LOCUS       sequence-338713-       16597 bp    DNA     circular SYN 14-APR-2025
DEFINITION  doxycycline inducible BE3 expression for human CLYBL locus knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16597)
  AUTHORS   Coelho MA, Cooper S, Strauss ME, Karakoc E, Bhosle S, Goncalves E, 
            Picco G, Burgold T, Cattaneo CM, Veninga V, Consonni S, Dincer C, 
            Vieira SF, Gibson F, Barthorpe S, Hardy C, Rein J, Thomas M, Marioni
            J, Voest EE, Bassett A, Garnett MJ
  TITLE     Base editing screens map mutations affecting interferon-gamma 
            signaling in cancer.
  JOURNAL   Cancer Cell. 2023 Feb 13;41(2):288-303.e6. doi: 
            10.1016/j.ccell.2022.12.009. Epub 2023 Jan 19.
  PUBMED    36669486
REFERENCE   2  (bases 1 to 16597)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.ccell.2022.12.009"; journalName: "Cancer Cell"; date: 
            "2023-02-13- 13"; volume: "41"; issue: "2"; pages: "288-303.e6"
COMMENT     Sequence Label: iBE3 CLYBL
FEATURES             Location/Qualifiers
     source          1..16597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     266..285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2052..2429
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2059..2077
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2095..2113
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2131..2149
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2167..2185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2203..2221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2239..2257
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2275..2293
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2338..2362
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2439..2448
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2451..2471
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2472..3155
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             join(3204..3227,3228..3230,3231..7304)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     RBS             7217..7225
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             7317..7565
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             7578..7598
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7605..7625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    7658..7779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7695..7714)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7749..7768
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7828..7844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7828..7844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7841..7863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7852..7868
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7876..7882,7883..7900,7901..7906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7921..7942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8059..8076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     8368..8387
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    8386..8441
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     8487..8506
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(8597..9343)
                     /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(9375..9394)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          9402..10419
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(9456..9478)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(10421..10698)
                     /label=chicken beta-actin promoter
     enhancer        10700..11079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    11085..11309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     11298..11315
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    11331..11364
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      12142..12151
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(12200..12226)
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             complement(12242..12268)
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             complement(12275..12328)
                     /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"
     LTR             12797..13065
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(13078..13289)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     polyA_signal    13325..13549
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(13554..14354)
                     /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="GPRIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(13665..13684)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     14275..14294
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(14349..14402)
                     /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    14426..14451
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    14452..14485
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(15343..15411,15412..16203)
                     /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(15561..15580)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(16374..16597,1..365)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
       61 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      121 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      181 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      241 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      301 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      361 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc gaattcgccc ttagagcaga
      421 gccaggaacc cctgtaggga aggggcagga gagccagggg catgagatgg tggacgagga
      481 agggggacag ggaagcctga gcgcctctcc tgggcttgcc aaggactcaa acccagaagc
      541 ccagagcagg gccttaggga agcgggacgg ctctgttgga gagtccagta ttgaattagt
      601 gatagataca cagaaaaaaa gcaaattgaa taggtgaatg gataaacaaa ctgtggaaca
      661 tccagacaat gaaatattca ccaataaaaa gaaatgagct atcaagccac attggctatg
      721 cccttcacat ttaaggagga accttaaatg catattgcca aattttaaaa gctagtctga
      781 aaatgctaca gactgtatga ttccaattat atgacatttt agaaaaggca aaactataga
      841 gatcagtggt tgccagcggc tcatggaggc aggatgaatt ggtggaacac agggatgctt
      901 tcagggtaat gaaaatatct tgtgtgatcc tgtaataatg gatacgtggc ataatgcatt
      961 tgttaaaacc tgtaaactag ccaggtatag tggctttttt gcacctatag tctcagctac
     1021 ttggtaggct gagacaggaa gatcacttga gcccaggact tcaagaccag cctgggtaat
     1081 atagtaagac cccatctcta ttttttaaag aaattaagaa agaaaagaaa cccatcaact
     1141 gtataacaca aagaatcgta atgtaaacta cagtcctgag taatgaaaca tttttaaaga
     1201 aataaataaa ttcaaggcaa ttattaactt caggaaaaac agtaaagtaa acataagtat
     1261 acaaatcaaa catggctcag ttgtgaaaat atttacaagg tcactttaag taacattgaa
     1321 tgttgatata atcccaaaat atatttatgt tggaaggatg aggagattta attaagggag
     1381 gtggtccctg cagttacgcc aatgataacc cccgccagaa aaatcttagt agccttccct
     1441 ttttgttttc cgtgccccaa ctcggcggat tgactcggcc ccttccggaa acacccgaat
     1501 caacttctag tcaaattatt gttcacgccg caatgaccca cccctggccc gcgtctgtgg
     1561 aactgacccc tggtgtacag gagagttcgc tgctgaaagt ggtcccaaag gggtactagt
     1621 ttttaagctc ccaactcccc ctcccccagc gtctggagga ttccacaccc tcgcaccgca
     1681 ggggcgagga agtgggcgga gtccggtttt ggcgccagcc gctgaggctg ccaagcagaa
     1741 aagccaccgc tgaggagact ccggtcactg tcctcgcccc gcctccccct tccctcccct
     1801 tggggaccac cgggcgccac gccgcgaacg gtaagtgccg cggtcgtcgg cgcctccgcc
     1861 ctccccctag ggccccaatt cccagcgggc gcggcgcgcg gcccctcccc ccgccgggcg
     1921 cgcgcccgct gccccgccct tcgtggccgc ccggcgtggg cggtgccacc cctccccccg
     1981 gcggccccgc gcgcagctcc cggctccctc ccccttcgga tgtggcttga gctgtaggcg
     2041 cggagggtac cagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta
     2101 tcagtgatag agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga
     2161 gtttactccc tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag
     2221 aacgtatcta cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta
     2281 tcagtgatag agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc
     2341 tcgtttagtg aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa
     2401 ctttccgtac cacttcctac cctcgtaaag gatccgccgc caccatggct cctaagaaaa
     2461 agcggaaggt gtctagcgag acaggccctg tggccgtgga tcctacactg cggagaagaa
     2521 tcgagcccca cgagttcgag gtgttcttcg accccagaga gctgcggaaa gagacatgcc
     2581 tgctgtacga gatcaactgg ggcggcagac actctatctg gcggcacaca agccagaaca
     2641 ccaacaagca cgtggaagtg aactttatcg agaagtttac gaccgagcgg tacttctgcc
     2701 ccaacaccag atgcagcatc acctggtttc tgagctggtc cccttgcggc gagtgcagca
     2761 gagccatcac cgagtttctg tccagatatc cccacgtgac cctgttcatc tatatcgccc
     2821 ggctgtacca ccacgccgat cctagaaata gacagggact gcgcgacctg atcagcagcg
     2881 gagtgaccat ccagatcatg accgagcaag agagcggcta ctgctggcgg aacttcgtga
     2941 actacagccc cagcaacgaa gcccactggc ctagatatcc tcacctgtgg gtccgactgt
     3001 acgtgctgga actgtactgc atcatcctgg gcctgcctcc atgcctgaac atcctgagaa
     3061 gaaagcagcc tcagctgacc ttcttcacaa tcgccctgca gagctgccac taccagagac
     3121 tgcctccaca catcctgtgg gccaccggac tgaagtctgg ctctgagaca cctggcacaa
     3181 gcgagtctgc cacacctgag tctgacaaga aatactcaat cgggctggcc atcggaacta
     3241 actcagtggg gtgggcagtc attactgacg agtacaaagt gccaagcaag aaatttaagg
     3301 tcctgggcaa caccgatagg cactccatca agaaaaatct gattggggcc ctgctgttcg
     3361 actctggaga gacagctgaa gcaactagac tgaaaaggac tgctagaagg cgctataccc
     3421 ggcgaaagaa tcgcatctgc tacctgcagg agattttctc taacgaaatg gccaaggtgg
     3481 acgatagttt ctttcatcgg ctggaggaat cattcctggt cgaggaagat aagaaacacg
     3541 agagacatcc tatctttgga aacattgtgg acgaggtcgc ttatcacgaa aaatacccca
     3601 ccatctatca tctgcgcaag aaactggtgg actctacaga taaagcagac ctgcggctga
     3661 tctatctggc cctggctcac atgattaagt tcagaggcca ttttctgatc gagggagatc
     3721 tgaacccaga caatagcgat gtggacaagc tgttcatcca gctggtccag acatacaatc
     3781 agctgtttga ggaaaaccct attaatgcat ctggcgtgga cgcaaaagcc atcctgagtg
     3841 ccaggctgtc taagagtaga aggctggaga acctgatcgc tcagctgcca ggcgaaaaga
     3901 aaaacggcct gtttggaaat ctgattgcac tgtcactggg actgacacct aacttcaaga
     3961 gcaattttga tctggccgag gacgctaaac tgcagctgag caaggacact tatgacgatg
     4021 acctggataa cctgctggct cagatcggag atcagtacgc agacctgttc ctggccgcta
     4081 agaatctgtc tgacgctatc ctgctgagtg atattctgcg ggtgaacacc gagattacaa
     4141 aagcccctct gtcagctagc atgatcaaga gatatgacga gcaccatcag gatctgaccc
     4201 tgctgaaggc actggtgcgc cagcagctgc ccgagaagta caaggaaatc ttctttgatc
     4261 agagtaagaa cgggtacgcc ggttatattg acggcggagc ttcacaggag gaattctaca
     4321 agtttatcaa acctattctg gagaagatgg acggcaccga ggaactgctg gtgaaactga
     4381 atcgcgagga cctgctgcgc aagcagcgga catttgataa cggctccatc ccccaccaga
     4441 ttcatctggg agagctgcac gcaatcctgc gacgacagga agacttctac ccatttctga
     4501 aggataaccg cgagaagatc gaaaaaattc tgaccttccg gatcccttac tatgtggggc
     4561 ccctggcaag gggtaattcc cgctttgcct ggatgacacg gaaatctgag gaaacaatca
     4621 ctccttggaa cttcgaggaa gtggtcgata agggagcttc cgcacagtct ttcatcgaga
     4681 gaatgacaaa cttcgacaaa aacctgccaa atgagaaagt gctgcctaag cacagtctgc
     4741 tgtacgagta tttcacagtc tataacgaac tgactaaggt gaaatacgtc accgagggga
     4801 tgaggaagcc cgccttcctg agcggtgaac agaagaaagc tatcgtggac ctgctgttta
     4861 aaaccaatcg caaggtgaca gtcaagcagc tgaaggagga ctacttcaag aaaattgaat
     4921 gtttcgattc tgtggagatc agtggcgtcg aagacagatt taacgcttct ctgggaacct
     4981 accacgatct gctgaagatc attaaggata aagacttcct ggacaacgag gaaaatgagg
     5041 atatcctgga agacattgtg ctgaccctga cactgtttga ggatcgcgaa atgatcgagg
     5101 aacggctgaa aacttatgcc catctgttcg atgacaaggt gatgaaacag ctgaagcgaa
     5161 gaaggtacac cggctgggga cgactgagca gaaagctgat caacggcatt cgggacaaac
     5221 agagtggaaa gactatcctg gactttctga aatcagatgg cttcgctaac agaaatttta
     5281 tgcagctgat tcacgatgac agcctgacct tcaaagagga tatccagaag gcacaggtgt
     5341 ccgggcaggg tgactctctg cacgagcata tcgcaaacct ggccgggtcc cccgccatca
     5401 agaaaggtat tctgcagacc gtgaaggtgg tcgatgagct ggtgaaagtc atgggcaggc
     5461 ataagccaga aaacatcgtg attgagatgg cccgcgaaaa tcagaccaca cagaaaggac
     5521 agaagaacag ccgcgagcgg atgaaaagga tcgaggaagg cattaaggaa ctgggatccc
     5581 agatcctgaa agagcaccct gtggaaaaca ctcagctgca gaatgagaag ctgtatctgt
     5641 actatctgca gaatgggcgg gatatgtacg tggaccagga gctggatatt aaccgactgt
     5701 ctgattacga cgtggatcat atcgtcccac agtcattcct gaaagatgac agcattgaca
     5761 ataaggtgct gacccggagt gacaaaaacc gaggaaagag tgataatgtc ccttcagagg
     5821 aagtggtcaa gaaaatgaag aactactgga gacagctgct gaatgccaaa ctgatcacac
     5881 agcgaaagtt tgataacctg actaaagctg agagaggggg tctgtcagaa ctggacaaag
     5941 caggcttcat caagcgacag ctggtggaga ccagacagat cacaaagcac gtcgctcaga
     6001 ttctggatag caggatgaac acaaagtacg atgagaatga caaactgatc cgcgaagtga
     6061 aggtcattac tctgaagtca aaacttgtga gcgacttcag aaaggatttc cagttctaca
     6121 aagtcaggga gatcaacaat tatcaccatg ctcatgacgc atacctgaac gcagtggtcg
     6181 ggaccgccct gattaagaaa taccccaaac tggagagcga attcgtgtac ggtgactata
     6241 aggtgtacga tgtcagaaaa atgatcgcca agagtgagca ggaaattgga aaagccaccg
     6301 ctaagtattt cttttactca aacatcatga atttctttaa gactgagatc accctggcaa
     6361 atggggaaat ccgaaagaga ccactgattg agactaacgg cgagaccgga gaaatcgtgt
     6421 gggacaaggg tagggatttt gccacagtgc gcaaggtcct gtccatgcct caagtgaata
     6481 ttgtcaagaa aacagaggtg cagactggcg gattcagtaa ggaatcaatt ctgcccaaac
     6541 ggaactctga taagctgatc gcccgaaaga aagactggga tcccaagaaa tatgggggtt
     6601 tcgactcccc aacagtggct tactctgtcc tggtggtcgc aaaggtggag aaggggaaaa
     6661 gcaagaaact gaaatccgtc aaggagctgc tgggtatcac tattatggag aggagctcct
     6721 tcgagaagaa ccccatcgat tttctggagg ctaaaggcta taaggaagtg aagaaagacc
     6781 tgatcattaa actgccaaag tacagcctgt ttgagctgga aaacggaagg aagcgaatgc
     6841 tggcatccgc aggagagctg cagaagggta atgaactggc cctgccttct aagtacgtga
     6901 acttcctgta tctggctagc cactacgaga agctgaaagg ctcccccgag gataacgaac
     6961 agaaacagct gtttgtggag cagcacaagc attatctgga cgagatcatt gaacagatta
     7021 gcgagttctc caaaagagtg atcctggctg acgcaaatct ggataaggtc ctgagcgcat
     7081 acaacaaaca cagagataag ccaatcaggg agcaggccga aaatatcatt catctgttca
     7141 ctctgaccaa cctgggagcc cctgcagcct tcaagtattt tgacactacc atcgatcgga
     7201 aacgatacac atccactaag gaggtgctgg acgctaccct gattcaccag agcattaccg
     7261 gcctgtatga aacaaggatt gacctgtctc agctgggggg cgactctggc ggcagcacca
     7321 acctgagcga catcatcgag aaagagacag gcaagcagct ggtcatccaa gagtccatcc
     7381 tgatgctgcc tgaagaggtg gaagaagtga tcggcaacaa gcccgagtcc gacatcctgg
     7441 tgcacaccgc ctacgatgag agcaccgacg agaacgtgat gctgctgacc tctgacgccc
     7501 ctgagtacaa gccttgggct ctcgtgatcc aggacagcaa cggcgagaac aagatcaaga
     7561 tgctgagcgg cggcagccct aagaagaagc ggaaggtcga ggaccccaag aaaaaacgga
     7621 aggtgtgata aaaactcgcg aatcgatcgc ccaccccaac ttgtttattg cagcttataa
     7681 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     7741 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     7801 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     7861 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     7921 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     7981 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     8041 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     8101 agcggtatca gctcactcaa aggacggccg cacatgtaag gaaaatttta gggatgttaa
     8161 agaaaaaaat aacacaaaac aaaatataaa aaaaatctaa cctcaagtca aggcttttct
     8221 atggaataag gaatggacag cagggggctg tttcatatac tgatgacctc tttatagcca
     8281 acctttgttc atggcagcca gcatatgggc atatgttgcc aaactctaaa ccaaatactc
     8341 attctgatgt tttaaatgat ttgccctccc atatgtcctt ccgagtgaga gacacaaaaa
     8401 attccaacac actattgcaa tgaaaataaa tttcctttat tagccagaag tcagatgctc
     8461 aaggggcttc atgatgtccc cataattttt ggcagaggga aaaagatctc agtggtattt
     8521 gtgagccagg gcattggcca caccagccac caccttctga taggcagcct gcacctgagg
     8581 agtgaattca cgcgttttag cctggcagca tatcgagatc gaagtcatcg agagcgtcag
     8641 cagggagcat atccagatca aaatcatcca gggcatcggc gggcagcatg tccaggtcga
     8701 agtcgtccag ggcgtcggtg ggtccgccgc tctcgcactt cagctgcttc tcgaggccgc
     8761 agatgatcag ttccaggccg aacaggaagg cgggctcggc gccctgtctg tcgaacagct
     8821 cgatggcctg cttcagcagg gggggcatgc tgtcggtggt gggtgtctct ctttcctctt
     8881 tggcgacctg gtgttcctgt tcttccagca cgcagcccag ggtgaagtgg cccacggcgc
     8941 tcagggcgta cagggcgttt tccaggctga agccctgctg gcacaggaag gccagctggt
     9001 tttccagtgt ctcgtactgc ttctcggtgg gtctggtgcc caggtgcact ttggcgccgt
     9061 cccggtggct cagcagggcg catctgtagc tcttggcgtt gttccgcagg aagtcctgcc
     9121 agctctcgcc ttccagaggg cagctgtggg tgtggtgccg gtccagcatc tcgatgggca
     9181 gggcgtccag cagggcccgc ttgttcttca cgtgccagta cagggtgggc tgttccacgc
     9241 ccagtttctg ggccagcttc cgggtggtca ggccctcgat gcccacgccg ttcagcagtt
     9301 ccagggcgct gttgatcact ttgctcttgt ccagccggct catgtcgaca ttctttgcca
     9361 aaatgatgag acagcacaat aaccagcacg ttgcccagga gctgtaggaa aaagaagaag
     9421 gcatgaacat ggttagcaga ggctctagag ccgccggtca cacgccagaa gccgaacccc
     9481 gccctgcccc gtcccccccg aaggcagccg tccccccgcg gacagccccg aggctggaga
     9541 gggagaaggg gacggcggcg cggcgacgca cgaaggccct ccccgcccat ttccttcctg
     9601 ccggcgccgc accgcttcgc cccgcgcccg ctagaggggg tgcggcggcg cctcccagat
     9661 ttcggctccg cacagatttg ggacaaagga agtccctgcg ccctctcgca cgattaccat
     9721 aaaaggcaat ggctgcggct cgccgcgcct cgacagccgc cggcgctccg ggggccgccg
     9781 cgcccctccc ccgagccctc cccggcccga ggcggccccg ccccgcccgg cacccccacc
     9841 tgccgccacc ccccgcccgg cacggcgagc cccgcgccac gccccgtacg gagccccgca
     9901 cccgaagccg ggccgtgctc agcaactcgg ggaggggggt gcaggggggg ttgcagcccg
     9961 accgacgcgc ccacaccccc tgctcacccc cccacgcaca caccccgcac gcagcctttg
    10021 ttcccctcgc agcccccccc gcaccgcggg gcaccgcccc cggccgcgct cccctcgcgc
    10081 acactgcgga gcgcacaaag ccccgcgccg cgcccgcagc gctcacagcc gccgggcagc
    10141 gcggagccgc acgcggcgct ccccacgcac acacacacgc acgcaccccc cgagccgctc
    10201 cccccgcaca aagggccctc ccggagcccc tcaaggcttt cacgcagcca cagaaaagaa
    10261 acaagccgtc attaaaccaa gcgctaatta cagcccggag gagaagggcc gtcccgcccg
    10321 ctcacctgtg ggagtaacgc ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg
    10381 gagcggggca cggggcgaag gcagcgtcgc agcgactccc cgcccgccgc gcgcttcgct
    10441 ttttataggg ccgccgccgc cgccgcctcg ccataaaagg aaactttcgg agcgcgccgc
    10501 tctgattggc tgccgccgca cctctccgcc tcgccccgcc ccgcccctcg ccccgccccg
    10561 ccccgcctgg cgcgcgcccc cccccccccc ccgcccccat cgctgcacaa aataattaaa
    10621 aaataaataa atacaaaatt gggggtgggg agggggggga gatggggaga gtgaagcaga
    10681 acgtggggct cacctcgacc atggtaatag cgatgactaa tacgtagatg tactgccaag
    10741 taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat
    10801 tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag
    10861 tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg
    10921 gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc
    10981 catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg
    11041 taattgatta ctattaataa ctagtcaata atcaatgtcg agacccatag agcccaccgc
    11101 atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc
    11161 accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta
    11221 ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac
    11281 aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt ataacttcgt
    11341 ataatgtatg ctatacgaag ttatggtacc ttactcgtcc atgccgccgg tggagtggcg
    11401 gccctcggcg cgttcgtact gttccacgat ggtgttactt gtacagctcg tccattccgc
    11461 ctgtgctgtg tctgccttcg gctctctcgt actgttccac gatggtgtag tcctcgttgt
    11521 gggacacgat gtccagcttg atatccacga tgtaggcgcc aggcagctgc acgggtttct
    11581 tggccttgta ggtggttttc acttcggcgg catagtggcc gccatccttc agcttcagcc
    11641 gcttcttgat ctcggacttc agggcgccat cctcagggta cattctttcc tcgctggctt
    11701 cccagcccat ggtctttttc tgcatcacgg ggccatcaga gggaaagttg gtgccccgca
    11761 gcttcacttt gtagatgaac acgccgtcct gcagagagct atcctggttc acgtggatga
    11821 tgccgccgtc ctcgaagttc atcactctct cccatctgaa gccctcgggg aagctcagct
    11881 tgaagtagtc gggaatgtcg gcggggtgct tgatgtacac cttgctgccg tacataaact
    11941 gagggctcag aatgtcccag gcaaaaggca gagggccgcc tttggtcact ttcagcttgg
    12001 cggtctgaaa agcctcgtag ggtctgccct cgccttcgcc ttcaatctcg aactcgtggc
    12061 cgttcacgct gccttccatg tgcaccttga accgcatgaa ttctttgatg atggccatgt
    12121 tgttctcttc gcccttggac accatggtgg cgaccggtag cgtggcgacc ggggtggcgg
    12181 tggctccccc tgaccctcca tccaacttaa cgcgctttgc agcaggacct ccagaaccgc
    12241 cgtcgagttt gacccgttta gcggcaggga ccggtgggcc aggattctcc tcgacgtcac
    12301 cgcatgttag cagacttcct ctgccctctc ccgatcccga tccatttcgg gtatatttga
    12361 gtggaatgag ttcttcaatc gtagttttga ctaacttgcc attcatttct attaacacaa
    12421 aacaatctgg tgcatagtct gaaatcaact ccctacacat accacaagga cttaccactc
    12481 gaatacttct atctacttcg tcagaataag ggtgtctaac agctacaatc gtgtcaaaat
    12541 ccttttgtcc attcgaaact gcactaccaa tcgcaatggc ttctgcacaa acagttactc
    12601 gtcctatata cgcttcaata tgtactgccg aaatgatttc tcctgttttc gtacgaattg
    12661 ccgctcccac atgatgttta ttatcctcat aaagcattgt aatcttctct gtcgctactt
    12721 ctactaattc tagatcctgc tgggagatgt tgaaggtctt catggtggcg ctagcgtagg
    12781 cgccggtcac agcttggatc tgtaacggcg cagaacagaa aacgaaacaa agacgtagag
    12841 ttgagcaagc agggtcaggc aaagcgtgga gagccggctg agtctaggta ggctccaagg
    12901 gagcgccgga caaaggcccg gtctcgacct gagctttaaa cttacctaga cggcggacgc
    12961 agttcaggag gcaccacagg cgggaggcgg cagaacgcga ctcaaccggc gtggatggcg
    13021 gcctcaggta gggcggcggg cgcgtgaagg agagatgcga gcccctcgaa gcttcagctg
    13081 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
    13141 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
    13201 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
    13261 ggggactgtg ggcgatgtgc gctctgccca ctgacgggca ccggagcgat cgcagatcat
    13321 gcatccatag agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc
    13381 ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag acaatgcgat
    13441 gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa
    13501 ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagg tttttagaag
    13561 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa
    13621 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc
    13681 aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa
    13741 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga
    13801 tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc
    13861 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct
    13921 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc
    13981 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac
    14041 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca
    14101 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc
    14161 tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc
    14221 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag
    14281 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt
    14341 tcaatcctag ggccgggatt ctcctccacg tcaccgcatg ttagaagact tcctctgccc
    14401 tctccgctgc cagatctctc gaggccctgt gggaggaaga gaagaggtca gataacttcg
    14461 tataatgtat gctatacgaa gttatgcggc cgctgggaga agggtaagag agctcaatcc
    14521 tcatcttaca tagaaggaag tcagtagata atatcttaca tttaaaagtc aagaaataac
    14581 agtctgacca tactatctag aaatatggaa gttaacagta aggaagttaa aacagttgaa
    14641 agtagtttcg tctagaaaga aagacttggg gaagaacaaa gaaggctaat tttcattatg
    14701 agctttattg taacagaaaa gagaccacta actcagctag gttttcccaa ttcctcaaca
    14761 ctttggggca cagtgtttag tcagagctgt gatcactctc tgagcacatg gattgtatct
    14821 taattttcat ctcattgaaa atatgtacca tttgtccaag tggaaactgg gctgaagcat
    14881 gactgtgacc accaccaaag aaatggctag actcgctaag gtctgctggg gtgacccgtg
    14941 attgtgttgt gcttgatctt gttgagcatg gctcttttaa gtcaccatac ctgcccctgc
    15001 caatgcacca aacaccaact taaagggact ttcagctttt gtaagtcagt ctcagcctta
    15061 cttctgaacc atttctttga cttctttcat gactctgatc ctgtcccagt tttaagcctc
    15121 ccagccacca gtcatgctgt tttgctctgc tatttacatt cttctctacc tactttggac
    15181 ttgatggtat gcctactata ttagttattt attgctgtgt aacaaaccac tctgaaacat
    15241 agtggcttac aaccacaacc attttatttt attatgaaac tgtgggtcag aatgtatatt
    15301 ggcccattgg catgattctc ctgctattga aaaaggaaga gtatgagtat tcaacatttc
    15361 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
    15421 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
    15481 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
    15541 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
    15601 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
    15661 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
    15721 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
    15781 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
    15841 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
    15901 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
    15961 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
    16021 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
    16081 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
    16141 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
    16201 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
    16261 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
    16321 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
    16381 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
    16441 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
    16501 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
    16561 tctgtagcac cgcctacata cctcgctctg ctaatcc
//
