LOCUS       sequence-218604-       13225 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Doxycycline inducible dCas9-KRAB targeting construct for AAVS1 
            locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13225)
  AUTHORS   Genga RMJ, Kernfeld EM, Parsi KM, Parsons TJ, Ziller MJ, Maehr R
  TITLE     Single-Cell RNA-Sequencing-Based CRISPRi Screening Resolves 
            Molecular Drivers of Early Human Endoderm Development.
  JOURNAL   Cell Rep. 2019 Apr 16;27(3):708-718.e10. doi: 
            10.1016/j.celrep.2019.03.076.
  PUBMED    30995470
REFERENCE   2  (bases 1 to 13225)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.03"; journalName: "Cell Rep"; date: 
            "2019-04-16- 16"; volume: "27"; issue: "3"; pages: "708-718.e10"
COMMENT     Sequence Label: pAAVS1-TetOn-dCas9-KRAB
FEATURES             Location/Qualifiers
     source          1..13225
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(54..122,123..914)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(272..291)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1085..1673
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1574..1593
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    1721..2557
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    2590..2608
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2626..2644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2662..2680
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2698..2716
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2734..2752
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2770..2788
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2806..2824
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2869..2893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2975..2999
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3050..3070
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      3119..3128
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3125..3151,3152..3154,3155..5641,5642..5644,
                     5645..7228)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3125..7228
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7232..7258
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7238..7264
                     /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="YDVPDYASL"
     CDS             7277..7297
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7304..7324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7394..7588
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     protein_bind    complement(7597..7621)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    7649..7770
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7686..7705)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7740..7759
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7819..7835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7819..7835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7832..7854)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7843..7859
                     /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(7867..7873,7874..7891,7892..7897))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7912..7933
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8050..8067)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8123..8156
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     8487..8506
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    8505..8560
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     8606..8625
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(8715..9461)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(9493..9512)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          9520..10537
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(9574..9596)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(10539..10816)
                     /label=chicken beta-actin promoter
     enhancer        10818..11197
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    11203..11427
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     11416..11433
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(11453..12256)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(11564..11583)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     12174..12193
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     regulatory      12253..12262
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(12278..12331)
                     /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"
     misc_feature    12355..12380
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    12387..12420
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    12422..13225
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 aagggcgaat tcaaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
       61 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
      421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1201 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1681 gcaacgcggc ctttttacgg ttcctggcga attcgccctt agagcagagc caggaacccc
     1741 tgtagggaag gggcaggaga gccaggggca tgagatggtg gacgaggaag ggggacaggg
     1801 aagcctgagc gcctctcctg ggcttgccaa ggactcaaac ccagaagccc agagcagggc
     1861 cttagggaag cgggaccctg ctctgggcgg aggaatatgt cccagatagc actggggact
     1921 ctttaaggaa agaaggatgg agaaagagaa agggagtaga ggcggccacg acctggtgaa
     1981 cacctaggac gcaccattct cacaaaggga gttttccaca cggacacccc cctcctcacc
     2041 acagccctgc caggacgggg ctggctactg gccttatctc acaggtaaaa ctgacgcacg
     2101 gaggaacaat ataaattggg gactagaaag gtgaagagcc aaagttagaa ctcaggacca
     2161 acttattctg attttgtttt tccaaactgc ttctcctctt gggaagtgta aggaagctgc
     2221 agcaccagga tcagtgaaac gcaccagaca gccgcgtcag agcagctcag gttctgggag
     2281 agggtagcgc agggtggcca ctgagaaccg ggcaggtcac gcatcccccc cttccctccc
     2341 accccctgcc aagctctccc tcccaggatc ctctctggct ccatcgtaag caaaccttag
     2401 aggttctggc aaggagagag atggctccag gaaatggggg tgtgtcacca gataaggaat
     2461 ctgcctaaca ggaggtgggg gttagaccca atatcaggag actaggaagg aggaggccta
     2521 aggatggggc ttttctgtca ccaatcctgt ccctagtaaa gctgatatct ccaccccaca
     2581 gtggtttact ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata
     2641 gagaacgtat gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc
     2701 ctatcagtga tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct
     2761 acagtttact ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata
     2821 gagaacgtat aagctttagg cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt
     2881 gaaccgtcag atcgcctgga gcaattccac aacacttttg tcttatacca actttccgta
     2941 ccacttccta ccctcgtaaa cttaaggtta atcaacaagt ttgtacaaaa aagcaggctg
     3001 gcgccggaac caattcagtc gacaggccta ggctacgcgc gccaccatgc ccaagaagaa
     3061 gcgcaaggtg ggacgcgtct gcaggatatc aagcttgcgg taccgcgggc ccgggatcgc
     3121 caccatggac aagaagtaca gcatcggcct ggccatcggc accaactctg tgggctgggc
     3181 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     3241 ccggcacagc atcaagaaga acctgatcgg cgccctgctg ttcgacagcg gagaaacagc
     3301 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     3361 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     3421 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     3481 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     3541 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     3601 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     3661 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     3721 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     3781 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     3841 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     3901 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     3961 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     4021 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     4081 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     4141 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     4201 cgccggctac atcgatggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     4261 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     4321 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     4381 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     4441 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     4501 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     4561 ggaagtggtg gacaagggcg ccagcgccca gagcttcatc gagcggatga ccaacttcga
     4621 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     4681 cgtgtacaac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     4741 cctgagcggc gagcagaaaa aagccatcgt ggacctgctg ttcaagacca accggaaagt
     4801 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     4861 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     4921 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     4981 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     5041 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     5101 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     5161 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     5221 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     5281 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     5341 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     5401 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     5461 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     5521 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     5581 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     5641 cgctatcgtg cctcagagct ttctgaagga cgactccatc gataacaaag tgctgactcg
     5701 gagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     5761 gaagaactac tggcgccagc tgctgaatgc caagctgatt acccagagga agttcgacaa
     5821 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     5881 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     5941 gaacactaag tacgacgaga acgacaaact gatccgggaa gtgaaagtga tcaccctgaa
     6001 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     6061 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     6121 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     6181 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     6241 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     6301 gcggcctctg atcgagacaa acggcgaaac aggcgagatc gtgtgggata agggccggga
     6361 ctttgccacc gtgcggaaag tgctgtctat gccccaagtg aatatcgtga aaaagaccga
     6421 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgacaagct
     6481 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     6541 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     6601 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     6661 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     6721 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     6781 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     6841 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     6901 ggaacagcac aaacactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     6961 agtgatcctg gccgacgcta atctggacaa ggtgctgagc gcctacaaca agcacagaga
     7021 caagcctatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     7081 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     7141 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     7201 gatcgacctg tctcagctgg gaggcgacgc ctatccctat gacgtgcccg attatgccag
     7261 cctgggcagc ggctccccca agaaaaaacg caaggtggaa gatcctaaga aaaagcggaa
     7321 agtggacggc attggtagtg ggagcaacgg cagcagcgga tccatggacg cgaaatcact
     7381 tacggcatgg tcgagaacac tggttacgtt caaggacgtg tttgtggact ttacacgtga
     7441 ggagtggaaa ttgctggata ctgcgcaaca aattgtgtat cgaaatgtca tgcttgagaa
     7501 ttacaagaac ctcgtcagtc tcggatacca gttgacgaaa ccggatgtga tccttaggct
     7561 cgaaaagggg gaagaacctt ggctggtata atctagaccc agctttcttg tacaaagtgg
     7621 ttgatggccg caatcgatcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     7681 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     7741 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     7801 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     7861 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     7921 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     7981 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     8041 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     8101 agctcactca aaggacggcc gcataacttc gtataatgta tgctatacga agttataagc
     8161 ttgggctgca ggtcgaggga tctccataag agaagaggga cagctatgac tgggagtagt
     8221 caggagagga ggaaaaatct ggctagtaaa acatgtaagg aaaattttag ggatgttaaa
     8281 gaaaaaaata acacaaaaca aaatataaaa aaaatctaac ctcaagtcaa ggcttttcta
     8341 tggaataagg aatggacagc agggggctgt ttcatatact gatgacctct ttatagccaa
     8401 cctttgttca tggcagccag catatgggca tatgttgcca aactctaaac caaatactca
     8461 ttctgatgtt ttaaatgatt tgccctccca tatgtccttc cgagtgagag acacaaaaaa
     8521 ttccaacaca ctattgcaat gaaaataaat ttcctttatt agccagaagt cagatgctca
     8581 aggggcttca tgatgtcccc ataatttttg gcagagggaa aaagatctca gtggtatttg
     8641 tgagccaggg cattggccac accagccacc accttctgat aggcagcctg cacctgagga
     8701 gtgaattcac gcgtttagcc tggcagcata tcgagatcga agtcatcgag agcgtcagca
     8761 gggagcatat ccagatcaaa atcatccagg gcatcggcgg gcagcatgtc caggtcgaag
     8821 tcgtccaggg cgtcggtggg tccgccgctc tcgcacttca gctgcttctc gaggccgcag
     8881 atgatcagtt ccaggccgaa caggaaggcg ggctcggcgc cctgtctgtc gaacagctcg
     8941 atggcctgct tcagcagggg gggcatgctg tcggtggtgg gtgtctctct ttcctctttg
     9001 gcgacctggt gttcctgttc ttccagcacg cagcccaggg tgaagtggcc cacggcgctc
     9061 agggcgtaca gggcgttttc caggctgaag ccctgctggc acaggaaggc cagctggttt
     9121 tccagtgtct cgtactgctt ctcggtgggt ctggtgccca ggtgcacttt ggcgccgtcc
     9181 cggtggctca gcagggcgca tctgtagctc ttggcgttgt tccgcaggaa gtcctgccag
     9241 ctctcgcctt ccagagggca gctgtgggtg tggtgccggt ccagcatctc gatgggcagg
     9301 gcgtccagca gggcccgctt gttcttcacg tgccagtaca gggtgggctg ttccacgccc
     9361 agtttctggg ccagcttccg ggtggtcagg ccctcgatgc ccacgccgtt cagcagttcc
     9421 agggcgctgt tgatcacttt gctcttgtcc agccggctca tgtcgacatt ctttgccaaa
     9481 atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa agaagaaggc
     9541 atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc cgaaccccgc
     9601 cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag gctggagagg
     9661 gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt ccttcctgcc
     9721 ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc tcccagattt
     9781 cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg attaccataa
     9841 aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg ggccgccgcg
     9901 cccctccccc gagccctccc cggcccgagg cggccccgcc ccgcccggca cccccacctg
     9961 ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga gccccgcacc
    10021 cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt gcagcccgac
    10081 cgacgcgccc acaccccctg ctcacccccc cacgcacaca ccccgcacgc agcctttgtt
    10141 cccctcgcag ccccccccgc accgcggggc accgcccccg gccgcgctcc cctcgcgcac
    10201 actgcggagc gcacaaagcc ccgcgccgcg cccgcagcgc tcacagccgc cgggcagcgc
    10261 ggagccgcac gcggcgctcc ccacgcacac acacacgcac gcaccccccg agccgctccc
    10321 cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca gaaaagaaac
    10381 aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt cccgcccgct
    10441 cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg cggcggcgga
    10501 gcggggcacg gggcgaaggc agcgtcgcag cgactccccg cccgccgcgc gcttcgcttt
    10561 ttatagggcc gccgccgccg ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc
    10621 tgattggctg ccgccgcacc tctccgcctc gccccgcccc gcccctcgcc ccgccccgcc
    10681 ccgcctggcg cgcgcccccc cccccccccc gcccccatcg ctgcacaaaa taattaaaaa
    10741 ataaataaat acaaaattgg gggtggggag gggggggaga tggggagagt gaagcagaac
    10801 gtggggctca cctcgaccat ggtaatagcg atgactaata cgtagatgta ctgccaagta
    10861 ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt accgtcattg
    10921 acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa gtgggcagtt
    10981 taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt tactatggga
    11041 acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca
    11101 tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa tgaccccgta
    11161 attgattact attaataact agtcaataat caatgtcgag acccatagag cccaccgcat
    11221 ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac
    11281 cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg
    11341 aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa
    11401 cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa actcagaaga
    11461 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
    11521 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
    11581 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
    11641 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
    11701 catcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
    11761 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
    11821 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
    11881 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
    11941 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
    12001 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
    12061 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
    12121 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
    12181 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
    12241 caatggccga tcccatggtg gcggtaccct cgagcctagg gccgggattc tcctccacgt
    12301 caccgcatgt tagaagactt cctctgccct ctccgctgcc agatctctcg aggccctgtg
    12361 ggaggaagag aagaggtcag aagcttataa cttcgtataa tgtatgctat acgaagttat
    12421 tgccccactg tggggtggag gggacagata aaagtaccca gaaccagagc cacattaacc
    12481 ggccctggga atataaggtg gtcccagctc ggggacacag gatccctgga ggcagcaaac
    12541 atgctgtcct gaagtggaca taggggcccg ggttggagga agaagactag ctgagctctc
    12601 ggacccctgg aagatgccat gacagggggc tggaagagct agcacagact agagaggtaa
    12661 ggggggtagg ggagctgccc aaatgaaagg agtgagaggt gacccgaatc cacaggagaa
    12721 cggggtgtcc aggcaaagaa agcaagagga tggagaggtg gctaaagcca gggagacggg
    12781 gtactttggg gttgtccaga aaaacggtga tgatgcaggc ctacaagaag gggaggcggg
    12841 acgcaaggga gacatccgtc ggagaaggcc atcctaagaa acgagagatg gcacaggccc
    12901 cagaaggaga aggaaaaggg aacccagcga gtgaagacgg catggggttg ggtgagggag
    12961 gagagatgcc cggagaggac ccagacacgg ggaggatccg ctcagaggac atcacgtggt
    13021 gcagcgccga gaaggaagtg ctccggaaag agcatccttg ggcagcaaca cagcagagag
    13081 caaggggaag agggagtgga ggaagacgga acctgaagga ggcggcaggg aaggatctgg
    13141 gccagccgta gaggtgaccc aggccacaag ctgcagacag aaagcggcac aggcccaggg
    13201 gagagaatgc tggtcagaga aagca
//
