LOCUS       sequence-391236-       11249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for genomic targeting of a Tetracycline-inducible 
            dCas9-KRAB cassette to the human AAVS1/PPP1R12C locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11249)
  AUTHORS   Luo R, Yan J, Oh JW, Xi W, Shigaki D, Wong W, Cho HS, Murphy D, 
            Cutler R, Rosen BP, Pulecio J, Yang D, Glenn RA, Chen T, Li QV, 
            Vierbuchen T, Sidoli S, Apostolou E, Huangfu D, Beer MA
  TITLE     Dynamic network-guided CRISPRi screen identifies 
            CTCF-loop-constrained nonlinear enhancer gene regulatory activity 
            during cell state transitions.
  JOURNAL   Nat Genet. 2023 Aug;55(8):1336-1346. doi: 
            10.1038/s41588-023-01450-7. Epub 2023 Jul 24.
  PUBMED    37488417
REFERENCE   2  (bases 1 to 11249)
  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: 
            "10.1038/s41588-023-01450-7"; journalName: "Nat Genet"; date: 
            "2023-08"; volume: "55"; issue: "8"; pages: "1336-1346"
COMMENT     Sequence Label: AAVS1-idCas9-KRAB-Hygro donor
FEATURES             Location/Qualifiers
     source          1..11249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..837
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        868..1182
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    876..894
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    912..930
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    947..965
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    983..1001
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1019..1037
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1054..1072
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1090..1108
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1155..1179
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1199..1223
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1248..1257
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1257..1322
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1329..1349
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1374..1397,1398..1400,1401..3887,3888..3890,
                     3891..5474)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1374..5474
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5475..5522
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5523..5549
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5553..5579
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5583..5609
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             5655..5840
                     /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"
     protein_bind    complement(5901..5925)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6087..6168
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6124..6143)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     polyA_signal    6175..6399
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6388..6405
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(6437..7462)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(6437..7462)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(7472..7525)
                     /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    7549..7574
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    7581..8384
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(8398..8417)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8399..8417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8427..8444)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8427..8443)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8436..8458)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8585..9040
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8672..8691)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8882..8903
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9066..9170
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9171..9239,9240..10031)
                     /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(9389..9408)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10202..10790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10691..10710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10944..10961
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11078..11099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11114..11119,11120..11137,11138..11144)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11152..11168
                     /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     11157..11179
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11176..11192
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11176..11192
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11211..11231
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11213..11231
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agagcagagc caggaacccc tgtagggaag gggcaggaga gccaggggca tgagatggtg
       61 gacgaggaag ggggacaggg aagcctgagc gcctctcctg ggcttgccaa ggactcaaac
      121 ccagaagccc agagcagggc cttagggaag cgggaccctg ctctgggcgg aggaatatgt
      181 cccagatagc actggggact ctttaaggaa agaaggatgg agaaagagaa agggagtaga
      241 ggcggccacg acctggtgaa cacctaggac gcaccattct cacaaaggga gttttccaca
      301 cggacacccc cctcctcacc acagccctgc caggacgggg ctggctactg gccttatctc
      361 acaggtaaaa ctgacgcacg gaggaacaat ataaattggg gactagaaag gtgaagagcc
      421 aaagttagaa ctcaggacca acttattctg attttgtttt tccaaactgc ttctcctctt
      481 gggaagtgta aggaagctgc agcaccagga tcagtgaaac gcaccagaca gccgcgtcag
      541 agcagctcag gttctgggag agggtagcgc agggtggcca ctgagaaccg ggcaggtcac
      601 gcatcccccc cttccctccc accccctgcc aagctctccc tcccaggatc ctctctggct
      661 ccatcgtaag caaaccttag aggttctggc aaggagagag atggctccag gaaatggggg
      721 tgtgtcacca gataaggaat ctgcctaaca ggaggtgggg gttagaccca atatcaggag
      781 actaggaagg aggaggccta aggatggggc ttttctgtca ccaatcctgt ccctagttta
      841 attaaccctc tagagcttag tactgtcgag tttactccct atcagtgata gagaacgtat
      901 gtcgagttta ctccctatca gtgatagaga acgatgtcga gtttactccc tatcagtgat
      961 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
     1021 cctatcagtg atagagaacg tatgtcgagt ttatccctat cagtgataga gaacgtatgt
     1081 cgagtttact ccctatcagt gatagagaac gtatgtcgag gtaggcgtgt acggtgggag
     1141 gcctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagaat tccgatcaac
     1201 aagtttgtac aaaaaagcag gctccgcggt tttcaggttg gaccggtgcc accatggact
     1261 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata
     1321 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     1381 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1441 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1501 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1561 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1621 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1681 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1741 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1801 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1861 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1921 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1981 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     2041 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     2101 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2161 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2221 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2281 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2341 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2401 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2461 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2521 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2581 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2641 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2701 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2761 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2821 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2881 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2941 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     3001 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3061 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3121 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3181 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3241 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3301 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3361 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3421 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3481 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3541 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3601 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3661 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3721 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3781 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3841 tggaccagga actggacatc aaccggctgt ccgactacga tgtggacgct atcgtgcctc
     3901 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3961 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     4021 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4081 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4141 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4201 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4261 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4321 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4381 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4441 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4501 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4561 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4621 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4681 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4741 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4801 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4861 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4921 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4981 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     5041 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5101 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5161 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5221 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5281 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5341 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5401 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5461 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     5521 agtatcccta tgatgtgcca gactatgctg gctatccata tgatgttcct gattatgctg
     5581 gataccctta tgatgtgcca gactatgccg gcggtggcgg agggatggat gctaagtcac
     5641 taactgcctg gtcccggaca ctggtgacct tcaaggatgt atttgtggac ttcaccaggg
     5701 aggagtggaa gctgctggac actgctcagc agatcgtgta cagaaatgtg atgctggaga
     5761 actataagaa cctggtttcc ttgggttatc agcttactaa gccagatgtg atcctccggt
     5821 tggagaaggg agaagagccc tagggccggc caggcaaaaa agaaaaagta agaattcaag
     5881 ggtgggcgcg gggcgcgccg acccagcttt cttgtacaaa gtggttgatc cggggatcct
     5941 ctagtcagcg tttatctgca gaattcgccc ttgtttaaac gatcataatc agccatacca
     6001 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     6061 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     6121 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcct cgacccatag
     6181 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
     6241 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct
     6301 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
     6361 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
     6421 aaacgggccc tctagactat tcctttgccc tcggacgagt gctggggcgt cggtttccac
     6481 tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt
     6541 gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc
     6601 aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc
     6661 ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
     6721 agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct
     6781 cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat
     6841 tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc
     6901 agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt
     6961 cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac
     7021 gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc
     7081 taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga acagcgggca
     7141 gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg
     7201 tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg
     7261 caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca
     7321 ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga
     7381 gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg
     7441 cggtgagttc aggctttttc atctgcagcc tagggccggg attctcctcc acgtcaccgc
     7501 atgttagaag acttcctctg ccctctccgc tgccagatct ctcgaggccc tgtgggagga
     7561 agagaagagg tcagggatct gccccactgt ggggtggagg ggacagataa aagtacccag
     7621 aaccagagcc acattaaccg gccctgggaa tataaggtgg tcccagctcg gggacacagg
     7681 atccctggag gcagcaaaca tgctgtcctg aagtggacat aggggcccgg gttggaggaa
     7741 gaagactagc tgagctctcg gacccctgga agatgccatg acagggggct ggaagagcta
     7801 gcacagacta gagaggtaag gggggtaggg gagctgccca aatgaaagga gtgagaggtg
     7861 acccgaatcc acaggagaac ggggtgtcca ggcaaagaaa gcaagaggat ggagaggtgg
     7921 ctaaagccag ggagacgggg tactttgggg ttgtccagaa aaacggtgat gatgcaggcc
     7981 tacaagaagg ggaggcggga cgcaagggag acatccgtcg gagaaggcca tcctaagaaa
     8041 cgagagatgg cacaggcccc agaaggagaa ggaaaaggga acccagcgag tgaagacggc
     8101 atggggttgg gtgagggagg agagatgccc ggagaggacc cagacacggg gaggatccgc
     8161 tcagaggaca tcacgtggtg cagcgccgag aaggaagtgc tccggaaaga gcatccttgg
     8221 gcagcaacac agcagagagc aaggggaaga gggagtggag gaagacggaa cctgaaggag
     8281 gcggcaggga aggatctggg ccagccgtag aggtgaccca ggccacaagc tgcagacaga
     8341 aagcggcaca ggcccagggg agagaatgct ggtcagagaa agcaggtacc caattcgccc
     8401 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     8461 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     8521 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     8581 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     8641 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     8701 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     8761 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     8821 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     8881 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     8941 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     9001 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     9061 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     9121 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     9181 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     9241 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     9301 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     9361 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     9421 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     9481 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     9541 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     9601 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     9661 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     9721 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     9781 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     9841 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     9901 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     9961 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
    10021 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
    10081 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
    10141 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
    10201 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
    10261 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
    10321 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
    10381 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
    10441 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
    10501 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
    10561 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
    10621 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
    10681 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
    10741 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
    10801 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
    10861 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
    10921 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
    10981 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
    11041 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
    11101 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
    11161 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
    11221 ctcactaaag ggaacaaaag ctggagctc
//
