LOCUS       sequence-247304-       13833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible CRISPR interference (CRISPRi) knock in construct into 
            the AAVS1 locus with mCherry marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13833)
  AUTHORS   Mandegar MA, Huebsch N, Frolov EB, Shin E, Truong A, Olvera MP, Chan
            AH, Miyaoka Y, Holmes K, Spencer CI, Judge LM, Gordon DE, Eskildsen 
            TV, Villalta JE, Horlbeck MA, Gilbert LA, Krogan NJ, Sheikh SP, 
            Weissman JS, Qi LS, So PL, Conklin BR
  TITLE     CRISPR Interference Efficiently Induces Specific and Reversible Gene
            Silencing in Human iPSCs.
  JOURNAL   Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 
            10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.
  PUBMED    26971820
REFERENCE   2  (bases 1 to 13833)
  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"; doi: 
            "10.1016/j.stem.2016.01.022"; journalName: "Cell Stem Cell"; date: 
            "2016-04-7- 7"; volume: "18"; issue: "4"; pages: "541-53"
COMMENT     Sequence Label: pAAVS1-NDi-CRISPRi (Gen1)
FEATURES             Location/Qualifiers
     source          1..13833
                     /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"
     regulatory      2977..2986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3013..3198
                     /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
                     QLTKPDVILRLEKGEEP"
     CDS             join(3220..3246,3247..3249,3250..5736,5737..5739,
                     5740..7323)
                     /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             3220..7323
                     /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             7327..7353
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7333..7359
                     /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             7372..7392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7399..7419
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7459..7515
                     /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             7516..8226
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(7661..7679)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7929..7948
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    8258..8379
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8295..8314)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8349..8368
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8428..8444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8428..8444)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8441..8463)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8452..8468
                     /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(8476..8482,8483..8500,8501..8506))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8521..8542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8659..8676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8732..8765
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     9096..9115
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    9114..9169
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     9215..9234
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(9325..10071)
                     /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(10103..10122)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          10130..11146
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(10184..10206)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(11147..11424)
                     /label=chicken beta-actin promoter
     enhancer        11426..11805
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    11811..12035
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     12024..12041
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(12061..12864)
                     /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(12172..12191)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     12782..12801
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     regulatory      12861..12870
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(12886..12939)
                     /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    12963..12988
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    12995..13028
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    13030..13833
                     /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 ggagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1201 gcttcagcag agcgcagata ccaaatactg ttcttctagt 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 attaacgcca ccatggatgc taagtcacta
     3001 actgcctggt cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag
     3061 gagtggaagc tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac
     3121 tataagaacc tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg
     3181 gagaagggag aagagcccgg cggttccggc ggagggtcga tggacaagaa gtacagcatc
     3241 ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     3301 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     3361 atcggcgccc tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc
     3421 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     3481 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     3541 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     3601 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     3661 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     3721 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     3781 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     3841 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     3901 cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc
     3961 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     4021 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     4081 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     4141 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     4201 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     4261 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc
     4321 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     4381 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     4441 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     4501 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     4561 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     4621 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc
     4681 gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg
     4741 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg
     4801 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc
     4861 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     4921 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     4981 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     5041 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     5101 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     5161 atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc
     5221 aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     5281 ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac
     5341 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg
     5401 gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc
     5461 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     5521 cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc
     5581 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag
     5641 aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa
     5701 ctggacatca accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg
     5761 aaggacgact ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc
     5821 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg
     5881 aatgccaagc tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc
     5941 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc
     6001 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac
     6061 aaactgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     6121 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc
     6181 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag
     6241 ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag
     6301 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     6361 accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc
     6421 gaaacaggcg agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg
     6481 tctatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     6541 gagtctatcc tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac
     6601 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     6661 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     6721 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     6781 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     6841 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     6901 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     6961 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac
     7021 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     7081 gacaaggtgc tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag
     7141 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     7201 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     7261 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     7321 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
     7381 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc
     7441 aacggcagca gcggatccgc tactaacttc agcctgctga agcaggctgg agacgtggag
     7501 gagaaccctg gacctatggt gagcaagggc gaggaggata acatggccat catcaaggag
     7561 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     7621 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     7681 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     7741 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     7801 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     7861 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     7921 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     7981 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     8041 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     8101 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     8161 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     8221 aagtaaaccg gtgcggccgc aatcgatcgc ccaccccaac ttgtttattg cagcttataa
     8281 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     8341 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     8401 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     8461 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     8521 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     8581 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     8641 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     8701 agcggtatca gctcactcaa aggacggccg cataacttcg tataatgtat gctatacgaa
     8761 gttataagct tgggctgcag gtcgagggat ctccataaga gaagagggac agctatgact
     8821 gggagtagtc aggagaggag gaaaaatctg gctagtaaaa catgtaagga aaattttagg
     8881 gatgttaaag aaaaaaataa cacaaaacaa aatataaaaa aaatctaacc tcaagtcaag
     8941 gcttttctat ggaataagga atggacagca gggggctgtt tcatatactg atgacctctt
     9001 tatagccaac ctttgttcat ggcagccagc atatgggcat atgttgccaa actctaaacc
     9061 aaatactcat tctgatgttt taaatgattt gccctcccat atgtccttcc gagtgagaga
     9121 cacaaaaaat tccaacacac tattgcaatg aaaataaatt tcctttatta gccagaagtc
     9181 agatgctcaa ggggcttcat gatgtcccca taatttttgg cagagggaaa aagatctcag
     9241 tggtatttgt gagccagggc attggccaca ccagccacca ccttctgata ggcagcctgc
     9301 acctgaggag tgaattcacg cgttttagcc tggcagcata tcgagatcga agtcatcgag
     9361 agcgtcagca gggagcatat ccagatcaaa atcatccagg gcatcggcgg gcagcatgtc
     9421 caggtcgaag tcgtccaggg cgtcggtggg tccgccgctc tcgcacttca gctgcttctc
     9481 gaggccgcag atgatcagtt ccaggccgaa caggaaggcg ggctcggcgc cctgtctgtc
     9541 gaacagctcg atggcctgct tcagcagggg gggcatgctg tcggtggtgg gtgtctctct
     9601 ttcctctttg gcgacctggt gttcctgttc ttccagcacg cagcccaggg tgaagtggcc
     9661 cacggcgctc agggcgtaca gggcgttttc caggctgaag ccctgctggc acaggaaggc
     9721 cagctggttt tccagtgtct cgtactgctt ctcggtgggt ctggtgccca ggtgcacttt
     9781 ggcgccgtcc cggtggctca gcagggcgca tctgtagctc ttggcgttgt tccgcaggaa
     9841 gtcctgccag ctctcgcctt ccagagggca gctgtgggtg tggtgccggt ccagcatctc
     9901 gatgggcagg gcgtccagca gggcccgctt gttcttcacg tgccagtaca gggtgggctg
     9961 ttccacgccc agtttctggg ccagcttccg ggtggtcagg ccctcgatgc ccacgccgtt
    10021 cagcagttcc agggcgctgt tgatcacttt gctcttgtcc agccggctca tgtcgacatt
    10081 ctttgccaaa atgatgagac agcacaataa ccagcacgtt gcccaggagc tgtaggaaaa
    10141 agaagaaggc atgaacatgg ttagcagagg ctctagagcc gccggtcaca cgccagaagc
    10201 cgaaccccgc cctgccccgt cccccccgaa ggcagccgtc cccccgcgga cagccccgag
    10261 gctggagagg gagaagggga cggcggcgcg gcgacgcacg aaggccctcc ccgcccattt
    10321 ccttcctgcc ggcgccgcac cgcttcgccc cgcgcccgct agagggggtg cggcggcgcc
    10381 tcccagattt cggctccgca cagatttggg acaaaggaag tccctgcgcc ctctcgcacg
    10441 attaccataa aaggcaatgg ctgcggctcg ccgcgcctcg acagccgccg gcgctccggg
    10501 ggccgccgcg cccctccccc gagccctccc cggcccgagg cggccccgcc ccgcccggca
    10561 cccccacctg ccgccacccc ccgcccggca cggcgagccc cgcgccacgc cccgtacgga
    10621 gccccgcacc cgaagccggg ccgtgctcag caactcgggg aggggggtgc agggggggtt
    10681 gcagcccgac cgacgcgccc acaccccctg ctcacccccc cacgcacaca ccccgcacgc
    10741 agcctttgtt cccctcgcag ccccccccgc accgcggggc accgcccccg gccgcgctcc
    10801 cctcgcgcac actgcggagc gcacaaagcc ccgcgccgcg cccgcagcgc tcacagccgc
    10861 cgggcagcgc ggagccgcac gcggcgctcc ccacgcacac acacacgcac gcaccccccg
    10921 agccgctccc cccgcacaaa gggccctccc ggagcccctc aaggctttca cgcagccaca
    10981 gaaaagaaac aagccgtcat taaaccaagc gctaattaca gcccggagga gaagggccgt
    11041 cccgcccgct cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg
    11101 cggcggcgga gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc
    11161 ttcgcttttt atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg
    11221 cgccgctctg attggctgcc gccgcacctc tccgcctccc cccccccccc cccccccccc
    11281 cccccccccc ccccggcgcg cgcccccccc ccccccccgc ccccatcgct gcacaaaata
    11341 attaaaaaat aaataaatac aaaattgggg gtggggaggg gggggagatg gggagagtga
    11401 agcagaacgt ggggctcacc tcgaccatgg taatagcgat gactaatacg tagatgtact
    11461 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
    11521 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
    11581 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
    11641 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
    11701 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
    11761 accccgtaat tgattactat taataactag tcaataatca atgtcgagac ccatagagcc
    11821 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
    11881 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
    11941 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
    12001 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
    12061 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
    12121 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
    12181 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
    12241 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
    12301 gacgagatca tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
    12361 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
    12421 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
    12481 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
    12541 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
    12601 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
    12661 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
    12721 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
    12781 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
    12841 atcttgttca atggccgatc ccatggtggc ggtaccctcg agcctagggc cgggattctc
    12901 ctccacgtca ccgcatgtta gaagacttcc tctgccctct ccgctgccag atctctcgag
    12961 gccctgtggg aggaagagaa gaggtcagaa gcttataact tcgtataatg tatgctatac
    13021 gaagttattg ccccactgtg gggtggaggg gacagataaa agtacccaga accagagcca
    13081 cattaaccgg ccctgggaat ataaggtggt cccagctcgg ggacacagga tccctggagg
    13141 cagcaaacat gctgtcctga agtggacata ggggcccggg ttggaggaag aagactagct
    13201 gagctctcgg acccctggaa gatgccatga cagggggctg gaagagctag cacagactag
    13261 agaggtaagg ggggtagggg agctgcccaa atgaaaggag tgagaggtga cccgaatcca
    13321 caggagaacg gggtgtccag gcaaagaaag caagaggatg gagaggtggc taaagccagg
    13381 gagacggggt actttggggt tgtccagaaa aacggtgatg atgcaggcct acaagaaggg
    13441 gaggcgggac gcaagggaga catccgtcgg agaaggccat cctaagaaac gagagatggc
    13501 acaggcccca gaaggagaag gaaaagggaa cccagcgagt gaagacggca tggggttggg
    13561 tgagggagga gagatgcccg gagaggaccc agacacgggg aggatccgct cagaggacat
    13621 cacgtggtgc agcgccgaga aggaagtgct ccggaaagag catccttggg cagcaacaca
    13681 gcagagagca aggggaagag ggagtggagg aagacggaac ctgaaggagg cggcagggaa
    13741 ggatctgggc cagccgtaga ggtgacccag gccacaagct gcagacagaa agcggcacag
    13801 gcccagggga gagaatgctg gtcagagaaa gca
//
