038/ncomms14370"
COMMENT     Sequence Label: DD-Cas9 with filler sequence and Venus (EDCPV)
FEATURES             Location/Qualifiers
     source          1..15327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4738..4813
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4875..5086
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      5100..5109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5106..5108,5109..5429)
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD
                     /note="L106P mutant of FKBP12"
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKPE"
     CDS             5439..9542
                     /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             5439..9542
                     /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             9543..9590
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             9591..9614
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             9624..9680
                     /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             join(9684..9686,9687..9689,9690..10403)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(9990..10009)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     10337..10358
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    10419..11007
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10472..10492)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10890..10901)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             11079..11312
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(11338..11355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11344..11568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11614..12042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11701..11720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11911..11932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12051..12071)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12056..12385
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12236..12371
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12298..12317
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12433..12480
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12499..12873
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    13003..13124
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13040..13059)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13094..13113
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13173..13189)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13173..13189)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13186..13208)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13197..13213
                     /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(13221..13227,13228..13245,13246..13251))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13266..13287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13404..13421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13575..14163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13655..13674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14334..15125,15126..15194))
                     /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     14957..14976
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15195..15299)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac
     2881 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
     2941 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
     3001 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
     3061 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
     3121 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
     3181 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
     3241 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
     3301 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
     3361 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
     3421 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
     3481 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
     3541 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
     3601 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
     3661 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
     3721 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
     3781 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
     3841 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
     3901 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
     3961 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
     4021 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
     4081 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
     4141 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
     4201 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
     4261 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
     4321 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
     4381 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
     4441 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
     4501 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
     4561 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
     4621 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
     4681 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
     4741 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     4801 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     4861 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     4921 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     4981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     5041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
     5101 ccaccatggg agtgcaggtg gaaaccatct ccccaggtga cgggcgcacc ttccccaagc
     5161 gcggccagac ctgtgtggtg cactacaccg ggatgcttga agatggaaag aaagtcgatt
     5221 cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag
     5281 gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc
     5341 cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg
     5401 tcttcgatgt ggagcttcta aaaccggaac cggctagcat ggacaagaag tacagcatcg
     5461 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     5521 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     5581 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     5641 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     5701 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     5761 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     5821 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     5881 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     5941 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     6001 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     6061 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     6121 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     6181 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     6241 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     6301 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     6361 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     6421 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     6481 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     6541 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     6601 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     6661 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     6721 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     6781 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     6841 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     6901 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     6961 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     7021 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     7081 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     7141 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     7201 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     7261 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     7321 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     7381 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     7441 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     7501 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     7561 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     7621 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     7681 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     7741 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     7801 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     7861 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     7921 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     7981 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     8041 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     8101 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     8161 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     8221 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     8281 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     8341 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     8401 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     8461 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     8521 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     8581 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     8641 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     8701 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     8761 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     8821 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     8881 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     8941 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     9001 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     9061 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     9121 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     9181 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     9241 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     9301 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     9361 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     9421 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     9481 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     9541 acaagcgacc tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag
     9601 acgatgacga taagggatcc ggagctacta acttcagcct gctgaagcag gctggggacg
     9661 tggaggagaa ccctggacct aggatggtga gcaagggcga ggagctgttc accggggtgg
     9721 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     9781 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc accaccggca
     9841 agctgcccgt gccctggccc accctcgtga ccaccctggg ctacggcctg cagtgcttcg
     9901 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     9961 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
    10021 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
    10081 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
    10141 tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc cacaacatcg
    10201 aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
    10261 ccgtgctgct gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca
    10321 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
    10381 gcatggacga gctgtacaag taaggatcct aagtcgacaa tcaacctctg gattacaaaa
    10441 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
    10501 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
    10561 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
    10621 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
    10681 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
    10741 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
    10801 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
    10861 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
    10921 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
    10981 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
    11041 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
    11101 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
    11161 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    11221 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    11281 acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc tgatcagcct
    11341 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
    11401 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
    11461 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
    11521 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
    11581 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
    11641 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
    11701 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
    11761 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
    11821 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
    11881 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
    11941 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
    12001 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
    12061 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
    12121 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
    12181 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
    12241 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
    12301 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
    12361 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
    12421 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
    12481 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
    12541 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
    12601 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
    12661 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
    12721 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
    12781 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
    12841 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc
    12901 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
    12961 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
    13021 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
    13081 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
    13141 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
    13201 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
    13261 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
    13321 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
    13381 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
    13441 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
    13501 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
    13561 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
    13621 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
    13681 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
    13741 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
    13801 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
    13861 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
    13921 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
    13981 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
    14041 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
    14101 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
    14161 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
    14221 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
    14281 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
    14341 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
    14401 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
    14461 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
    14521 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
    14581 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
    14641 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
    14701 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
    14761 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
    14821 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
    14881 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
    14941 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
    15001 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
    15061 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
    15121 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
    15181 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
    15241 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
    15301 acatttcccc gaaaagtgcc acctgac
//
