LOCUS       sequence-364403-       12994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dox-inducible CRISPR interference (CRISPRi) knock in construct into 
            the AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12994)
  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 12994)
  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 (Gen2)
FEATURES             Location/Qualifiers
     source          1..12994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          89..1105
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1029..1051
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1113..1132
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1164..1910
                     /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(2001..2020)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2066..2121
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2120..2139)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(2470..2503)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2559..2576
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2693..2714
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2729..2734,2735..2752,2753..2759)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2767..2783
                     /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)."
     primer_bind     2772..2794
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2791..2807
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2791..2807
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2856..2977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2867..2886)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2921..2940
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3051..3071)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3078..3098)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3111..3137)
                     /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             complement(3117..3143)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(3147..4730,4731..4733,4734..7220,7221..7223,
                     7224..7250))
                     /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             complement(3147..7250)
                     /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             complement(7272..7457)
                     /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"
     regulatory      7484..7493
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(7577..7601)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    7646..7664
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7682..7700
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7718..7736
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7754..7772
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7790..7808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7826..7844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7862..7880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    7913..8749
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(8797..9385)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8877..8896)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9556..10347,10348..10416))
                     /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     10179..10198
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    10470..11273
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(11275..11308)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    11315..11340
                     /label=SA
                     /note="splice acceptor site"
     CDS             11364..11417
                     /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"
     regulatory      11433..11442
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             11439..12242
                     /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(11502..11521)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     12112..12131
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(12262..12279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12268..12492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        12498..12877
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 saatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
       61 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
      121 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      181 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg
      241 gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg
      301 cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg
      361 ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg
      421 cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga
      481 ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc
      541 gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
      601 ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
      661 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
      721 ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
      781 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
      841 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
      901 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
      961 tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg
     1021 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt
     1081 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
     1141 tcattttggc aaagaatgtc gacatgagcc ggctggacaa gagcaaagtg atcaacagcg
     1201 ccctggaact gctgaacggc gtgggcatcg agggcctgac cacccggaag ctggcccaga
     1261 aactgggcgt ggaacagccc accctgtact ggcacgtgaa gaacaagcgg gccctgctgg
     1321 acgccctgcc catcgagatg ctggaccggc accacaccca cagctgccct ctggaaggcg
     1381 agagctggca ggacttcctg cggaacaacg ccaagagcta cagatgcgcc ctgctgagcc
     1441 accgggacgg cgccaaagtg cacctgggca ccagacccac cgagaagcag tacgagacac
     1501 tggaaaacca gctggccttc ctgtgccagc agggcttcag cctggaaaac gccctgtacg
     1561 ccctgagcgc cgtgggccac ttcaccctgg gctgcgtgct ggaagaacag gaacaccagg
     1621 tcgccaaaga ggaaagagag acacccacca ccgacagcat gccccccctg ctgaagcagg
     1681 ccatcgagct gttcgacaga cagggcgccg agcccgcctt cctgttcggc ctggaactga
     1741 tcatctgcgg cctcgagaag cagctgaagt gcgagagcgg cggacccacc gacgccctgg
     1801 acgacttcga cctggacatg ctgcccgccg atgccctgga tgattttgat ctggatatgc
     1861 tccctgctga cgctctcgat gacttcgatc tcgatatgct gccaggctaa aacgcgtgaa
     1921 ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     1981 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     2041 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     2101 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     2161 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     2221 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     2281 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     2341 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     2401 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     2461 cccaagctta taacttcgta tagcatacat tatacgaagt tatgcggccg tcctttgagt
     2521 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     2581 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2641 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2701 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2761 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2821 agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca
     2881 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     2941 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgatc gattgcggcc
     3001 gcaccggttt aggatccgct gctgccgttg ctcccactac caatgccgtc cactttccgc
     3061 tttttcttag gatcttccac cttgcgtttt ttcttggggg agccgctgcc caggctggca
     3121 taatcgggca cgtcataggg ataggcgtcg cctcccagct gagacaggtc gatccgtgtc
     3181 tcgtacaggc cggtgatgct ctggtggatc agggtggcgt ccagcacctc tttggtgctg
     3241 gtgtacctct tccggtcgat ggtggtgtca aagtacttga aggcggcagg ggctcccaga
     3301 ttggtcaggg taaacaggtg gatgatattc tcggcctgct ctctgatagg cttgtctctg
     3361 tgcttgttgt aggcgctcag caccttgtcc agattagcgt cggccaggat cactctcttg
     3421 gagaactcgc tgatctgctc gatgatctcg tccaggtagt gtttgtgctg ttccacaaac
     3481 agctgtttct gctcattatc ctcgggggag cccttcagct tctcatagtg gctggccagg
     3541 tacaggaagt tcacatattt ggagggcagg gccagttcgt ttcccttctg cagttcgccg
     3601 gcagaggcca gcattctctt ccggccgttt tccagctcga acagggagta cttaggcagc
     3661 ttgatgatca ggtccttttt cacttctttg tagcccttgg cttccagaaa gtcgatggga
     3721 ttcttctcga agctgcttct ttccatgatg gtgatcccca gcagctcttt cacactcttc
     3781 agtttcttgg acttgccctt ttccactttg gccaccacca gcacagaata ggccacggtg
     3841 gggctgtcga agccgccgta cttcttaggg tcccagtcct tctttctggc gatcagcttg
     3901 tcgctgttcc tcttgggcag gatagactct ttgctgaagc cgcctgtctg cacctcggtc
     3961 tttttcacga tattcacttg gggcatagac agcactttcc gcacggtggc aaagtcccgg
     4021 cccttatccc acacgatctc gcctgtttcg ccgtttgtct cgatcagagg ccgcttccgg
     4081 atctcgccgt tggccagggt aatctcggtc ttgaaaaagt tcatgatgtt gctgtagaag
     4141 aagtacttgg cggtagcctt gccgatttcc tgctcgctct tggcgatcat cttccgcacg
     4201 tcgtacacct tgtagtcgcc gtacacgaac tcgctttcca gcttagggta ctttttgatc
     4261 agggcggttc ccacgacggc gttcaggtag gcgtcgtggg cgtggtggta gttgttgatc
     4321 tcgcgcactt tgtaaaactg gaaatccttc cggaaatcgg acaccagctt ggacttcagg
     4381 gtgatcactt tcacttcccg gatcagtttg tcgttctcgt cgtacttagt gttcatccgg
     4441 gagtccagga tctgtgccac gtgctttgtg atctgccggg tttccaccag ctgtctcttg
     4501 atgaagccgg ccttatccag ttcgctcagg ccgcctctct cggccttggt cagattgtcg
     4561 aacttcctct gggtaatcag cttggcattc agcagctggc gccagtagtt cttcatcttc
     4621 ttcacgacct cttcggaggg cacgttgtcg ctcttgcccc ggttcttgtc gctccgagtc
     4681 agcactttgt tatcgatgga gtcgtccttc agaaagctct gaggcacgat agcgtccaca
     4741 tcgtagtcgg acagccggtt gatgtccagt tcctggtcca cgtacatatc ccgcccattc
     4801 tgcaggtagt acaggtacag cttctcgttc tgcagctggg tgttttccac ggggtgttct
     4861 ttcaggatct ggctgcccag ctctttgatg ccctcttcga tccgcttcat tctctcgcgg
     4921 ctgttcttct gtcccttctg ggtggtctgg ttctctctgg ccatttcgat cacgatgttc
     4981 tcgggcttgt gccggcccat cactttcacg agctcgtcca ccaccttcac tgtctgcagg
     5041 atgcccttct taatggcggg gctgccggcc agattggcaa tgtgctcgtg caggctatcg
     5101 ccctggccgg acacctgggc tttctggatg tcctctttaa aggtcaggct gtcgtcgtgg
     5161 atcagctgca tgaagtttct gttggcgaag ccgtcggact tcaggaaatc caggattgtc
     5221 ttgccggact gcttgtcccg gatgccgttg atcagcttcc ggctcagcct gccccagccg
     5281 gtgtatctcc gccgcttcag ctgcttcatc actttgtcgt cgaacaggtg ggcataggtt
     5341 ttcagccgtt cctcgatcat ctctctgtcc tcaaacagtg tcagggtcag cacgatatct
     5401 tccagaatgt cctcgttttc ctcattgtcc aggaagtcct tgtccttgat aattttcagc
     5461 agatcgtggt atgtgcccag ggaggcgttg aaccgatctt ccacgccgga gatttccacg
     5521 gagtcgaagc actcgatttt cttgaagtag tcctctttca gctgcttcac ggtcactttc
     5581 cggttggtct tgaacagcag gtccacgatg gcttttttct gctcgccgct caggaaggcg
     5641 ggctttctca ttccctcggt cacgtatttc actttggtca gctcgttgta cacggtgaag
     5701 tactcgtaca gcaggctgtg cttgggcagc accttctcgt tgggcaggtt cttatcgaag
     5761 ttggtcatcc gctcgatgaa gctctgggcg ctggcgccct tgtccaccac ttcctcgaag
     5821 ttccaggggg tgatggtttc ctcgctcttt ctggtcatcc aggcgaatct gctgtttccc
     5881 ctggccagag ggcccacgta gtaggggatg cggaaggtca ggatcttctc gatcttttcc
     5941 cggttgtcct tcaggaatgg gtaaaaatct tcctgccgcc gcagaatggc gtgcagctct
     6001 cccaggtgga tctggtgggg gatgctgccg ttgtcgaagg tccgctgctt ccgcagcagg
     6061 tcctctctgt tcagcttcac gagcagttcc tcggtgccgt ccatcttttc caggatgggc
     6121 ttgatgaact tgtagaactc ttcctggctg gctccgccat cgatgtagcc ggcgtagccg
     6181 ttcttgctct ggtcgaagaa aatctctttg tacttctcag gcagctgctg ccgcacgaga
     6241 gctttcagca gggtcaggtc ctggtggtgc tcgtcgtatc tcttgatcat agaggcgctc
     6301 aggggggcct tggtgatctc ggtgttcact ctcaggatgt cgctcagcag gatggcgtcg
     6361 gacaggttct tggcggccag aaacaggtcg gcgtactggt cgccgatctg ggccagcagg
     6421 ttgtccaggt cgtcgtcgta ggtgtccttg ctcagctgca gtttggcatc ctcggccagg
     6481 tcgaagttgc tcttgaagtt gggggtcagg cccaggctca gggcaatcag gttgccgaac
     6541 aggccattct tcttctcgcc gggcagctgg gcgatcagat tttccagccg tctgctcttg
     6601 ctcagtctgg cagacaggat ggccttggcg tccacgccgc tggcgttgat ggggttttcc
     6661 tcgaacagct ggttgtaggt ctgcaccagc tggatgaaca gcttgtccac gtcgctgttg
     6721 tcggggttca ggtcgccctc gatcaggaag tggccccgga acttgatcat gtgggccagg
     6781 gccagataga tcagccgcag gtcggccttg tcggtgctgt ccaccagttt ctttctcagg
     6841 tggtagatgg tggggtactt ctcgtggtag gccacctcgt ccacgatgtt gccgaagatg
     6901 gggtgccgct cgtgcttctt atcctcttcc accaggaagg actcttccag tctgtggaag
     6961 aagctgtcgt ccaccttggc catctcgttg ctgaagatct cttgcagata gcagatccgg
     7021 ttcttccgtc tggtgtatct tcttctggcg gttctcttca gccgggtggc ctcggctgtt
     7081 tctccgctgt cgaacagcag ggcgccgatc aggttcttct tgatgctgtg ccggtcggtg
     7141 ttgcccagca ccttgaattt cttgctgggc accttgtact cgtcggtgat cacggcccag
     7201 cccacagagt tggtgccgat ggccaggccg atgctgtact tcttgtccat cgaccctccg
     7261 ccggaaccgc cgggctcttc tcccttctcc aaccggagga tcacatctgg cttagtaagc
     7321 tgataaccca aggaaaccag gttcttatag ttctccagca tcacatttct gtacacgatc
     7381 tgctgagcag tgtccagcag cttccactcc tccctggtga agtccacaaa tacatccttg
     7441 aaggtcacca gtgtccggga ccaggcagtt agtgacttag catccatggt ggcgttaatt
     7501 aaccttaagt ttacgagggt aggaagtggt acggaaagtt ggtataagac aaaagtgttg
     7561 tggaattgct ccaggcgatc tgacggttca ctaaacgagc tctgctttta taggcgccca
     7621 ccgtacacgc ctaaagctta tacgttctct atcactgata gggagtaaac tggatatacg
     7681 ttctctatca ctgataggga gtaaactgta gatacgttct ctatcactga tagggagtaa
     7741 actggtcata cgttctctat cactgatagg gagtaaactc cttatacgtt ctctatcact
     7801 gatagggagt aaagtctgca tacgttctct atcactgata gggagtaaac tcttcatacg
     7861 ttctctatca ctgataggga gtaaaccact gtggggtgga gatatcagct ttactaggga
     7921 caggattggt gacagaaaag ccccatcctt aggcctcctc cttcctagtc tcctgatatt
     7981 gggtctaacc cccacctcct gttaggcaga ttccttatct ggtgacacac ccccatttcc
     8041 tggagccatc tctctccttg ccagaacctc taaggtttgc ttacgatgga gccagagagg
     8101 atcctgggag ggagagcttg gcagggggtg ggagggaagg gggggatgcg tgacctgccc
     8161 ggttctcagt ggccaccctg cgctaccctc tcccagaacc tgagctgctc tgacgcggct
     8221 gtctggtgcg tttcactgat cctggtgctg cagcttcctt acacttccca agaggagaag
     8281 cagtttggaa aaacaaaatc agaataagtt ggtcctgagt tctaactttg gctcttcacc
     8341 tttctagtcc ccaatttata ttgttcctcc gtgcgtcagt tttacctgtg agataaggcc
     8401 agtagccagc cccgtcctgg cagggctgtg gtgaggaggg gggtgtccgt gtggaaaact
     8461 ccctttgtga gaatggtgcg tcctaggtgt tcaccaggtc gtggccgcct ctactccctt
     8521 tctctttctc catccttctt tccttaaaga gtccccagtg ctatctggga catattcctc
     8581 cgcccagagc agggtcccgc ttccctaagg ccctgctctg ggcttctggg tttgagtcct
     8641 tggcaagccc aggagaggcg ctcaggcttc cctgtccccc ttcctcgtcc accatctcat
     8701 gcccctggct ctcctgcccc ttccctacag gggttcctgg ctctgctcta agggcgaatt
     8761 cgccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     8821 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     8881 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     8941 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     9001 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     9061 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     9121 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     9181 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     9241 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     9301 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     9361 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     9421 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     9481 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     9541 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     9601 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     9661 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     9721 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     9781 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     9841 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     9901 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     9961 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
    10021 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
    10081 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
    10141 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
    10201 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
    10261 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
    10321 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
    10381 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
    10441 aagcatttat cagggttgaa ttcgcccttt gctttctctg accagcattc tctcccctgg
    10501 gcctgtgccg ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat
    10561 ccttccctgc cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct
    10621 gctgtgttgc tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg
    10681 tgatgtcctc tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct
    10741 cacccaaccc catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct
    10801 gtgccatctc tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc
    10861 tccccttctt gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc
    10921 tccctggctt tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc
    10981 tgtggattcg ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct
    11041 ctctagtctg tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc
    11101 tcagctagtc ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc
    11161 tgcctccagg gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa
    11221 tgtggctctg gttctgggta cttttatctg tcccctccac cccacagtgg ggcaataact
    11281 tcgtatagca tacattatac gaagttataa gcttctgacc tcttctcttc ctcccacagg
    11341 gcctcgagag atctggcagc ggagagggca gaggaagtct tctaacatgc ggtgacgtgg
    11401 aggagaatcc cggccctagg ctcgagggta ccgccaccat gggatcggcc attgaacaag
    11461 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
    11521 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
    11581 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
    11641 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
    11701 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
    11761 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
    11821 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
    11881 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
    11941 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gatgatctcg
    12001 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
    12061 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
    12121 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
    12181 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
    12241 gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
    12301 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
    12361 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
    12421 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
    12481 tgggctctat gggtctcgac attgattatt gactagttat taatagtaat caattacggg
    12541 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
    12601 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
    12661 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
    12721 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
    12781 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
    12841 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
    12901 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
    12961 attattttgt gcagcgatgg gggcgggggg gggg
//
