LOCUS       sequence-243534-       13224 bp    DNA     circular SYN 15-DEC-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 13224)
  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 13224)
  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.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..13224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          1..1017
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     941..963
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1025..1044
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1076..1822
                     /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(1913..1932)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1978..2033
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2032..2051)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    complement(2382..2415)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2471..2488
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2605..2626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2641..2646,2647..2664,2665..2671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2679..2695
                     /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     2684..2706
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2703..2719
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2703..2719
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2768..2889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2779..2798)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2833..2852
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    2917..2941
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2950..3144)
                     /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"
     CDS             complement(3214..3234)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3241..3261)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3274..3300)
                     /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(3280..3306)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(3310..4893,4894..4896,4897..7383,7384..7386,
                     7387..7413))
                     /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(3310..7413)
                     /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"
     regulatory      7410..7419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(7468..7488)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(7539..7563)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(7645..7669)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    7714..7732
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7750..7768
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7786..7804
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7822..7840
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7858..7876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7894..7912
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7930..7948
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    7981..8817
                     /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(8865..9453)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8945..8964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9624..10415,10416..10484))
                     /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     10247..10266
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    10538..11341
                     /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(11343..11376)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    11383..11408
                     /label=SA
                     /note="splice acceptor site"
     CDS             11432..11485
                     /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      11501..11510
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             11507..12310
                     /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(11570..11589)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     12180..12199
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(12330..12347)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12336..12560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        12566..12945
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12947..13224
                     /label=chicken beta-actin promoter
ORIGIN
        1 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
       61 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      121 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      181 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      241 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      301 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
      361 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
      421 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
      481 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
      541 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
      601 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
      661 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
      721 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
      781 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
      841 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
      901 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
      961 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1021 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatg tcgacatgag
     1081 ccggctggac aagagcaaag tgatcaacag cgccctggaa ctgctgaacg gcgtgggcat
     1141 cgagggcctg accacccgga agctggccca gaaactgggc gtggaacagc ccaccctgta
     1201 ctggcacgtg aagaacaagc gggccctgct ggacgccctg cccatcgaga tgctggaccg
     1261 gcaccacacc cacagctgcc ctctggaagg cgagagctgg caggacttcc tgcggaacaa
     1321 cgccaagagc tacagatgcg ccctgctgag ccaccgggac ggcgccaaag tgcacctggg
     1381 caccagaccc accgagaagc agtacgagac actggaaaac cagctggcct tcctgtgcca
     1441 gcagggcttc agcctggaaa acgccctgta cgccctgagc gccgtgggcc acttcaccct
     1501 gggctgcgtg ctggaagaac aggaacacca ggtcgccaaa gaggaaagag agacacccac
     1561 caccgacagc atgccccccc tgctgaagca ggccatcgag ctgttcgaca gacagggcgc
     1621 cgagcccgcc ttcctgttcg gcctggaact gatcatctgc ggcctcgaga agcagctgaa
     1681 gtgcgagagc ggcggaccca ccgacgccct ggacgacttc gacctggaca tgctgcccgc
     1741 cgatgccctg gatgattttg atctggatat gctccctgct gacgctctcg atgacttcga
     1801 tctcgatatg ctgccaggct aaaacgcgtg aattcactcc tcaggtgcag gctgcctatc
     1861 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     1921 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     1981 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     2041 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     2101 caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     2161 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     2221 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     2281 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     2341 ctcttctctt atggagatcc ctcgacctgc agcccaagct tataacttcg tatagcatac
     2401 attatacgaa gttatgcggc cgtcctttga gtgagctgat accgctcgcc gcagccgaac
     2461 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     2521 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2581 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     2641 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     2701 cacaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca
     2761 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     2821 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     2881 aaacaagttg gggtgggcga tcgattgcgg ccatcaacca ctttgtacaa gaaagctggg
     2941 tctagattat accagccaag gttcttcccc cttttcgagc ctaaggatca catccggttt
     3001 cgtcaactgg tatccgagac tgacgaggtt cttgtaattc tcaagcatga catttcgata
     3061 cacaatttgt tgcgcagtat ccagcaattt ccactcctca cgtgtaaagt ccacaaacac
     3121 gtccttgaac gtaaccagtg ttctcgacca tgccgtaagt gatttcgcgt ccatggatcc
     3181 gctgctgccg ttgctcccac taccaatgcc gtccactttc cgctttttct taggatcttc
     3241 caccttgcgt tttttcttgg gggagccgct gcccaggctg gcataatcgg gcacgtcata
     3301 gggataggcg tcgcctccca gctgagacag gtcgatccgt gtctcgtaca ggccggtgat
     3361 gctctggtgg atcagggtgg cgtccagcac ctctttggtg ctggtgtacc tcttccggtc
     3421 gatggtggtg tcaaagtact tgaaggcggc aggggctccc agattggtca gggtaaacag
     3481 gtggatgata ttctcggcct gctctctgat aggcttgtct ctgtgcttgt tgtaggcgct
     3541 cagcaccttg tccagattag cgtcggccag gatcactctc ttggagaact cgctgatctg
     3601 ctcgatgatc tcgtccaggt agtgtttgtg ctgttccaca aacagctgtt tctgctcatt
     3661 atcctcgggg gagcccttca gcttctcata gtggctggcc aggtacagga agttcacata
     3721 tttggagggc agggccagtt cgtttccctt ctgcagttcg ccggcagagg ccagcattct
     3781 cttccggccg ttttccagct cgaacaggga gtacttaggc agcttgatga tcaggtcctt
     3841 tttcacttct ttgtagccct tggcttccag aaagtcgatg ggattcttct cgaagctgct
     3901 tctttccatg atggtgatcc ccagcagctc tttcacactc ttcagtttct tggacttgcc
     3961 cttttccact ttggccacca ccagcacaga ataggccacg gtggggctgt cgaagccgcc
     4021 gtacttctta gggtcccagt ccttctttct ggcgatcagc ttgtcgctgt tcctcttggg
     4081 caggatagac tctttgctga agccgcctgt ctgcacctcg gtctttttca cgatattcac
     4141 ttggggcata gacagcactt tccgcacggt ggcaaagtcc cggcccttat cccacacgat
     4201 ctcgcctgtt tcgccgtttg tctcgatcag aggccgcttc cggatctcgc cgttggccag
     4261 ggtaatctcg gtcttgaaaa agttcatgat gttgctgtag aagaagtact tggcggtagc
     4321 cttgccgatt tcctgctcgc tcttggcgat catcttccgc acgtcgtaca ccttgtagtc
     4381 gccgtacacg aactcgcttt ccagcttagg gtactttttg atcagggcgg ttcccacgac
     4441 ggcgttcagg taggcgtcgt gggcgtggtg gtagttgttg atctcgcgca ctttgtaaaa
     4501 ctggaaatcc ttccggaaat cggacaccag cttggacttc agggtgatca ctttcacttc
     4561 ccggatcagt ttgtcgttct cgtcgtactt agtgttcatc cgggagtcca ggatctgtgc
     4621 cacgtgcttt gtgatctgcc gggtttccac cagctgtctc ttgatgaagc cggccttatc
     4681 cagttcgctc aggccgcctc tctcggcctt ggtcagattg tcgaacttcc tctgggtaat
     4741 cagcttggca ttcagcagct ggcgccagta gttcttcatc ttcttcacga cctcttcgga
     4801 gggcacgttg tcgctcttgc cccggttctt gtcgctccga gtcagcactt tgttatcgat
     4861 ggagtcgtcc ttcagaaagc tctgaggcac gatagcgtcc acatcgtagt cggacagccg
     4921 gttgatgtcc agttcctggt ccacgtacat atcccgccca ttctgcaggt agtacaggta
     4981 cagcttctcg ttctgcagct gggtgttttc cacggggtgt tctttcagga tctggctgcc
     5041 cagctctttg atgccctctt cgatccgctt cattctctcg cggctgttct tctgtccctt
     5101 ctgggtggtc tggttctctc tggccatttc gatcacgatg ttctcgggct tgtgccggcc
     5161 catcactttc acgagctcgt ccaccacctt cactgtctgc aggatgccct tcttaatggc
     5221 ggggctgccg gccagattgg caatgtgctc gtgcaggcta tcgccctggc cggacacctg
     5281 ggctttctgg atgtcctctt taaaggtcag gctgtcgtcg tggatcagct gcatgaagtt
     5341 tctgttggcg aagccgtcgg acttcaggaa atccaggatt gtcttgccgg actgcttgtc
     5401 ccggatgccg ttgatcagct tccggctcag cctgccccag ccggtgtatc tccgccgctt
     5461 cagctgcttc atcactttgt cgtcgaacag gtgggcatag gttttcagcc gttcctcgat
     5521 catctctctg tcctcaaaca gtgtcagggt cagcacgata tcttccagaa tgtcctcgtt
     5581 ttcctcattg tccaggaagt ccttgtcctt gataattttc agcagatcgt ggtatgtgcc
     5641 cagggaggcg ttgaaccgat cttccacgcc ggagatttcc acggagtcga agcactcgat
     5701 tttcttgaag tagtcctctt tcagctgctt cacggtcact ttccggttgg tcttgaacag
     5761 caggtccacg atggcttttt tctgctcgcc gctcaggaag gcgggctttc tcattccctc
     5821 ggtcacgtat ttcactttgg tcagctcgtt gtacacggtg aagtactcgt acagcaggct
     5881 gtgcttgggc agcaccttct cgttgggcag gttcttatcg aagttggtca tccgctcgat
     5941 gaagctctgg gcgctggcgc ccttgtccac cacttcctcg aagttccagg gggtgatggt
     6001 ttcctcgctc tttctggtca tccaggcgaa tctgctgttt cccctggcca gagggcccac
     6061 gtagtagggg atgcggaagg tcaggatctt ctcgatcttt tcccggttgt ccttcaggaa
     6121 tgggtaaaaa tcttcctgcc gccgcagaat ggcgtgcagc tctcccaggt ggatctggtg
     6181 ggggatgctg ccgttgtcga aggtccgctg cttccgcagc aggtcctctc tgttcagctt
     6241 cacgagcagt tcctcggtgc cgtccatctt ttccaggatg ggcttgatga acttgtagaa
     6301 ctcttcctgg ctggctccgc catcgatgta gccggcgtag ccgttcttgc tctggtcgaa
     6361 gaaaatctct ttgtacttct caggcagctg ctgccgcacg agagctttca gcagggtcag
     6421 gtcctggtgg tgctcgtcgt atctcttgat catagaggcg ctcagggggg ccttggtgat
     6481 ctcggtgttc actctcagga tgtcgctcag caggatggcg tcggacaggt tcttggcggc
     6541 cagaaacagg tcggcgtact ggtcgccgat ctgggccagc aggttgtcca ggtcgtcgtc
     6601 gtaggtgtcc ttgctcagct gcagtttggc atcctcggcc aggtcgaagt tgctcttgaa
     6661 gttgggggtc aggcccaggc tcagggcaat caggttgccg aacaggccat tcttcttctc
     6721 gccgggcagc tgggcgatca gattttccag ccgtctgctc ttgctcagtc tggcagacag
     6781 gatggccttg gcgtccacgc cgctggcgtt gatggggttt tcctcgaaca gctggttgta
     6841 ggtctgcacc agctggatga acagcttgtc cacgtcgctg ttgtcggggt tcaggtcgcc
     6901 ctcgatcagg aagtggcccc ggaacttgat catgtgggcc agggccagat agatcagccg
     6961 caggtcggcc ttgtcggtgc tgtccaccag tttctttctc aggtggtaga tggtggggta
     7021 cttctcgtgg taggccacct cgtccacgat gttgccgaag atggggtgcc gctcgtgctt
     7081 cttatcctct tccaccagga aggactcttc cagtctgtgg aagaagctgt cgtccacctt
     7141 ggccatctcg ttgctgaaga tctcttgcag atagcagatc cggttcttcc gtctggtgta
     7201 tcttcttctg gcggttctct tcagccgggt ggcctcggct gtttctccgc tgtcgaacag
     7261 cagggcgccg atcaggttct tcttgatgct gtgccggtcg gtgttgccca gcaccttgaa
     7321 tttcttgctg ggcaccttgt actcgtcggt gatcacggcc cagcccacag agttggtgcc
     7381 gatggccagg ccgatgctgt acttcttgtc catggtggcg atcccgggcc cgcggtaccg
     7441 caagcttgat atcctgcaga cgcgtcccac cttgcgcttc ttcttgggca tggtggcgcg
     7501 cgtagcctag gcctgtcgac tgaattggtt ccggcgccag cctgcttttt tgtacaaact
     7561 tgttgattaa ccttaagttt acgagggtag gaagtggtac ggaaagttgg tataagacaa
     7621 aagtgttgtg gaattgctcc aggcgatctg acggttcact aaacgagctc tgcttttata
     7681 ggcgcccacc gtacacgcct aaagcttata cgttctctat cactgatagg gagtaaactg
     7741 gatatacgtt ctctatcact gatagggagt aaactgtaga tacgttctct atcactgata
     7801 gggagtaaac tggtcatacg ttctctatca ctgataggga gtaaactcct tatacgttct
     7861 ctatcactga tagggagtaa agtctgcata cgttctctat cactgatagg gagtaaactc
     7921 ttcatacgtt ctctatcact gatagggagt aaaccactgt ggggtggaga tatcagcttt
     7981 actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc
     8041 ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc
     8101 ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc
     8161 cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg
     8221 acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg
     8281 acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag
     8341 aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc
     8401 tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag
     8461 ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt
     8521 ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct
     8581 actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca
     8641 tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt
     8701 tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac
     8761 catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag
     8821 ggcgaattcg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     8881 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     8941 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     9001 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     9061 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     9121 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     9181 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     9241 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     9301 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     9361 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     9421 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     9481 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     9541 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     9601 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     9661 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     9721 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     9781 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     9841 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     9901 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     9961 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    10021 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    10081 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    10141 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    10201 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    10261 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    10321 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    10381 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    10441 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    10501 tattattgaa gcatttatca gggttgaatt cgccctttgc tttctctgac cagcattctc
    10561 tcccctgggc ctgtgccgct ttctgtctgc agcttgtggc ctgggtcacc tctacggctg
    10621 gcccagatcc ttccctgccg cctccttcag gttccgtctt cctccactcc ctcttcccct
    10681 tgctctctgc tgtgttgctg cccaaggatg ctctttccgg agcacttcct tctcggcgct
    10741 gcaccacgtg atgtcctctg agcggatcct ccccgtgtct gggtcctctc cgggcatctc
    10801 tcctccctca cccaacccca tgccgtcttc actcgctggg ttcccttttc cttctccttc
    10861 tggggcctgt gccatctctc gtttcttagg atggccttct ccgacggatg tctcccttgc
    10921 gtcccgcctc cccttcttgt aggcctgcat catcaccgtt tttctggaca accccaaagt
    10981 accccgtctc cctggcttta gccacctctc catcctcttg ctttctttgc ctggacaccc
    11041 cgttctcctg tggattcggg tcacctctca ctcctttcat ttgggcagct cccctacccc
    11101 ccttacctct ctagtctgtg ctagctcttc cagccccctg tcatggcatc ttccaggggt
    11161 ccgagagctc agctagtctt cttcctccaa cccgggcccc tatgtccact tcaggacagc
    11221 atgtttgctg cctccaggga tcctgtgtcc ccgagctggg accaccttat attcccaggg
    11281 ccggttaatg tggctctggt tctgggtact tttatctgtc ccctccaccc cacagtgggg
    11341 caataacttc gtatagcata cattatacga agttataagc ttctgacctc ttctcttcct
    11401 cccacagggc ctcgagagat ctggcagcgg agagggcaga ggaagtcttc taacatgcgg
    11461 tgacgtggag gagaatcccg gccctaggct cgagggtacc gccaccatgg gatcggccat
    11521 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
    11581 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
    11641 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
    11701 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
    11761 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
    11821 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
    11881 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
    11941 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
    12001 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
    12061 tgatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
    12121 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
    12181 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
    12241 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
    12301 gttcttctga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
    12361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
    12421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
    12481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
    12541 ggatgcggtg ggctctatgg gtctcgacat tgattattga ctagttatta atagtaatca
    12601 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
    12661 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
    12721 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
    12781 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
    12841 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
    12901 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc
    12961 acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt
    13021 attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccrggs
    13081 gggggggggs ggggsggggg gsggggsggg gcgaggcgga gaggtgcggc ggcagccaat
    13141 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
    13201 aaaaagcgaa gcgcgcggcg ggcg
//
