LOCUS       sequence-373524-       13906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AID degron-tagged dCas9 fused with tagRFPt, P2A site and tagBFP, 
            under EF1-alpha promoter in a piggyBac plasmid. Expresses 
            Blasticidin resistance, T2A site and osTIR1 under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13906)
  AUTHORS   Noviello G, Gjaltema RAF, Schulz EG
  TITLE     CasTuner is a degron and CRISPR/Cas-based toolkit for analog tuning 
            of endogenous gene expression.
  JOURNAL   Nat Commun. 2023 Jun 3;14(1):3225. doi: 10.1038/s41467-023-38909-4.
  PUBMED    37270532
REFERENCE   2  (bases 1 to 13906)
  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: "2023-06-3"; volume: "14(1):3225. doi"; pages: " 
            10.1038/s41467-023-38909-4"
COMMENT     Sequence Label: 
            pSLPB3B-AID-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin-T2A-osTIR1
FEATURES             Location/Qualifiers
     source          1..13906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     412..431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(650..754)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(780..1235)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(917..938)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1129..1148
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1362..1384
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1376..1393
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1377..1393
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1403..1422
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1403..1421
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1437..1471
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1793..2004
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2017..2285
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2337..2357
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2367..3050
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="MGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNN
                     EPANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSG
                     GPEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMN
                     ERKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAME
                     KCKSRA"
     CDS             join(3057..3080,3081..3083,3084..5570,5571..5573,
                     5574..7157)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3057..7157
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7158..7178
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7179..7196
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7251..7979
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     CDS             7998..8054
                     /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             8055..8756
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    8771..8995
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     9334..9353
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             9409..9804
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             9814..9867
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             9868..11589
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein"
                     /label=OsTIR1
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="TYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFVG
                     NCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEEL
                     RMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEVE
                     DRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTLA
                     KILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYPL
                     CAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELRV
                     FPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRLC
                     ILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSIA
                     FAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKGC
                     QVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     CDS             11599..11628
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_feature    11632..12220
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11685..11705)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12103..12114)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   12403..12465
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(12495..12515)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(12495..12513)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12534..12550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12534..12550)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12547..12569)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12558..12574
                     /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(12582..12588,12589..12606,12607..12612))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12627..12648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12765..12782)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12936..13524)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13016..13035)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13695..13906,1..580,581..649))
                     /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"
ORIGIN
        1 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
       61 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
      121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
      181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
      241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
      301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta
      781 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
      841 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
      901 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
      961 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1021 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1081 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1141 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1201 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     1261 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     1321 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     1381 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg ggctaaccct
     1441 agaaagatag tctgcgtaaa attgacgcat gcattcttga aatattgctc tctctttcta
     1501 aatagcgcga atccgtcgct gtgcatttag gacatctcag tcgccgcttg gagctcccgt
     1561 gaggcgtgct tgtcaatgcg gtaagtgtca ctgattttga actataacga ccgcgtgagt
     1621 caaaatgacg catgattatc ttttacgtga cttttaagat ttaactcata cgataattat
     1681 attgttattt catgttctac ttacgtgata acttattata tatatatttt cttgttatag
     1741 atatcaacta gaatacgcgt aaggatctgc gatcgctccg gtgcccgtca gtgggcagag
     1801 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc
     1861 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     1921 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     1981 aacgggtttg ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc
     2041 gcccgccgcc ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc
     2101 gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga
     2161 ccgggccttt gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt
     2221 tgcctgaccc tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac
     2281 agatccaagc tgtgaccggc gcctactcta gagctagcga attcgccgcc accatgccta
     2341 agaaaaagag gaaggtgagc ggccgcatgg gcagtgtcga gctgaatctg agggagactg
     2401 agctgtgtct tggtcttccc ggtggagata cagtggctcc ggtaaccgga aacaagagag
     2461 ggttctcaga gacggttgat ctgaagctaa atctgaataa tgagcctgca aacaaggaag
     2521 gatctacgac tcatgacgtc gtgacttttg attccaagga gaagagtgct tgtcctaaag
     2581 atccagccaa acctccggcc aaggcacaag ttgtgggatg gccaccggtg agatcatacc
     2641 ggaagaacgt gatggtttcc tgccaaaaat caagcggtgg cccggaggcg gcggcgttcg
     2701 tgaaggtatc aatggacgga gcaccgtact tgaggaaaat cgatttgagg atgtataaaa
     2761 gctacgatga gctttctaat gctttgtcca acatgttcag ctcttttacc atgggcaaac
     2821 atggaggaga agaaggaatg atagacttca tgaatgagag gaaattgatg gatttggtga
     2881 atagctggga ctatgttccc tcttatgaag acaaagacgg tgattggatg ctcgtcggcg
     2941 acgttccttg gccaatgttc gtcgatacat gcaagcgttt acgtctcatg aaaggatcgg
     3001 atgccattgg tctcgctccg agggcgatgg agaagtgcaa gagcagagct ggatccgaca
     3061 agaagtacag catcggcctg gccatcggca ccaactctgt gggctgggcc gtgatcaccg
     3121 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     3181 tcaagaagaa cctgatcggc gccctgctgt tcgacagcgg agaaacagcc gaggccaccc
     3241 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     3301 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     3361 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     3421 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     3481 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     3541 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     3601 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     3661 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     3721 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcggc aacctgattg
     3781 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     3841 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     3901 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     3961 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     4021 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     4081 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     4141 tcgatggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     4201 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     4261 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     4321 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     4381 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     4441 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     4501 acaagggcgc cagcgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     4561 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtacaacg
     4621 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     4681 agcagaaaaa agccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     4741 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     4801 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     4861 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     4921 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     4981 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     5041 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     5101 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     5161 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     5221 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     5281 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     5341 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     5401 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     5461 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     5521 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac gctatcgtgc
     5581 ctcagagctt tctgaaggac gactccatcg ataacaaagt gctgactcgg agcgacaaga
     5641 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     5701 ggcgccagct gctgaatgcc aagctgatta cccagaggaa gttcgacaat ctgaccaagg
     5761 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     5821 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     5881 acgacgagaa cgacaaactg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     5941 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     6001 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     6061 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     6121 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     6181 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     6241 tcgagacaaa cggcgaaaca ggcgagatcg tgtgggataa gggccgggac tttgccaccg
     6301 tgcggaaagt gctgtctatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     6361 gcggcttcag caaagagtct atcctgccca agaggaacag cgacaagctg atcgccagaa
     6421 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     6481 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     6541 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     6601 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     6661 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     6721 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     6781 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     6841 aacactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     6901 ccgacgctaa tctggacaag gtgctgagcg cctacaacaa gcacagagac aagcctatca
     6961 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     7021 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     7081 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     7141 ctcagctggg aggcgacccc aagaagaagc gcaaggtgca ccaccaccac caccacggag
     7201 gaggtggaag cggaggagga ggaagcggag gaggaggtag ctggcgcgcc gtgtctaagg
     7261 gcgaagagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtgaaca
     7321 accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     7381 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacca
     7441 gcttcatgta cggcagcaga accttcatca accacaccca gggcatcccc gacttcttta
     7501 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     7561 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     7621 tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     7681 aggccaacac cgagatgctg taccccgctg acggcggcct ggaaggcaga accgacatgg
     7741 ccctgaagct cgtgggcggg ggccacctga tctgcaactt caagaccaca tacagatcca
     7801 agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggaccac agactggaaa
     7861 gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct gtggccagat
     7921 actgcgacct ccctagcaaa ctggggcaca aacttaatgg catggacgag ctgtacaagt
     7981 taattaacgg aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     8041 agaaccctgg acctatgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg
     8101 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     8161 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     8221 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     8281 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     8341 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     8401 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     8461 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     8521 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga
     8581 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     8641 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     8701 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aattagacgc
     8761 gtattaacga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     8821 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     8881 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     8941 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcctct
     9001 ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc
     9061 ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg
     9121 cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca
     9181 gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct
     9241 ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg
     9301 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     9361 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcaat ggccaagcct
     9421 ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa cagcatcccc
     9481 atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg catcttcact
     9541 ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt gctgggcact
     9601 gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa tgagaacagg
     9661 ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca tcctgggatc
     9721 aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg tgaattgctg
     9781 ccctctggtt atgtgtggga gggcggaagc ggagagggca gaggaagtct tctaacatgc
     9841 ggtgacgtgg aggagaatcc cggccctaca tactttcctg aagaggtcgt cgaacacatt
     9901 tttagcttcc tgcctgcaca gagagataga aacacagtga gcctggtctg caaagtgtgg
     9961 tacgagatcg aacgcctgag ccggagagga gtgttcgtcg gcaactgcta tgctgtgaga
    10021 gcaggcaggg tcgccgctag gtttccaaat gtgcgcgcac tgaccgtcaa ggggaaaccc
    10081 cacttcgccg actttaacct ggtgccccct gattggggag gatacgccgg cccttggatc
    10141 gaggcagccg ctcgcggctg tcatggactg gaggaactgc gcatgaagcg aatggtggtc
    10201 tctgacgaaa gtctggagct gctggctcgg agcttcccta ggtttcgcgc actggtgctg
    10261 atttcttgcg aaggcttcag caccgatgga ctggcagccg tggcctccca ctgtaagctg
    10321 ctgcgggagc tggacctcca ggagaatgaa gtggaggata gaggccccag atggctgtct
    10381 tgcttcccag actcatgtac cagcctggtg tccctgaact ttgcctgcat caaaggcgaa
    10441 gtgaatgctg ggtccctgga gcggctggtc tcaagaagcc ccaacctgag gtctctgcgg
    10501 ctgaaccgga gcgtgagcgt ggacactctg gctaagattc tgctgagaac ccctaacctg
    10561 gaggatctgg gaaccggcaa tctgacagac gatttccaga cagaatccta ctttaaactg
    10621 acttctgccc tggagaagtg taaaatgctg aggagtctgt caggattctg ggatgcttca
    10681 cccgtgtgcc tgagctttat ctaccctctg tgtgcacagc tgacaggcct gaacctgagc
    10741 tatgcaccaa ccctggacgc cagtgatctg acaaagatga tctcacgctg cgtgaaactc
    10801 cagcgactgt gggtgctgga ctgtatttcc gataaggggc tccaggtggt cgccagctcc
    10861 tgcaaggacc tccaggagct gagagtgttc ccatctgatt tttacgtggc cggatatagt
    10921 gctgtcactg aggaaggcct ggtggcagtc tcactgggat gcccaaagct gaacagcctg
    10981 ctgtatttct gtcatcagat gactaatgct gcactggtga ccgtcgccaa gaactgccct
    11041 aatttcaccc gatttcggct gtgtattctg gaaccaggca aacccgacgt ggtcacatcc
    11101 cagccactgg atgaagggtt tggagctatc gtgagagagt gcaagggact ccagaggctg
    11161 agcatttccg gcctgctgac agacaaagtg ttcatgtaca tcggcaagta tgctaagcag
    11221 ctggagatgc tgagcattgc atttgccgga gactccgata agggcatgat gcacgtgatg
    11281 aacgggtgta agaatctgcg aaaactggaa atccgggaca gccctttcgg ggatgccgct
    11341 ctgctgggaa actttgccag atacgagaca atgaggagcc tgtggatgtc tagttgcaat
    11401 gtgactctga agggctgtca ggtcctggct agtaaaatgc ctatgctgaa cgtggaagtc
    11461 attaatgagc gggacgggtc taacgaaatg gaggaaaatc atggcgacct gccaaaggtg
    11521 gagaaactgt atgtgtatcg gaccaccgca ggggcaagag atgatgctcc caactttgtg
    11581 aagattctgg gaagcggaga acaaaaactc atctcagaag aggatctgtg aaatcaacct
    11641 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
    11701 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
    11761 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
    11821 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
    11881 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
    11941 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
    12001 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    12061 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    12121 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct ccgccttcgc
    12181 cctcagacga gtcggatctc cctttgggcc gcctccccgc cggtacctaa aagttttgtt
    12241 actttataga agaaattttg agtttttgtt tttttttaat aaataaataa acataaataa
    12301 attgtttgtt gaatttatta ttagtatgta agtgtaaata taataaaact taatatctat
    12361 tcaaattaat aaataaacct cgatatacag accgataaaa cacatgcgtc aattttacgc
    12421 atgattatct ttaacgtacg tcacaatatg attatctttc tagggttaat ctagtattaa
    12481 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
    12541 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
    12601 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
    12661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
    12721 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
    12781 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
    12841 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
    12901 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
    12961 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
    13021 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
    13081 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
    13141 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
    13201 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
    13261 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
    13321 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
    13381 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
    13441 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
    13501 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
    13561 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
    13621 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
    13681 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
    13741 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
    13801 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
    13861 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtg
//
