pLX-TRE-dCas9-KRAB-MeCP2-BSD

LOCUS       pLX-TRE-dCas9-KRAB-MeCP2-BSD               16734 bp ds-DNA     circular SYN 17-NOV-2023
DEFINITION  Inducible knockdown of gene expression in human cells for pooled 
            scRNA-seq experiments.
ACCESSION   .
VERSION     .
KEYWORDS    pLX-TRE-dCas9-KRAB-MeCP2-BSD
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16734)
  TITLE     A Tet-ON 3G inducible enhanced CRISPR-repressor (dCas9-KRAB-MeCP2) 
            in a lentiviral vector with blasticidin selection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 16734)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 17, 2023 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..16734
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2050..2167
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2929..3307
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    2937..2955
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2973..2991
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3009..3027
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3045..3063
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3081..3099
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3117..3135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3153..3171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3216..3240
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3334..3343
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3340..7443
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7447..7473
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7492..7512
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7519..7539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7579..7764
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             7798..7818
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7828..8685
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(8740..8759)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8784..8832
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        9063..9274
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             9320..10063
                     /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"
     CDS             10064..10117
                     /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"
     CDS             10139..10537
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    10598..11186
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10651..10671)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11069..11080)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(11189..11205)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11190..11206)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11296..11529
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(11554..11572)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(11555..11572)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(11862..11881)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(12095..12114)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12214..12236
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(12274..12292)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        12360..12464
                     /gene="bla"
                     /label=AmpR promoter
     CDS             12465..13325
                     /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(12683..12702)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      13496..14084
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13985..14004
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     14238..14255
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(14325..14345)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        14330..14659
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      14510..14645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     14572..14591
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     intron          15793..15858
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             15988..16008
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    16433..16567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(16470..16489)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     16524..16543
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccaaa tggcagtatt
     2041 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2101 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2161 aaattttcgg gtttattaca gggacagcag agatccagtt tggatcgata agcttgatat
     2221 cgaattcggg aggtggtccc tgcagttacg ccaatgataa cccccgccag aaaaatctta
     2281 gtagccttcc ctttttgttt tccgtgcccc aactcggcgg attgactcgg ccccttccgg
     2341 aaacacccga atcaacttct agtcaaatta ttgttcacgc cgcaatgacc cacccctggc
     2401 ccgcgtctgt ggaactgacc cctggtgtac aggagagttc gctgctgaaa gtggtcccaa
     2461 aggggtacta gtttttaagc tcccaactcc ccctccccca gcgtctggag gattccacac
     2521 cctcgcaccg caggggcgag gaagtgggcg gagtccggtt ttggcgccag ccgctgaggc
     2581 tgccaagcag aaaagccacc gctgaggaga ctccggtcac tgtcctcgcc ccgcctcccc
     2641 cttccctccc cttggggacc accgggcgcc acgccgcgaa cggtaagtgc cgcggtcgtc
     2701 ggcgcctccg ccctccccct agggccccaa ttcccagcgg gcgcggcgcg cggccccgcg
     2761 cgcagctccc ggctccctcc cccttcggat gtggcttgag ctgtaggcgc ggagtcctgc
     2821 agccccgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc
     2881 cacctgtagg tttggcaagc tagctgcatt cgtcttcaag aattcctcga gtttactccc
     2941 tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag aacgtatgca
     3001 gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta tcagtgatag
     3061 agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca gtttactccc
     3121 tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag aacgtataag
     3181 ctttaggcgt gtacggtggg cgcctataaa agcagagctc gtttagtgaa ccgtcagatc
     3241 gcctggagca attccacaac acttttgtct tataccaact ttccgtacca cttcctaccc
     3301 tcgtaaaggt ctagagctag cgaattcgaa tttgccacca tggacaagaa gtactccatt
     3361 gggctcgcta tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg
     3421 ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc
     3481 attggcgccc tcctgttcga ctccggggag acggccgaag ccacgcggct caaaagaaca
     3541 gcacggcgca gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt
     3601 aatgagatgg ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg
     3661 gaggaggata aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg
     3721 taccatgaaa agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat
     3781 aaggctgact tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac
     3841 ttcctcatcg agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa
     3901 ctggttcaga cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac
     3961 gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca
     4021 cagctccctg gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg
     4081 ctgaccccca actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc
     4141 aaagacacct acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca
     4201 gacctttttt tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga
     4261 gtgaacacgg agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag
     4321 caccaccaag acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac
     4381 aaggaaattt tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca
     4441 agccaggagg aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag
     4501 gagctgctgg taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat
     4561 ggaagcatcc cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag
     4621 gatttctacc cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg
     4681 ataccctact atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc
     4741 aaatcagaag agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct
     4801 gcccagtcct tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg
     4861 cttcctaaac actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc
     4921 aaatacgtca cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct
     4981 atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac
     5041 tatttcaaaa agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc
     5101 aacgcatccc tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg
     5161 gacaatgagg agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa
     5221 gatagggaga tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc
     5281 atgaaacagc tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc
     5341 aatgggatcc gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga
     5401 tttgccaacc ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac
     5461 atccagaaag cacaagtttc tggccagggg gacagtcttc acgagcacat cgctaatctt
     5521 gcaggtagcc cagctatcaa aaagggaata ctgcagaccg ttaaggtcgt ggatgaactc
     5581 gtcaaagtaa tgggaaggca taagcccgag aatatcgtta tcgagatggc ccgagagaac
     5641 caaactaccc agaagggaca gaagaacagt agggaaagga tgaagaggat tgaagagggt
     5701 ataaaagaac tggggtccca aatccttaag gaacacccag ttgaaaacac ccagcttcag
     5761 aatgagaagc tctacctgta ctacctgcag aacggcaggg acatgtacgt ggatcaggaa
     5821 ctggacatca atcggctctc cgactacgac gtggctgcta tcgtgcccca gtcttttctc
     5881 aaagatgatt ctattgataa taaagtgttg acaagatccg ataaagctag agggaagagt
     5941 gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa attattggcg gcagctgctg
     6001 aacgccaaac tgatcacaca acggaagttc gataatctga ctaaggctga acgaggtggc
     6061 ctgtctgagt tggataaagc cggcttcatc aaaaggcagc ttgttgagac acgccagatc
     6121 accaagcacg tggcccaaat tctcgattca cgcatgaaca ccaagtacga tgaaaatgac
     6181 aaactgattc gagaggtgaa agttattact ctgaagtcta agctggtctc agatttcaga
     6241 aaggactttc agttttataa ggtgagagag atcaacaatt accaccatgc gcatgatgcc
     6301 tacctgaatg cagtggtagg cactgcactt atcaaaaaat atcccaagct tgaatctgaa
     6361 tttgtttacg gagactataa agtgtacgat gttaggaaaa tgatcgcaaa gtctgagcag
     6421 gaaataggca aggccaccgc taagtacttc ttttacagca atattatgaa ttttttcaag
     6481 accgagatta cactggccaa tggagagatt cggaagcgac cacttatcga aacaaacgga
     6541 gaaacaggag aaatcgtgtg ggacaagggt agggatttcg cgacagtccg gaaggtcctg
     6601 tccatgccgc aggtgaacat cgttaaaaag accgaagtac agaccggagg cttctccaag
     6661 gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg cacgcaaaaa agattgggac
     6721 cccaagaaat acggcggatt cgattctcct acagtcgctt acagtgtact ggttgtggcc
     6781 aaagtggaga aagggaagtc taaaaaactc aaaagcgtca aggaactgct gggcatcaca
     6841 atcatggagc gatcaagctt cgaaaaaaac cccatcgact ttctcgaggc gaaaggatat
     6901 aaagaggtca aaaaagacct catcattaag cttcccaagt actctctctt tgagcttgaa
     6961 aacggccgga aacgaatgct cgctagtgcg ggcgagctgc agaaaggtaa cgagctggca
     7021 ctgccctcta aatacgttaa tttcttgtat ctggccagcc actatgaaaa gctcaaaggg
     7081 tctcccgaag ataatgagca gaagcagctg ttcgtggaac aacacaaaca ctaccttgat
     7141 gagatcatcg agcaaataag cgaattctcc aaaagagtga tcctcgccga cgctaacctc
     7201 gataaggtgc tttctgctta caataagcac agggataagc ccatcaggga gcaggcagaa
     7261 aacattatcc acttgtttac tctgaccaac ttgggcgcgc ctgcagcctt caagtacttc
     7321 gacaccacca tagacagaaa gcggtacacc tctacaaagg aggtcctgga cgccacactg
     7381 attcatcagt caattacggg gctctatgaa acaagaatcg acctctctca gctcggtgga
     7441 gacgcctatc cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa
     7501 aaacgcaagg tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc
     7561 aacggcagca gcggatcccg gacactggtg accttcaagg atgtatttgt ggacttcacc
     7621 agggaggagt ggaagctgct ggacactgct cagcagatcg tgtacagaaa tgtgatgctg
     7681 gagaactata agaacctggt ttccttgggt tatcagctta ctaagccaga tgtgatcctc
     7741 cggttggaga agggagaaga gccctcggga ggtggttcgg gtggctctgg atcaagccca
     7801 aagaagaaac ggaaggtgga agcctcagtg caggtgaaaa gggtgctgga aaaatccccc
     7861 ggcaaactcc tcgtgaagat gcccttccag gcttcccctg gcggaaaagg tgaagggggt
     7921 ggcgcaacca catctgccca ggtcatggtc atcaagcgac ctggaaggaa aagaaaggcc
     7981 gaggctgacc ctcaggccat tccaaagaaa cggggacgca agccagggtc cgtggtcgca
     8041 gctgcagcag ctgaggctaa gaaaaaggca gtgaaggaaa gctccatccg cagtgtgcag
     8101 gagactgtcc tgcccatcaa gaagaggaag actagggaga ccgtgtccat cgaggtcaaa
     8161 gaagtggtca agcccctgct cgtgtccacc ctgggcgaaa aatctggaaa ggggctcaaa
     8221 acatgcaagt cacctggacg gaaaagcaag gagtctagtc caaaggggcg ctcaagctcc
     8281 gcttctagtc cccctaaaaa ggaacaccat caccatcacc atcacgccga gtctcctaag
     8341 gctcctatgc cactgctccc accacctcca ccacctgagc cacagtcaag cgaagacccc
     8401 atcagcccac ccgagcctca ggatctgtcc tctagtattt gcaaagagga aaagatgccc
     8461 agagcaggca gcctggagag tgatggctgt ccaaaagaac ccgccaagac ccagcctatg
     8521 gtggcagccg ctgcaactac caccacaacc acaactacca cagtggccga aaaatacaag
     8581 catcgcggcg agggcgaacg aaaggacatt gtgtcaagct ccatgcccag acctaaccgg
     8641 gaggaaccag tcgatagtag gacacccgtg actgagagag tctcatgaaa gggttcgatc
     8701 cctaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga aggagacaat
     8761 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acgggtgttg
     8821 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     8881 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     8941 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcag
     9001 atctgcgaaa tcggatccag ctaaatctaa gtggatctgc gatcgctccg gtgcccgtca
     9061 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     9121 aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     9181 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     9241 tctttttcgc aacgggtttg ccgccagaac acagctgaag cttcgataag ctgtgaccgg
     9301 cgcctactct agagccgcca tgtctagact ggacaagagc aaagtcataa actctgctct
     9361 ggaattactc aatggagtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct
     9421 gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc
     9481 cctgccaatc gagatgctgg acaggcatca tacccactcc tgccccctgg aaggcgagtc
     9541 atggcaagac tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg
     9601 cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga
     9661 aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct
     9721 gtccgccgtg ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc
     9781 aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga aacaagcaat
     9841 tgagctgttc gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat
     9901 atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccgaccgacg cccttgacga
     9961 ttttgactta gacatgctcc cagccgatgc ccttgacgac tttgaccttg atatgctgcc
    10021 tgctgacgct cttgacgatt ttgaccttga catgctcccc ggggagggca gaggaagtct
    10081 tctaacatgc ggtgacgtgg aggagaatcc cggccctgaa ttcatggata gatccggaat
    10141 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
    10201 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
    10261 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
    10321 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
    10381 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
    10441 tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg
    10501 tgaattgctg ccctctggtt atgtgtggga gggctaataa ggccgcgact ctagagtcga
    10561 cctgcaggca tgcaagcttg atatcaagct tatcgataat caacctctgg attacaaaat
    10621 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
    10681 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
    10741 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
    10801 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
    10861 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
    10921 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
    10981 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
    11041 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
    11101 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
    11161 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgaggga attaattcga
    11221 gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
    11281 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
    11341 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
    11401 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
    11461 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
    11521 tctctagcag catctagaat taattccgtg tattctatag tgtcacctaa atcgtatgtg
    11581 tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata tgtacaagcc
    11641 taattgtgta gcatctggct tactgaagca gaccctatca tctctctcgt aaactgccgt
    11701 cagagtcggt ttggttggac gaaccttctg agtttctggt aacgccgtcc cgcacccgga
    11761 aatggtcagc gaaccaatca gcagggtcat cgctagccag atcctctacg ccggacgcat
    11821 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
    11881 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
    11941 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
    12001 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
    12061 gcataaggga gagcgtcgaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
    12121 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
    12181 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
    12241 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
    12301 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
    12361 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
    12421 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
    12481 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
    12541 aaacgctggt gaaagtaaaa gatgctgaag atcaattggg tgcacgagtg ggttacatcg
    12601 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
    12661 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
    12721 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
    12781 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
    12841 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
    12901 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
    12961 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
    13021 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
    13081 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
    13141 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
    13201 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
    13261 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
    13321 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
    13381 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
    13441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
    13501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
    13561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
    13621 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
    13681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
    13741 gtggcgataa gtcgtgtctt acctggttgg actcaagacg atagttaccg gataaggcgc
    13801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
    13861 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
    13921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
    13981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
    14041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
    14101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
    14161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
    14221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
    14281 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgtggaatg tgtgtcagtt
    14341 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
    14401 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
    14461 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
    14521 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
    14581 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
    14641 aggcctaggc ttttgcaaaa agcttggaca caagacaggc ttgcgagata tgtttgagaa
    14701 taccacttta tcccgcgtca gggagaggca gtgcgtaaaa agacgcggac tcatgtgaaa
    14761 tactggtttt tagtgcgcca gatctctata atctcgcgca acctattttc ccctcgaaca
    14821 ctttttaagc cgtagataaa caggctggga cacttcacat gagcgaaaaa tacatcgtca
    14881 cctgggacat gttgcagatc catgcacgta aactcgcaag ccgactgatg ccttctgaac
    14941 aatggaaagg cattattgcc gtaagccgtg gcggtctgta ccgggtgcgt tactggcgcg
    15001 tgaactgggt attcgtcatg tcgataccgt ttgtatttcc agctacgatc acgacaacca
    15061 gcgcgagctt aaagtgctga aacgcgcaga aggcgatggc gaaggcttca tcgttattga
    15121 tgacctggtg gataccggtg gtactgcggt tgcgattcgt gaaatgtatc caaaagcgca
    15181 ctttgtcacc atcttcgcaa aaccggctgg tcgtccgctg gttgatgact atgttgttga
    15241 tatcccgcaa gatacctgga ttgaacagcc gtgggatatg ggcgtcgtat tcgtcccgcc
    15301 aatctccggt cgctaatctt ttcaacgcct ggcactgccg ggcgttgttc tttttaactt
    15361 caggcgggtt acaatagttt ccagtaagta ttctggaggc tgcatccatg acacaggcaa
    15421 acctgagcga aaccctgttc aaaccccgct ttaaacatcc tgaaacctcg acgctagtcc
    15481 gccgctttaa tcacggtgca caaccgcctg tgcagtcggc ccttgatggt aaaaccatcc
    15541 ctcactggta tcgcatgatt aaccgtctga tgtggatctg gcgcggcatt gacccacgcg
    15601 aaatcctcga cgtccaggca cgtattgtga tgagcgatgc cgaacgtacc gacgatgatt
    15661 tatacgatac ggtgattggc taccgtggcg gcaactggat ttatgagtgg gccccggatc
    15721 tttgtgaagg aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt
    15781 taaagctcta aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta
    15841 attgtttgtg tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat
    15901 gcctttaatg aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct
    15961 actgctgact ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag
    16021 gactttcctt cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt
    16081 gcttgctttg ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg
    16141 gaaaaatatt ctgtaacctt tataagtagg cataacagtt ataatcataa catactgttt
    16201 tttcttactc cacacaggca tagagtgtct gctattaata actatgctca aaaattgtgt
    16261 acctttagct ttttaatttg taaaggggtt aataaggaat atttgatgta tagtgccttg
    16321 actagagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
    16381 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
    16441 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
    16501 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
    16561 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc
    16621 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt
    16681 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagt
//